Biogeosciences 15 797ndash808 2018httpsdoiorg105194bg-15-797-2018copy Author(s) 2018 This work is distributed underthe Creative Commons Attribution 40 License

Effects of storage temperature on the physiological characteristicsand vegetative propagation of desiccation-tolerant mossesYuewei Guo and Yunge ZhaoState Key Laboratory of Soil Erosion and Dry-land Farming on the Loess Plateau Institute of Soil and Water ConservationNorthwest A amp F University Yangling 712100 Shaanxi China

Correspondence Yunge Zhao (zyungemsiswcaccn)

Received 29 August 2017 ndash Discussion started 11 September 2017Revised 24 December 2017 ndash Accepted 4 January 2018 ndash Published 8 February 2018

Abstract Mosses as major components of later succes-sional biological soil crusts (biocrusts) play many criti-cal roles in arid and semiarid ecosystems Recently somespecies of desiccation-tolerant mosses have been artifi-cially cultured with the aim of accelerating the recovery ofbiocrusts Revealing the factors that influence the vegeta-tive propagation of mosses which is an important reproduc-tive mode of mosses in dry habitats will benefit the restora-tion of moss crusts In this study three air-dried desiccation-tolerant mosses (Barbula unguiculata Didymodon vinealisand Didymodon tectorum) were hermetically sealed andstored at five temperature levels (0 4 17 25 and 30 C) for40 days Then the vegetative propagation and physiologi-cal characteristics of the three mosses were investigated todetermine the influence of storage temperature on the veg-etative propagation of desiccation-tolerant mosses and themechanism The results showed that the vegetative propaga-tion of the three mosses varied with temperature The mostvariation in vegetative propagation among storage tempera-tures was observed in D tectorum followed by the varia-tion observed in B unguiculata In contrast no significantdifference in propagation among temperatures was found inD vinealis The regenerative capacity of the three mosses in-creased with increasing temperature from 0 to 17 C accom-panied by a decrease in malondialdehyde (MDA) contentand decreased thereafter As the temperature increased thechlorophyll and soluble protein contents increased in B un-guiculata but decreased in D vinealis and D tectorum Asto storage the MDA and soluble sugar contents increased af-ter storage The MDA content of the three mosses increasedat each of the investigated temperatures by more than 50 from the initial values and the soluble sugar content became

higher than before in the three mosses The integrity of cellsand cell membranes is likely the most important factor in-fluencing the vegetative propagation of desiccation-tolerantmosses A 40-day storage period caused cell injury Our re-sults suggest that storage temperature can enhance or sup-press such injury and change the regenerative capacity ofthe three mosses The data indicate that the suitable storagetemperature is 4 C for B unguiculata and 17 C for bothD vinealis and D tectorum

1 Introduction

Biological soil crusts (biocrusts) are composed of micro-scopic (cyanobacteria algae fungi and bacteria) and macro-scopic (lichens mosses) poikilohydric organisms (Belnapet al 2016) Biocrusts are widely distributed in arid andsemiarid ecosystems and play important roles in soil surfacestabilization soil fertility enhancement and soil hydrologyregulation (Belnap and Lange 2003) As major componentsof later successional biocrusts mosses exert much strongerecological functions than cyanobacteria (Seppelt et al 2016Gao et al 2017 Lan et al 2012) Thus some researcherssuggest artificially culturing moss biocrusts on degraded soilsurfaces to accelerate the recovery of degraded arid and semi-arid ecosystems (Belnap and Eldridge 2003 Zhao et al2016) Recently some mosses have been investigated byculturing gametophytes (Jones and Rosentreter 2006 Xiaoet al 2015) However cultivation research on moss crustsremains tentative potentially due to the lack of knowledgeregarding the vegetative propagation of mosses

Published by Copernicus Publications on behalf of the European Geosciences Union

798 Y Guo and Y Zhao Effects of storage temperature on desiccation-tolerant mosses

Vegetative propagation is an important reproduction modeof bryophytes (hornworts liverworts and mosses) in dryhabitats and gametophyte fragments may serve as the dom-inant inoculum in mosses (Mishler 1988 Tian et al 2005)To date several moss cultivation experiments have been con-ducted in which gametophyte fragments are used to estab-lish new colonies in the laboratory and field (Cleavitt 2002Jones and Rosentreter 2006 Xiao et al 2015) All of theseexperiments have demonstrated that artificial cultivation canaccelerate the succession process of moss crusts For exam-ple Antoninka et al (2016) found that the coverage andbiomass of mosses on an artificially inoculated soil surfaceincreased more rapidly than they did on uninoculated soilSome researchers have suggested that inoculation materialshould be mass-produced by vegetative regeneration withrapid development (Jones and Rosentreter 2006 Mishler1988) because of the need for moss biocrusts to inoculatelarge areas The factors that influence the tissue cultivationof mosses have been investigated for many years (Duckettet al 2004 Hoffman 1966 Sabovljevic et al 2003) how-ever the mechanism of moss regeneration remains unclear

After mosses regenerate protonema and gametophytes suf-fer desiccation stress desiccation tolerance (DT) has a criti-cal influence on their survival and restoration abilities (Proc-tor et al 2007) Adult gametophytes of some species canrecover physiological activities and generate new shoots af-ter being stored for more than 10 years in a desiccatedstate (Stark et al 2017 Keever 1957) Desiccation-tolerantmosses can suspend metabolism and maintain cell integrityduring dry periods (Mansour and Hallet 1981 Platt et al1994) then within a few minutes to a few hours after be-ing rehydrated they can resume cellular activity and returnto a normal hydrated state (Platt et al 1994 Pressel et al2006) However the decline and disappearance of the re-generative capacity of Syntrichia ruralis showed that long-term desiccation can cause irreversible damage despite vi-ability differences among individuals (Stark et al 2017) Itremains unclear why the potential for vegetative propagationin mosses can be altered by storage and why recovery abil-ity following drought-induced dormancy varies among mossspecies The lack of knowledge in these areas has impededthe study of moss cultivation

Investigations of DT in mosses have primarily focused onthe mechanism and evolutionary history (Proctor et al 2007Oliver et al 2000) with fewer investigations addressing DTin artificial cultivation However many studies suggest thatDT research can help improve artificial cultivation methodsFor example the impact of desiccation stress on moss re-generation varies with drying time and storage temperature(Keever 1957 Burch 2003) and an understanding of theserelationships may guide research on the regenerative mecha-nism of mosses upon desiccation and their asexual propaga-tion Furthermore DT plays essential roles in moss regener-ation in dry habitats highlighting the potential value of in-vestigating the relationships between the physiological char-

acteristics of mosses and their vegetative propagation Basedon the above observations it can be hypothesized that (1) drystorage impacts the vegetative propagation of desiccation-tolerant mosses (2) changes in vegetative propagation afterstorage involve the influences of storage on the physiologicalcharacteristics of mosses and (3) the degree to which storageaffects vegetative propagation and physiological characteris-tics is related to the storage temperature

In this study three desiccation-tolerant mosses Barbulaunguiculata Didymodon vinealis and Didymodon tectorumwhich are the dominant mosses in biocrust communities inthe Loess Plateau region were stored at five temperatures (04 17 25 and 30 C) for 40 days Then (1) the effect of stor-age temperature on the vegetative propagation of each mossand (2) the changes in physiological indices from before toafter storage including the contents of chlorophyll solublesugar soluble protein and malondialdehyde (MDA) wereinvestigated to reveal the influences of storage temperatureon the vegetative propagation of mosses and the mechanism

2 Materials and methods

21 Study site and moss species

The study was conducted in Ansai Country ShaanxiProvence China (3651prime N 10919prime E) which is located inthe central part of the Loess Plateau The elevation of thesampling plot varies from 1068 to 1309 m The plot hasa typical semiarid continental climate with an average an-nual temperature of 88 C and its average temperature inJanuary and July is minus72 and 228 C respectively The av-erage annual precipitation is 500 mm with 60 or moreof the precipitation falling between June and September(Zhang et al 2011) For the month of November when themoss crusts were collected the average monthly precipita-tion was 1198 mm and the average monthly temperaturewas 988 C (high) to minus364 C (low) (Chinese Central Me-teorological Station 2017) Cyanobacteria and mosses dom-inate the biocrust communities in this region and the cov-erage of moss-dominated biocrusts can reach approximately80 on north-facing slopes in the study region (Zhao et al2014)

The moss taxa used in the study were Barbula unguicu-lata Didymodon vinealis and Didymodon tectorum whichdominated the moss crusts in the plot B unguiculata dom-inated in woodland areas and was found in shaded areasand under vegetation coverage D vinealis was widely dis-tributed in the study site among different water and light en-vironments and the species were collected from croplandsthat had been abandoned for more than 10 years The dom-inant vegetation of the croplands was grasses thus mostD vinealis was exposed to sunlight in the winter D tecto-rum grew on side slopes and was occasionally collected fromunder the shade of vascular plants

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Y Guo and Y Zhao Effects of storage temperature on desiccation-tolerant mosses 799

22 Experimental design

Some of the three moss crusts were used to measure initialvalues of physiological indices (chlorophyll content solublesugar content soluble protein content and MDA content)and germination parameters (gametophyte germination ga-metophyte increment and gametophyte vigor index) imme-diately following their transport to the laboratory The rest ofthe moss crusts were stored at one of five temperature levelsie 0 4 17 25 and 30 C Each temperature was controlledwithin plusmn1 C around the target On the 41st day of storagethe moss crusts were removed and the physiological indicesand germination parameters described above were measured

23 Moss crusts storage and mosses collection

The crusts of three species of mosses were collected frommany colonies and then air-dried in the shade for 24ndash48 hmost of crust samples were dried in the field Then the sam-ples were transported to the laboratory of the State Key Lab-oratory of Soil Erosion and Dry-land Farming on the LoessPlateau in Yangling Shaanxi Province Samples were storedin one of two refrigerators (at 0 or 4 C) or one of threegrowth chambers (at 17 25 and 30 C) Before storage themoss crusts had been placed in resealable plastic bags to pre-vent changes in water content The samples were stored in thedark under light-blocking fabric The water content measure-ments of the moss gametophytes were all less than 10 andthe equilibrating relative humidity during storage was 55 After the 40-day dry period subsamples of desiccated game-tophytes were collected to measure the physiological indicesand germination parameters

24 Measurement of the physiological indices andgermination parameters

241 Physiological indices

Living mature gametophytes of B unguiculata D vinealisand D tectorum were collected from the moss crusts Shortlyafter being rehydrated and washed with deionized water thegametophytes were measured for the contents of chlorophyllsoluble sugar soluble protein and MDA Approximately01 g fresh mass of gametophytes was used to measure thecontents of soluble sugar soluble protein and MDA in eachreplicate whereas the measurements of chlorophyll contentused approximately 005 g fresh mass per replicate The fourindicators were measured by using the following protocolswith three replications

The chlorophyll was extracted by 95 (vv) ethanol andthe solution was boiled at 85 C for 5 min After being cen-trifuged at 4000 rpm for 10 min the chlorophyll in the super-natant was measured at absorbances of 665 and 649 nm witha spectrophotometer (UV-2300 Techcomp Shanghai ChinaWellburn and Lichtenthaler 1984)

After the soluble protein was extracted in ice-cold50 mmolLminus1 phosphate buffer (pH 78) the suspension wascentrifuged at 8000 rpm for 30 min at 4 C and the super-natant was collected The soluble protein was stained withCoomassie brilliant blue G-250 and the absorbance was readat 595 nm (Bradford 1976)

MDA and soluble protein were extracted and centrifugedThen the supernatant was homogenized with 06 (WV )thiobarbituric acid dissolved by 1 molLminus1 NaOH and 10 (WV ) trichloroacetic acid The mixed solution was heatedat 100 C for 20 min and then the absorbance was read at450 523 and 600 nm (Hodges et al 1999) The TechcompUV-2300 spectrophotometer was used to measure the ab-sorbance of the MDA and soluble protein

Soluble sugar was extracted by distilled water at 100 Cfor 30 min After being filtered and diluted the extract wasadded to an anthronendashsulfuric acid solution The mixed so-lution was used to measure the absorbance at 620 nm witha spectrophotometer (UV-1601 Shimadzu Kyoto JapanMorris 1948)

The fresh weight of gametophytes was measured shortlyafter rehydration and dry weight was measured after ovendrying to a constant weight at 70 C (Schonfeld et al 1988)The fresh and dry weights were used to calculate the fourphysiological indices on a dry basis

242 Germination parameters

At the same time as the physiological indices was measuredsome gametophytes of each of the three moss species werecollected to measure the germination parameters The loes-sial soil (uniform soil texture of Calciustepts) collected fromthe study region was used to culture the mosses The soilwas sieved through a 025 mm mesh and placed in each poreof a six-well plate each pore had a diameter of 35 mm anda depth of 12 mm Then the soil water content was adjustedto 23 (WW ) (the field water-holding capacity of the soil)by adding deionized water and the surface was flattened be-fore inoculation Five inocula representing the top 2 mm ofliving mature gametophytes of the mosses were cut rehy-drated washed and placed in each well Thirty inocula wereplaced in each six-well plate as one replication Three six-well plates were established for each moss species In total90 experimental inoculations were established for the mea-surement of germination parameters before and after storageat each of the five temperature levels for each moss speciesMeanwhile three six-well plates without inoculated mosseswere set up as experimental controls for the effect of otherpropagules such as spores in the experimental soil Thesix-well plates were wrapped tightly with transparent plasticfilm to retain the soil moisture Next they were placed intoa growth chamber (AGC-D003N Qiushi Hangzhou China)to incubate The parameters of the growth chamber were setto a 12 h photoperiod (4500ndash5500 Lux) a constant temper-ature of 17 C (plusmn1 C) and a relative humidity of 60ndash70

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800 Y Guo and Y Zhao Effects of storage temperature on desiccation-tolerant mosses

During the incubation period deionized water was suppliedto maintain the soil moisture at 23 The new gametophyteswere counted every 5 days beginning on the day they werefound Five observations were made over the subsequent25 days This paper reports the results of cultivation at thefifth observation No new gametophytes were found in theblank six-well plates during the entire incubation period Itwas difficult to distinguish protonemal germination betweenthe underside of original inocula and the soil substrate there-fore protonemal growth was not quantified

By analogy with seed germination the vegetative propa-gation of moss gametophytes was described by three germi-nation parameters gametophyte germination gametophyteincrement and the gametophyte vigor index In this papergametophyte germination is defined as the percent of mossinocula that germinated Gametophyte increment is the aver-age number of new gametophytes per six-well plate The ga-metophyte vigor index is analogous to the seed vigor indexwhich is calculated by multiplying the seed germination per-centage by the length of the hypocotyl (Abdul-baki and An-derson 1973) Here the seed germination percentage and thelength of hypocotyl were replaced by the gametophyte ger-mination and gametophyte increment respectively and usedto calculate the gametophyte vigor index Thus the germina-tion parameters were calculated by using Eqs (1)ndash(3)

gametophyte germination

=number of germinated inocula

number of total inoculatimes 100 (1)

gametophyte increment=number of new gametophyte

number of total inocula(2)

gametophyte vigor index= gametophyte germinationtimes gametophyte increment (3)

According to Eqs (1)ndash(3) the gametophyte vigor index sum-marizes the vegetative propagation of the mosses

25 Statistical analyses

The differences in physiological indices and germination pa-rameters among treatments and mosses were tested usingone-way analysis of variance (ANOVA) with Fisherrsquos leastsignificant difference post hoc test (LSD) at P lt 005 Therelationships between the physiological indices and germi-nation parameters of the three moss species were quantifiedby calculating Pearson correlation coefficients These statis-tical analyses were completed using SPSS 220

The effects of physiological characteristics on vegetativepropagation were analyzed by a gray incidence analysis inMicrosoft Excel 2010 (Deng 1982 Lin et al 2009) Thegray incidence degree between each of the reference se-quences (physiological indices) and the compared sequence(gametophyte vigor index) was calculated by using Eqs (4)ndash

(6)

1i(k)= |y(k)minus xi(k)| k = 12 n i = 1234 (4)

ξi (XiY )=minimink1i(k)+ ρmaximaxk1i(k)

1i (k)+ ρmaximaxk1i(k)

k = 12 n i = 1234 (5)

ri =1n

sumn

k=1ξi(k) k = 12 n i = 1234 (6)

where 1i (k) and ξi (XiY ) are the absolute difference andthe gray relational coefficient respectively between Xi(physiological index i) and Y (gametophyte vigor index) atpoint k The gray relational coefficient (ri) is between the ithphysiological index and its gametophyte vigor index whenthe distinguishing coefficient (ρ) is 05

The gray incidence degree is the sum of the gray relationalcoefficients

3 Results

31 The initial measurement values of the mosses

The three moss species began to germinate new gameto-phytes from the original inocula at different times whereasno gametophyte germinated in the control groups as of thefinal (fifth) observation B unguiculata germinated on the11th day of inoculation and the entire length of its cultiva-tion period was 35 days D vinealis and D tectorum eachgerminated on the sixth day with a 30-day cultivation pe-riod The initial values of the physiological indices and ger-mination parameters of the three mosses are shown in Ta-ble 1 It can be seen that the four physiological indices andgametophyte germination of D vinealis were significantlyhigher than those of the other two species The largest val-ues of gametophyte increment and gametophyte vigor indexwere found in D tectorum and the lowest germination pa-rameter values were found in B unguiculata However nosignificant differences in the contents of chlorophyll solubleprotein and MDA between D tectorum and B unguiculatawere found

32 Effect of storage temperature on the vegetativepropagation of mosses

The germination times of each of the three mosses after stor-age at each temperature did not differ significantly from theinitial values whereas controls still had no gametophyte Atthe fifth observation the gametophyte germination of eachof the three species had changed from the initial value byno more than 20 (Fig 1a Table 1) The highest gameto-phyte germination of B unguiculata was 9444 at 17 CNo significant difference was found between the maximumvalue and minimum value (7556 at 0 C) In D vinealisgametophyte germination did not significantly differ amongthe storage temperatures and ranged from 9556 (0 C) to

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Y Guo and Y Zhao Effects of storage temperature on desiccation-tolerant mosses 801

Figure 1 Data (averageplusmn1 SE) for the three moss species on (a) gametophyte germination and (b) gametophyte increment after the 40-daystorage period at each of the five temperatures Different letters indicate significant differences (P lt 005) among the five temperatureswithin the same species Dotted lines represent the approximate values of the two germination parameters before storage for each species(the true values are shown in Table 1)

Table 1 Initial values of physiological indices and germination parameters in the three mosses

Index B unguiculata D vinealis D tectorum

Chlorophyll content (mggminus1) 153plusmn 013a 333plusmn 018b 219plusmn 044aSoluble sugar content (mggminus1) 3002plusmn 367a 4413plusmn 341b 1419plusmn 177cSoluble protein content (mggminus1) 628plusmn 140a 1224plusmn 026b 792plusmn 046aMDA content (micromolgminus1) 2402plusmn 047a 3507plusmn 312b 2368plusmn 050aGametophyte germination () 8293plusmn 1000a 10000plusmn 000a 9833plusmn 236aGametophyte increment 154plusmn 018a 182plusmn 040ab 237plusmn 005bGametophyte vigor index 128plusmn 015a 182plusmn 040ab 233plusmn 005b

Data are averageplusmn1 SE and different letters indicate significant differences (P lt 005) among the three species

9889 (17 C) The only significant difference in gameto-phyte germination was observed in D tectorum and was be-tween 8192 and 100 after storage at 0 and 25 C respec-tively

The changes in gametophyte increment were all more than20 after storage except in D tectorum at 30 C for whicha slight decrease of 657 was observed (Fig 1b Table 1)After storage the largest gametophyte increment of B un-guiculata was 111 at 4 C whereas the smallest gameto-phyte increment was 081 at 25 C Except for a significantdifference between 4 and 25 C no significant difference ingametophyte increment was found among the storage tem-peratures in B unguiculata Similarly no significant differ-ence in the gametophyte increment of D vinealis was ob-served among the storage temperatures The maximum andminimum gametophyte increments after storage were 103and 123 at 0 and 17 C respectively for D vinealis Largerdifferences in gametophyte increment among the storagetemperatures were observed in D tectorum except for the dif-ference in gametophyte increment between 0 and 4 C The

maximum gametophyte increment of D tectorum was 374at 17 C after storage and the minimum value was 132 at0 C

The gametophyte vigor index of the three moss speciesshowed significant changes over the 40-day storage period(Table 2) The largest changes in gametophyte vigor indexafter storage were observed in D tectorum with the indexranging from a 5336 decrease (0 C) from the initial valueto a 5732 increase (17 C) No significant difference inthe gametophyte vigor index among the five temperatureswas observed in D vinealis However the index values wereall significantly lower than the initial value (before storage)representing decreases of 3286 (17 C) to 4565 (0 C)After storage the gametophyte vigor index values of B un-guiculata decreased the least by 1881 at 4 C and the mostby 4920 at 25 C representing changes between those ofD vinealis and D tectorum

After the 40-day storage at the five temperatures the high-est gametophyte germination percentages of B unguiculataand D vinealis were at 17 C whereas the highest percent-

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802 Y Guo and Y Zhao Effects of storage temperature on desiccation-tolerant mosses

Table 2 Gametophyte vigor index of the three mosses under treat-ments

Treatment B unguiculata D vinealis D tectorum

Initial value 128plusmn 015a 182plusmn 040a 233plusmn 005a0 C 068plusmn 022b 099plusmn 017b 109plusmn 010b4 C 104plusmn 002ac 111plusmn 013b 126plusmn 003b17 C 095plusmn 005c 122plusmn 010b 366plusmn 035c25 C 065plusmn 006b 117plusmn 013b 290plusmn 046a30 C 086plusmn 018bc 115plusmn 017b 204plusmn 033a

Data are averageplusmn 1 SE and different letters indicate significant differences(P lt 005) among treatments within the same species

age in D tectorum was at 25 C The highest gametophyteincrement of B unguiculata was at 4 C The highest game-tophyte increment values in D vinealis and D tectorum wereboth at 17 C as observed for the gametophyte vigor indexvalues of these two species

33 Effects of storage temperature on the physiologicalindices of mosses

As shown in Table 1 and Fig 2a the chlorophyll content ofB unguiculata increased after storage at four of the five tem-peratures ie all but 0 C The chlorophyll content of B un-guiculata showed an increasing trend with increasing stor-age temperature with the maximum increase of 7308 ob-served at 30 C The smallest change in chlorophyll contentwas observed in D vinealis which showed a maximum de-crease of 1789 at 4 C and a minimum decrease of 239 at 17 C The chlorophyll content of D tectorum after storagewas decreased by 3151 at 17 C and increased by 1850 at 25 C yielding the highest and lowest content values re-spectively

A similar increasing trend with temperature was found forsoluble sugar content (Fig 2b) The soluble sugar contentwas consistently higher after storage than before except inB unguiculata in which sugar content was decreased by5652 and 4047 at 0 and 4 C respectively (Fig 2b Ta-ble 1) The soluble sugar content of D vinealis showed lessvariation than the other species No significant difference wasfound between the minimum and maximum increases whichwere 992 at 0 C and 2314 at 25 C respectively Thegreatest changes in soluble sugar content with greater than65 increases at all storage temperatures occurred in D tec-torum

MDA content showed greater variation than sugar contentincreasing by more than 50 in all stored gametophytes(Fig 2d Table 1) The MDA content of both B unguicu-lata and D tectorum decreased as the temperature increasedfrom 0 to 17 C the minimum value of MDA content (at17 C) was 170 times and 206 times the initial value respec-tively However the MDA content of D vinealis was 154 to

298 times the initial value after storage and continuously de-creased with increasing temperature

Some temperatures caused the soluble protein content tochange significantly (Fig 2c Table 1) The soluble proteincontent of B unguiculata increased abruptly from a 3179 decrease from the initial value to a 4006 increase with in-creasing temperature In contrast soluble protein showed theopposite trend in D vinealis and D tectorum Both speciespresented a maximum increase at 0 C which was 1664 inD vinealis and 2365 in D tectorum The lowest solubleprotein content of D vinealis and D tectorum representeda decrease of 1600 at 25 C and a decrease of 2138 at30 C respectively

Our results indicated that the sharpest changes in chloro-phyll content and soluble protein content with increasingtemperature were observed in B unguiculata furthermoresoluble sugar content and MDA content changed morerapidly with increasing temperature in this species than inD vinealis and D tectorum (Fig 2andashd Table 1) D vinealisshowed slower changes in chlorophyll soluble sugar andsoluble protein contents with increasing temperature thanthe other two species MDA content however varied widelywith temperature The largest increases in soluble sugar con-tent and MDA content after 40 days of storage were observedin D tectorum In all three moss species the greatest changeswere observed in MDA content followed by soluble sugarcontent (Fig 2b and d Table 1)

34 Relationships between physiological characteristicsand the vegetative propagation of mosses

After analyzing the correlations between the physiologi-cal indices and germination parameters of the desiccation-tolerant mosses a significant correlation (P lt 001) wasfound between each physiological index except for chloro-phyll content and MDA content (Table 3) Gametophytegermination was significantly correlated (P lt 005) withsoluble protein content and highly significantly correlated(P lt 001) with both chlorophyll content and soluble sugarcontent MDA content was significantly negatively correlated(P lt 005) with both gametophyte increment and gameto-phyte vigor index

At a distinguishing coefficient of 05 the gray incidencedegrees between the physiological indices (X1 chlorophyllcontent X2 soluble sugar content X3 soluble protein con-tent X4 MDA content) and the gametophyte vigor indexin the three moss species were (1) X4gtX1gtX2=X3 inB unguiculata (2) X3gtX4gtX2gtX1 in D vinealis and(3) X4gtX3gtX1gtX2 in D tectorum (Table 4)

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Y Guo and Y Zhao Effects of storage temperature on desiccation-tolerant mosses 803

Figure 2 (andashd) Data (averageplusmn 1 SE) for the three moss species on (a) chlorophyll content (b) soluble sugar content (c) soluble proteincontent and (d) MDA content after the 40-day storage period at each of the five temperatures Different letters indicate significant differences(P lt 005) among the five temperatures within the same species Dotted lines represent the approximate values of the two germinationparameters before storage for each species (the true values are shown in Table 1)

Table 3 Correlation coefficients between physiological indices and germination parameters across all mosses and treatments

Variables Chlorophyll Sugar Protein MDA Germination Increment

Sugar 0762lowastlowast

Protein 0747lowastlowast 0781lowastlowast

MDA 0220 0402lowastlowast 0510lowastlowast

Germination 0473lowastlowast 0414lowastlowast 0313lowast minus0022Increment minus0239 minus0187 minus0249 minus0344lowast 0388lowastlowast

Vigor index minus0158 minus0122 minus0191 minus0328lowast 0441lowastlowast 0995lowastlowast

Chlorophyll chlorophyll content sugar soluble sugar content protein soluble protein content MDA MDA contentgermination gametophyte germination increment gametophyte increment vigor index gametophyte vigor indexThe lowast symbol indicates a significant correlation at P lt 005 lowastlowast indicates a significant correlation at P lt 001

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804 Y Guo and Y Zhao Effects of storage temperature on desiccation-tolerant mosses

Table 4 Gray incidence degree between physiological indices and the gametophyte vigor index across all treatments

Reference sequences B unguiculata D vinealis D tectorum

Chlorophyll content (X1) 060plusmn 020 055plusmn 027 066plusmn 021Soluble sugar content (X2) 057plusmn 020 062plusmn 023 062plusmn 017Soluble protein content (X3) 057plusmn 022 074plusmn 028 070plusmn 025MDA content (X4) 077plusmn 020 073plusmn 022 076plusmn 027

4 Discussion

41 Effects of storage temperature on the vegetativepropagation of mosses

For more than a century researchers have studied many as-pects of mosses such as inocula pretreatment (eg stor-age and sterilization) culture methods and culture condi-tions (Duckett et al 2004 Hoffman 1966) Some of thesestudies have implied that the physiological characteristics ofmoss gametophytes are closely related to the success of ar-tificial cultivation for example pretreatment with sucroseandor abscisic acid can improve the viability of mosses byincreasing DT (Burch and Wilkinson 2002) In line with pre-vious studies this study found that gametophyte regenera-tion within the same species after desiccation varied amongdifferent temperatures (Fig 1a and b Table 2) which islikely related to species-specific DT The regenerative capac-ity of mosses can be summarily described by the gameto-phyte vigor index on the basis of Eqs (1)ndash(3) and Table 3The gametophyte vigor index most sensitive to storage tem-perature was that of D tectorum whereas that of D vinealisvaried little with storage temperature with no significant dif-ferences among temperatures (Table 2) Thus the effect ofstorage temperature on regenerative capacity was strongestin D tectorum and weakest in D vinealis

The 40-day storage period adversely affected regenera-tion in most gametophytes (Fig 1a and b Table 1) how-ever some gametophytes of D tectorum stored at 17 and25 C produced more new shoots than before It is not clearwhether this enhanced regeneration was associated with thelow-temperature tolerance of D tectorum D tectorum possi-bly suffered low-temperature stress in early winter Further-more higher temperatures (eg 30 C) injured the gameto-phytes of D tectorum as did the lower temperatures of 0and 4 C These findings suggest that extreme temperaturesare unsuitable for storing this moss species Further stud-ies are warranted on the impact of the storage environmenton desiccation-tolerant mosses For example Burch (2003)found that the survival and regeneration of dehydrated pro-tonemata were reduced after cryopreservation due to dam-age caused by intracellular ice crystals The desiccationtime can also affect the restorability of vegetative propaga-tion in desiccation-tolerant mosses and their physiologicalcharacteristics (Keever 1957 Proctor 2001) Environmental

changes or variation in the dormancy period of cells mightinfluence the restoration results after rehydration

42 Effects of storage temperature on the physiologicalcharacteristics of mosses

MDA an important product of membrane lipid peroxidationincreased in all mosses over the storage period This find-ing indicated that the 40-day storage period caused cell dam-age (Fig 2d Table 1) Accordingly the soluble sugar con-tent increased to protect the membranes and proteins in thedried gametophytes (Fig 2b Table 1) Sugars are the mainsubstance used to stabilize protein structures in desiccation-tolerant cells (Hoekstra et al 2001) However the solublesugar content of B unguiculata stored at 0 and 4 C was de-creased relative to the initial value This result might havebeen due to the low temperatures preventing the conversionfrom starch to soluble sugar (Pressel et al 2006) Whenmosses suffered oxidative damage the increases in chloro-phyll content and soluble protein content in some gameto-phytes were related to the recovery ability of desiccation-tolerant cells (Fig 2a and c Table 1) In previous studiesthe chlorophyll content of mosses increased during desicca-tion and their photosynthetic capacity recovered rapidly af-ter rewetting (Alpert 1988 Csintalan et al 1999) Similarlyprotein synthesis recovered after rehydration (Oliver 1991)since cellular recovery is an important part of DT (Proctoret al 2007)

The recovery of photosynthesis and protein synthesis inB unguiculata was facilitated by higher temperatures (notmore than 30 C Fig 2a and c) This finding is inconsis-tent with the pattern in other mosses in which viabilitytends to be lower at increased temperatures (Hearnshaw andProctor 1982) However the increasing trend of MDA con-tent from 17 to 30 C suggests that more extensive mem-brane damage may be caused by storage temperatures above30 C (Fig 2d) The adverse effects of the higher tempera-tures in D vinealis and D tectorum were clearly reflectedby the slower recovery of photosynthesis and protein syn-thesis (Fig 2a and c) The changes in the MDA content inD vinealis suggested more rapid repair of cell membranewith increasing temperature however the species possiblyhad stronger tolerance under the protection of abundant sug-ars when the recovery of photosynthesis and protein synthe-sis was slower (Fig 2andashd)

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Y Guo and Y Zhao Effects of storage temperature on desiccation-tolerant mosses 805

The responses of the physiological characteristics of thethree species to temperature reflected species variation inrestoration ability over a short rehydration time Because therewetting periods were longer than 30 days in the cultiva-tion the vegetative propagation results can be considered asreflecting the long-term recovery of mosses Thus the long-term effect of cell recovery during short-term rehydration canbe explained by the relationships between the physiologi-cal characteristics and vegetative propagation of desiccation-tolerant mosses

43 Relationships between physiological characteristicsand the vegetative propagation of mosses

Before storage the four physiological indices of gameto-phytes showed significant differences between D vinealisand D tectorum However no significant differences be-tween the two species were observed in regard to the threegermination parameters (Table 1) Mosses of similar fertilityshowed significant differences in physiological characteris-tics Species differences in DT led to larger differences invegetative propagation among species than before as evi-denced by the values of the gametophyte vigor indices withinthe same treatment (Tables 1 and 2) Therefore the recoveryability of dried mosses with respect to development and re-generation might be more informative for screening suitableinocula than using fresh mosses in dry habitats Many stud-ies have indicated that desiccation-tolerant mosses can re-cover from drying once they are rehydrated (Csintalan et al1999 Pressel et al 2006) However long periods of desic-cation would impede the reuse of moss specimens and therestoration of dried biocrusts This study showed that cellswere subjected to oxidative damage after the 40-day desicca-tion period (Fig 2d Table 1) Over this period the regener-ative capacity of the three species declined (Table 2) whichsuggested that membrane integrity andor other factors af-fected the vegetative propagation of the desiccation-tolerantmosses

Based on the correlation coefficients among the physio-logical indices and germination parameters of desiccation-tolerant mosses (Table 3) gametophyte germination was sig-nificantly and positively correlated with chlorophyll contentsoluble sugar content and soluble protein content In addi-tion gametophyte increment and gametophyte vigor indexwere significantly and negatively correlated with MDA con-tent These findings are in accordance with the observationsthat metabolic repair is favorable to the germination of newgametophytes and that long-term recovery is more dependenton cell integrity than metabolic repair Therefore to quan-titatively compare the effects of the four physiological in-dices on vegetative propagation the gray incidence degreebetween the physiological indices and the gametophyte vigorindex for each of the three moss species was calculated byusing Eqs (4)ndash(6) As shown in Table 4 the effect of MDAcontent on the gametophyte vigor index was the strongest

in B unguiculata and D tectorum and the incidence degreeof MDA (073) in D vinealis was similar to the maximum(074) In all three mosses MDA content increased as stor-age temperature decreased from 17 to 0 C Smaller gameto-phyte vigor index values were observed for D vinealis andD tectorum at 0 and 4 C than at 25 and 30 C (Fig 2d Ta-ble 2) This result indicated that the greater membrane dam-age incurred at low temperatures caused the decline in regen-erative capacity In addition the higher gametophyte vigorindex values of D tectorum at 17 and 25 C than before stor-age were possibly related to the reduced formation of intra-cellular ice crystals at these temperatures during the storageperiod (Burch 2003) which facilitated more rapid recoveryupon rehydration (Table 2) However the number of nega-tive effects on physiological characteristics increased withincreasing temperature (Fig 2andashc) The high temperatureswere unfavorable to the recovery of the mosses (Hearnshawand Proctor 1982) When cells suffered damage under desic-cation and temperature stress the protection provided by ad-ditional sugars was important for maintaining cell integrityin the dry state (Fig 2d Table 1) D vinealis showed no sig-nificant difference in regenerative capacity among tempera-tures potentially because the level of cellular protection wasequivalent among the different temperatures

Researchers have summarized the recovery mechanisms ofmosses upon rehydration such as the rapid recovery of pho-tosynthesis respiration and protein synthesis within min-utes to hours (Proctor et al 2007) However recovery ofthe carbon balance cell cycle and the cytoskeleton requiremore than 24 h (Alpert and Oechel 1985 Mansour and Hal-let 1981 Pressel et al 2006) Based on these results it hasbeen speculated that cell integrity is more difficult to recoverthan physiological reactions and that cell integrity greatlylimits the recovery and regenerative capacity of desiccation-tolerant mosses Over long-term desiccation the cumulativedamage affects cell function and integrity (Proctor 2001)different temperatures might enhance or suppress such celldamage Thus the effects of temperature on the ecology ofDT in bryophytes warrant investigation especially during thedry season in semiarid and arid areas The greater sensitiv-ity of D tectorum observed here might provide insight intowhy this species is not a widely distributed species such asD vinealis in the study region Furthermore the ecologi-cal niche requirements of different mosses in both dry andwet periods will influence the choice of moss inocula for ar-tificial cultivation and biocrust restoration Field studies areneeded to better understand the ecological requirements ofdried mosses Furthermore a precise description of micro-climates and the application of quantitative methods wouldbe helpful

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806 Y Guo and Y Zhao Effects of storage temperature on desiccation-tolerant mosses

5 Conclusions

The conducted experiment explored the effect of storage tem-perature on the vegetative propagation of desiccation-tolerantmosses and influencing factors The results indicated that thedecline in regenerative capacity in mosses observed follow-ing storage was related to cell damage caused by dehydra-tion during storage The storage temperature during dehydra-tion influenced the vegetative propagation of mosses throughchanges in moss cell activity Further analysis showed thatthe factor with the strongest effect on vegetative propagationwas membrane damage During storage soluble sugars in-creased to protect the cells highlighting the important role ofcell integrity in influencing the physiological characteristicsand vegetative propagation of desiccation-tolerant mosses Inthis study the optimal storage temperature of D vinealis andD tectorum was 17 C whereas the optimal temperature forB unguiculata was 4 C Different responses to temperatureamong the three moss species were associated with speciesdifferences in DT These findings can potentially guide futureresearch on suitable storage methods for inoculation materialto improve the artificial cultivation of moss biocrusts

In general the properties of inoculation material are keyfactors affecting the development and recovery of mossbiocrusts such as species physiological features andorother factors The results provide insight into the factors thatinfluence the vegetative propagation of desiccation-tolerantmosses and highlight the potential applicability of a rapidexperimental approach for screening suitable inocula

Data availability Currently data can only be accessed in the formof Excel sheets via contact with the corresponding author

Competing interests The authors declare that they have no conflictof interest

Special issue statement This article is part of the special issue ldquoBi-ological soil crusts and their role in biogeochemical processes andcyclingrdquo It is a result of the BIOCRUST3 conference Moab USA26 to 30 September 2016

Acknowledgements The research was supported by the NationalNatural Science Foundation of China (grant nos 4157126841271298) We also express our gratitude to the anonymousreviewers and editors for their constructive comments and sugges-tions

Edited by Bettina WeberReviewed by three anonymous referees

References

Abdul-baki A A and Anderson J D Relation-ship between decarboxylation of glutamic-acid andvigor in soybean seed Crop Sci 13 227ndash232httpsdoiorg102135cropsci19730011183X001300020023x1973

Alpert P Survival of a desiccation-tolerant moss Grimmia laevi-gata beyond its observed microdistributional limits J Bryol15 219ndash227 httpsdoiorg101179jbr1988151219 1988

Alpert P and Oechel W C Carbon balance limits microdistribu-tion of Grimmia laevigata a desiccation-tolerant plant Ecology66 660ndash669 httpsdoiorg1023071940527 1985

Antoninka A Bowker M A Reed S C and Doherty K Pro-duction of greenhouse-grown biocrust mosses and associatedcyanobacteria to rehabilitate dryland soil function Restor Ecol24 324ndash335 httpsdoiorg101111rec12311 2016

Belnap J and Eldridge D Disturbance and recovery of biologicalsoil crusts in Biological Soil Crusts Structure Function andManagement edited by Belnap J and Lange O L SpringerBerlin Germany 363ndash383 2003

Belnap J and Lange O L Structure and functioning of biolog-ical soil crusts a synthesis in Biological Soil Crusts Struc-ture Function and Management edited by Belnap J andLange O L Springer Berlin Germany 471ndash479 2003

Belnap J Weber B and Buumldel B Biological soil crusts as an or-ganizing principle in drylands in Biological Soil Crusts An Or-ganizing Principle in Drylands edited by Weber B Buumldel Band Belnap J Springer Berlin Germany 3ndash13 2016

Bradford M M A rapid and sensitive method for the quantifi-cation of microgram quantities of protein utilizing the prin-ciple of protein dye binding Anal Biochem 72 248ndash254httpsdoiorg1010160003-2697(76)90527-3 1976

Burch J Some mosses survive cryopreserva-tion without prior pretreatment Bryologist106 270ndash277 httpsdoiorg1016390007-2745(2003)106[0270SMSCWP]20CO2 2003

Burch J and Wilkinson T Cryopreservation of protonemataof Ditrichum cornubicum (Paton) comparing the effectivenessof four cryoprotectant pretreatments Cryoletters 23 197ndash2082002

Chinese Central Meteorological Station httpwwwnmccnpublishforecastASNansaihtml last access 2 August 2017

Cleavitt N L Stress tolerance of rare and common moss speciesin relation to their occupied environments and asexual dispersalpotential J Ecol 90 785ndash795 httpsdoiorg101046j1365-2745200200713x 2002

Csintalan Z Proctor M C F and Tuba Z Chlorophyll fluo-rescence during drying and rehydration in the mosses Rhytidi-adelphus loreus (Hedw) Warnst Anomodon viticulosus (Hedw)Hook amp Tayl and Grimmia pulvinata (Hedw) Sm Ann Bot-London 84 235ndash244 httpsdoiorg101006anbo199909191999

Deng J L Control problems of grey systems Syst Control Lett1 288ndash294 httpsdoiorg101016S0167-6911(82)80025-X1982

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798 Y Guo and Y Zhao Effects of storage temperature on desiccation-tolerant mosses

Vegetative propagation is an important reproduction modeof bryophytes (hornworts liverworts and mosses) in dryhabitats and gametophyte fragments may serve as the dom-inant inoculum in mosses (Mishler 1988 Tian et al 2005)To date several moss cultivation experiments have been con-ducted in which gametophyte fragments are used to estab-lish new colonies in the laboratory and field (Cleavitt 2002Jones and Rosentreter 2006 Xiao et al 2015) All of theseexperiments have demonstrated that artificial cultivation canaccelerate the succession process of moss crusts For exam-ple Antoninka et al (2016) found that the coverage andbiomass of mosses on an artificially inoculated soil surfaceincreased more rapidly than they did on uninoculated soilSome researchers have suggested that inoculation materialshould be mass-produced by vegetative regeneration withrapid development (Jones and Rosentreter 2006 Mishler1988) because of the need for moss biocrusts to inoculatelarge areas The factors that influence the tissue cultivationof mosses have been investigated for many years (Duckettet al 2004 Hoffman 1966 Sabovljevic et al 2003) how-ever the mechanism of moss regeneration remains unclear

After mosses regenerate protonema and gametophytes suf-fer desiccation stress desiccation tolerance (DT) has a criti-cal influence on their survival and restoration abilities (Proc-tor et al 2007) Adult gametophytes of some species canrecover physiological activities and generate new shoots af-ter being stored for more than 10 years in a desiccatedstate (Stark et al 2017 Keever 1957) Desiccation-tolerantmosses can suspend metabolism and maintain cell integrityduring dry periods (Mansour and Hallet 1981 Platt et al1994) then within a few minutes to a few hours after be-ing rehydrated they can resume cellular activity and returnto a normal hydrated state (Platt et al 1994 Pressel et al2006) However the decline and disappearance of the re-generative capacity of Syntrichia ruralis showed that long-term desiccation can cause irreversible damage despite vi-ability differences among individuals (Stark et al 2017) Itremains unclear why the potential for vegetative propagationin mosses can be altered by storage and why recovery abil-ity following drought-induced dormancy varies among mossspecies The lack of knowledge in these areas has impededthe study of moss cultivation

Investigations of DT in mosses have primarily focused onthe mechanism and evolutionary history (Proctor et al 2007Oliver et al 2000) with fewer investigations addressing DTin artificial cultivation However many studies suggest thatDT research can help improve artificial cultivation methodsFor example the impact of desiccation stress on moss re-generation varies with drying time and storage temperature(Keever 1957 Burch 2003) and an understanding of theserelationships may guide research on the regenerative mecha-nism of mosses upon desiccation and their asexual propaga-tion Furthermore DT plays essential roles in moss regener-ation in dry habitats highlighting the potential value of in-vestigating the relationships between the physiological char-

acteristics of mosses and their vegetative propagation Basedon the above observations it can be hypothesized that (1) drystorage impacts the vegetative propagation of desiccation-tolerant mosses (2) changes in vegetative propagation afterstorage involve the influences of storage on the physiologicalcharacteristics of mosses and (3) the degree to which storageaffects vegetative propagation and physiological characteris-tics is related to the storage temperature

In this study three desiccation-tolerant mosses Barbulaunguiculata Didymodon vinealis and Didymodon tectorumwhich are the dominant mosses in biocrust communities inthe Loess Plateau region were stored at five temperatures (04 17 25 and 30 C) for 40 days Then (1) the effect of stor-age temperature on the vegetative propagation of each mossand (2) the changes in physiological indices from before toafter storage including the contents of chlorophyll solublesugar soluble protein and malondialdehyde (MDA) wereinvestigated to reveal the influences of storage temperatureon the vegetative propagation of mosses and the mechanism

2 Materials and methods

21 Study site and moss species

The study was conducted in Ansai Country ShaanxiProvence China (3651prime N 10919prime E) which is located inthe central part of the Loess Plateau The elevation of thesampling plot varies from 1068 to 1309 m The plot hasa typical semiarid continental climate with an average an-nual temperature of 88 C and its average temperature inJanuary and July is minus72 and 228 C respectively The av-erage annual precipitation is 500 mm with 60 or moreof the precipitation falling between June and September(Zhang et al 2011) For the month of November when themoss crusts were collected the average monthly precipita-tion was 1198 mm and the average monthly temperaturewas 988 C (high) to minus364 C (low) (Chinese Central Me-teorological Station 2017) Cyanobacteria and mosses dom-inate the biocrust communities in this region and the cov-erage of moss-dominated biocrusts can reach approximately80 on north-facing slopes in the study region (Zhao et al2014)

The moss taxa used in the study were Barbula unguicu-lata Didymodon vinealis and Didymodon tectorum whichdominated the moss crusts in the plot B unguiculata dom-inated in woodland areas and was found in shaded areasand under vegetation coverage D vinealis was widely dis-tributed in the study site among different water and light en-vironments and the species were collected from croplandsthat had been abandoned for more than 10 years The dom-inant vegetation of the croplands was grasses thus mostD vinealis was exposed to sunlight in the winter D tecto-rum grew on side slopes and was occasionally collected fromunder the shade of vascular plants

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Y Guo and Y Zhao Effects of storage temperature on desiccation-tolerant mosses 799

22 Experimental design

Some of the three moss crusts were used to measure initialvalues of physiological indices (chlorophyll content solublesugar content soluble protein content and MDA content)and germination parameters (gametophyte germination ga-metophyte increment and gametophyte vigor index) imme-diately following their transport to the laboratory The rest ofthe moss crusts were stored at one of five temperature levelsie 0 4 17 25 and 30 C Each temperature was controlledwithin plusmn1 C around the target On the 41st day of storagethe moss crusts were removed and the physiological indicesand germination parameters described above were measured

23 Moss crusts storage and mosses collection

The crusts of three species of mosses were collected frommany colonies and then air-dried in the shade for 24ndash48 hmost of crust samples were dried in the field Then the sam-ples were transported to the laboratory of the State Key Lab-oratory of Soil Erosion and Dry-land Farming on the LoessPlateau in Yangling Shaanxi Province Samples were storedin one of two refrigerators (at 0 or 4 C) or one of threegrowth chambers (at 17 25 and 30 C) Before storage themoss crusts had been placed in resealable plastic bags to pre-vent changes in water content The samples were stored in thedark under light-blocking fabric The water content measure-ments of the moss gametophytes were all less than 10 andthe equilibrating relative humidity during storage was 55 After the 40-day dry period subsamples of desiccated game-tophytes were collected to measure the physiological indicesand germination parameters

24 Measurement of the physiological indices andgermination parameters

241 Physiological indices

Living mature gametophytes of B unguiculata D vinealisand D tectorum were collected from the moss crusts Shortlyafter being rehydrated and washed with deionized water thegametophytes were measured for the contents of chlorophyllsoluble sugar soluble protein and MDA Approximately01 g fresh mass of gametophytes was used to measure thecontents of soluble sugar soluble protein and MDA in eachreplicate whereas the measurements of chlorophyll contentused approximately 005 g fresh mass per replicate The fourindicators were measured by using the following protocolswith three replications

The chlorophyll was extracted by 95 (vv) ethanol andthe solution was boiled at 85 C for 5 min After being cen-trifuged at 4000 rpm for 10 min the chlorophyll in the super-natant was measured at absorbances of 665 and 649 nm witha spectrophotometer (UV-2300 Techcomp Shanghai ChinaWellburn and Lichtenthaler 1984)

After the soluble protein was extracted in ice-cold50 mmolLminus1 phosphate buffer (pH 78) the suspension wascentrifuged at 8000 rpm for 30 min at 4 C and the super-natant was collected The soluble protein was stained withCoomassie brilliant blue G-250 and the absorbance was readat 595 nm (Bradford 1976)

MDA and soluble protein were extracted and centrifugedThen the supernatant was homogenized with 06 (WV )thiobarbituric acid dissolved by 1 molLminus1 NaOH and 10 (WV ) trichloroacetic acid The mixed solution was heatedat 100 C for 20 min and then the absorbance was read at450 523 and 600 nm (Hodges et al 1999) The TechcompUV-2300 spectrophotometer was used to measure the ab-sorbance of the MDA and soluble protein

Soluble sugar was extracted by distilled water at 100 Cfor 30 min After being filtered and diluted the extract wasadded to an anthronendashsulfuric acid solution The mixed so-lution was used to measure the absorbance at 620 nm witha spectrophotometer (UV-1601 Shimadzu Kyoto JapanMorris 1948)

The fresh weight of gametophytes was measured shortlyafter rehydration and dry weight was measured after ovendrying to a constant weight at 70 C (Schonfeld et al 1988)The fresh and dry weights were used to calculate the fourphysiological indices on a dry basis

242 Germination parameters

At the same time as the physiological indices was measuredsome gametophytes of each of the three moss species werecollected to measure the germination parameters The loes-sial soil (uniform soil texture of Calciustepts) collected fromthe study region was used to culture the mosses The soilwas sieved through a 025 mm mesh and placed in each poreof a six-well plate each pore had a diameter of 35 mm anda depth of 12 mm Then the soil water content was adjustedto 23 (WW ) (the field water-holding capacity of the soil)by adding deionized water and the surface was flattened be-fore inoculation Five inocula representing the top 2 mm ofliving mature gametophytes of the mosses were cut rehy-drated washed and placed in each well Thirty inocula wereplaced in each six-well plate as one replication Three six-well plates were established for each moss species In total90 experimental inoculations were established for the mea-surement of germination parameters before and after storageat each of the five temperature levels for each moss speciesMeanwhile three six-well plates without inoculated mosseswere set up as experimental controls for the effect of otherpropagules such as spores in the experimental soil Thesix-well plates were wrapped tightly with transparent plasticfilm to retain the soil moisture Next they were placed intoa growth chamber (AGC-D003N Qiushi Hangzhou China)to incubate The parameters of the growth chamber were setto a 12 h photoperiod (4500ndash5500 Lux) a constant temper-ature of 17 C (plusmn1 C) and a relative humidity of 60ndash70

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800 Y Guo and Y Zhao Effects of storage temperature on desiccation-tolerant mosses

During the incubation period deionized water was suppliedto maintain the soil moisture at 23 The new gametophyteswere counted every 5 days beginning on the day they werefound Five observations were made over the subsequent25 days This paper reports the results of cultivation at thefifth observation No new gametophytes were found in theblank six-well plates during the entire incubation period Itwas difficult to distinguish protonemal germination betweenthe underside of original inocula and the soil substrate there-fore protonemal growth was not quantified

By analogy with seed germination the vegetative propa-gation of moss gametophytes was described by three germi-nation parameters gametophyte germination gametophyteincrement and the gametophyte vigor index In this papergametophyte germination is defined as the percent of mossinocula that germinated Gametophyte increment is the aver-age number of new gametophytes per six-well plate The ga-metophyte vigor index is analogous to the seed vigor indexwhich is calculated by multiplying the seed germination per-centage by the length of the hypocotyl (Abdul-baki and An-derson 1973) Here the seed germination percentage and thelength of hypocotyl were replaced by the gametophyte ger-mination and gametophyte increment respectively and usedto calculate the gametophyte vigor index Thus the germina-tion parameters were calculated by using Eqs (1)ndash(3)

gametophyte germination

=number of germinated inocula

number of total inoculatimes 100 (1)

gametophyte increment=number of new gametophyte

number of total inocula(2)

gametophyte vigor index= gametophyte germinationtimes gametophyte increment (3)

According to Eqs (1)ndash(3) the gametophyte vigor index sum-marizes the vegetative propagation of the mosses

25 Statistical analyses

The differences in physiological indices and germination pa-rameters among treatments and mosses were tested usingone-way analysis of variance (ANOVA) with Fisherrsquos leastsignificant difference post hoc test (LSD) at P lt 005 Therelationships between the physiological indices and germi-nation parameters of the three moss species were quantifiedby calculating Pearson correlation coefficients These statis-tical analyses were completed using SPSS 220

The effects of physiological characteristics on vegetativepropagation were analyzed by a gray incidence analysis inMicrosoft Excel 2010 (Deng 1982 Lin et al 2009) Thegray incidence degree between each of the reference se-quences (physiological indices) and the compared sequence(gametophyte vigor index) was calculated by using Eqs (4)ndash

(6)

1i(k)= |y(k)minus xi(k)| k = 12 n i = 1234 (4)

ξi (XiY )=minimink1i(k)+ ρmaximaxk1i(k)

1i (k)+ ρmaximaxk1i(k)

k = 12 n i = 1234 (5)

ri =1n

sumn

k=1ξi(k) k = 12 n i = 1234 (6)

where 1i (k) and ξi (XiY ) are the absolute difference andthe gray relational coefficient respectively between Xi(physiological index i) and Y (gametophyte vigor index) atpoint k The gray relational coefficient (ri) is between the ithphysiological index and its gametophyte vigor index whenthe distinguishing coefficient (ρ) is 05

The gray incidence degree is the sum of the gray relationalcoefficients

3 Results

31 The initial measurement values of the mosses

The three moss species began to germinate new gameto-phytes from the original inocula at different times whereasno gametophyte germinated in the control groups as of thefinal (fifth) observation B unguiculata germinated on the11th day of inoculation and the entire length of its cultiva-tion period was 35 days D vinealis and D tectorum eachgerminated on the sixth day with a 30-day cultivation pe-riod The initial values of the physiological indices and ger-mination parameters of the three mosses are shown in Ta-ble 1 It can be seen that the four physiological indices andgametophyte germination of D vinealis were significantlyhigher than those of the other two species The largest val-ues of gametophyte increment and gametophyte vigor indexwere found in D tectorum and the lowest germination pa-rameter values were found in B unguiculata However nosignificant differences in the contents of chlorophyll solubleprotein and MDA between D tectorum and B unguiculatawere found

32 Effect of storage temperature on the vegetativepropagation of mosses

The germination times of each of the three mosses after stor-age at each temperature did not differ significantly from theinitial values whereas controls still had no gametophyte Atthe fifth observation the gametophyte germination of eachof the three species had changed from the initial value byno more than 20 (Fig 1a Table 1) The highest gameto-phyte germination of B unguiculata was 9444 at 17 CNo significant difference was found between the maximumvalue and minimum value (7556 at 0 C) In D vinealisgametophyte germination did not significantly differ amongthe storage temperatures and ranged from 9556 (0 C) to

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Y Guo and Y Zhao Effects of storage temperature on desiccation-tolerant mosses 801

Figure 1 Data (averageplusmn1 SE) for the three moss species on (a) gametophyte germination and (b) gametophyte increment after the 40-daystorage period at each of the five temperatures Different letters indicate significant differences (P lt 005) among the five temperatureswithin the same species Dotted lines represent the approximate values of the two germination parameters before storage for each species(the true values are shown in Table 1)

Table 1 Initial values of physiological indices and germination parameters in the three mosses

Index B unguiculata D vinealis D tectorum

Chlorophyll content (mggminus1) 153plusmn 013a 333plusmn 018b 219plusmn 044aSoluble sugar content (mggminus1) 3002plusmn 367a 4413plusmn 341b 1419plusmn 177cSoluble protein content (mggminus1) 628plusmn 140a 1224plusmn 026b 792plusmn 046aMDA content (micromolgminus1) 2402plusmn 047a 3507plusmn 312b 2368plusmn 050aGametophyte germination () 8293plusmn 1000a 10000plusmn 000a 9833plusmn 236aGametophyte increment 154plusmn 018a 182plusmn 040ab 237plusmn 005bGametophyte vigor index 128plusmn 015a 182plusmn 040ab 233plusmn 005b

Data are averageplusmn1 SE and different letters indicate significant differences (P lt 005) among the three species

9889 (17 C) The only significant difference in gameto-phyte germination was observed in D tectorum and was be-tween 8192 and 100 after storage at 0 and 25 C respec-tively

The changes in gametophyte increment were all more than20 after storage except in D tectorum at 30 C for whicha slight decrease of 657 was observed (Fig 1b Table 1)After storage the largest gametophyte increment of B un-guiculata was 111 at 4 C whereas the smallest gameto-phyte increment was 081 at 25 C Except for a significantdifference between 4 and 25 C no significant difference ingametophyte increment was found among the storage tem-peratures in B unguiculata Similarly no significant differ-ence in the gametophyte increment of D vinealis was ob-served among the storage temperatures The maximum andminimum gametophyte increments after storage were 103and 123 at 0 and 17 C respectively for D vinealis Largerdifferences in gametophyte increment among the storagetemperatures were observed in D tectorum except for the dif-ference in gametophyte increment between 0 and 4 C The

maximum gametophyte increment of D tectorum was 374at 17 C after storage and the minimum value was 132 at0 C

The gametophyte vigor index of the three moss speciesshowed significant changes over the 40-day storage period(Table 2) The largest changes in gametophyte vigor indexafter storage were observed in D tectorum with the indexranging from a 5336 decrease (0 C) from the initial valueto a 5732 increase (17 C) No significant difference inthe gametophyte vigor index among the five temperatureswas observed in D vinealis However the index values wereall significantly lower than the initial value (before storage)representing decreases of 3286 (17 C) to 4565 (0 C)After storage the gametophyte vigor index values of B un-guiculata decreased the least by 1881 at 4 C and the mostby 4920 at 25 C representing changes between those ofD vinealis and D tectorum

After the 40-day storage at the five temperatures the high-est gametophyte germination percentages of B unguiculataand D vinealis were at 17 C whereas the highest percent-

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802 Y Guo and Y Zhao Effects of storage temperature on desiccation-tolerant mosses

Table 2 Gametophyte vigor index of the three mosses under treat-ments

Treatment B unguiculata D vinealis D tectorum

Initial value 128plusmn 015a 182plusmn 040a 233plusmn 005a0 C 068plusmn 022b 099plusmn 017b 109plusmn 010b4 C 104plusmn 002ac 111plusmn 013b 126plusmn 003b17 C 095plusmn 005c 122plusmn 010b 366plusmn 035c25 C 065plusmn 006b 117plusmn 013b 290plusmn 046a30 C 086plusmn 018bc 115plusmn 017b 204plusmn 033a

Data are averageplusmn 1 SE and different letters indicate significant differences(P lt 005) among treatments within the same species

age in D tectorum was at 25 C The highest gametophyteincrement of B unguiculata was at 4 C The highest game-tophyte increment values in D vinealis and D tectorum wereboth at 17 C as observed for the gametophyte vigor indexvalues of these two species

33 Effects of storage temperature on the physiologicalindices of mosses

As shown in Table 1 and Fig 2a the chlorophyll content ofB unguiculata increased after storage at four of the five tem-peratures ie all but 0 C The chlorophyll content of B un-guiculata showed an increasing trend with increasing stor-age temperature with the maximum increase of 7308 ob-served at 30 C The smallest change in chlorophyll contentwas observed in D vinealis which showed a maximum de-crease of 1789 at 4 C and a minimum decrease of 239 at 17 C The chlorophyll content of D tectorum after storagewas decreased by 3151 at 17 C and increased by 1850 at 25 C yielding the highest and lowest content values re-spectively

A similar increasing trend with temperature was found forsoluble sugar content (Fig 2b) The soluble sugar contentwas consistently higher after storage than before except inB unguiculata in which sugar content was decreased by5652 and 4047 at 0 and 4 C respectively (Fig 2b Ta-ble 1) The soluble sugar content of D vinealis showed lessvariation than the other species No significant difference wasfound between the minimum and maximum increases whichwere 992 at 0 C and 2314 at 25 C respectively Thegreatest changes in soluble sugar content with greater than65 increases at all storage temperatures occurred in D tec-torum

MDA content showed greater variation than sugar contentincreasing by more than 50 in all stored gametophytes(Fig 2d Table 1) The MDA content of both B unguicu-lata and D tectorum decreased as the temperature increasedfrom 0 to 17 C the minimum value of MDA content (at17 C) was 170 times and 206 times the initial value respec-tively However the MDA content of D vinealis was 154 to

298 times the initial value after storage and continuously de-creased with increasing temperature

Some temperatures caused the soluble protein content tochange significantly (Fig 2c Table 1) The soluble proteincontent of B unguiculata increased abruptly from a 3179 decrease from the initial value to a 4006 increase with in-creasing temperature In contrast soluble protein showed theopposite trend in D vinealis and D tectorum Both speciespresented a maximum increase at 0 C which was 1664 inD vinealis and 2365 in D tectorum The lowest solubleprotein content of D vinealis and D tectorum representeda decrease of 1600 at 25 C and a decrease of 2138 at30 C respectively

Our results indicated that the sharpest changes in chloro-phyll content and soluble protein content with increasingtemperature were observed in B unguiculata furthermoresoluble sugar content and MDA content changed morerapidly with increasing temperature in this species than inD vinealis and D tectorum (Fig 2andashd Table 1) D vinealisshowed slower changes in chlorophyll soluble sugar andsoluble protein contents with increasing temperature thanthe other two species MDA content however varied widelywith temperature The largest increases in soluble sugar con-tent and MDA content after 40 days of storage were observedin D tectorum In all three moss species the greatest changeswere observed in MDA content followed by soluble sugarcontent (Fig 2b and d Table 1)

34 Relationships between physiological characteristicsand the vegetative propagation of mosses

After analyzing the correlations between the physiologi-cal indices and germination parameters of the desiccation-tolerant mosses a significant correlation (P lt 001) wasfound between each physiological index except for chloro-phyll content and MDA content (Table 3) Gametophytegermination was significantly correlated (P lt 005) withsoluble protein content and highly significantly correlated(P lt 001) with both chlorophyll content and soluble sugarcontent MDA content was significantly negatively correlated(P lt 005) with both gametophyte increment and gameto-phyte vigor index

At a distinguishing coefficient of 05 the gray incidencedegrees between the physiological indices (X1 chlorophyllcontent X2 soluble sugar content X3 soluble protein con-tent X4 MDA content) and the gametophyte vigor indexin the three moss species were (1) X4gtX1gtX2=X3 inB unguiculata (2) X3gtX4gtX2gtX1 in D vinealis and(3) X4gtX3gtX1gtX2 in D tectorum (Table 4)

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Y Guo and Y Zhao Effects of storage temperature on desiccation-tolerant mosses 803

Figure 2 (andashd) Data (averageplusmn 1 SE) for the three moss species on (a) chlorophyll content (b) soluble sugar content (c) soluble proteincontent and (d) MDA content after the 40-day storage period at each of the five temperatures Different letters indicate significant differences(P lt 005) among the five temperatures within the same species Dotted lines represent the approximate values of the two germinationparameters before storage for each species (the true values are shown in Table 1)

Table 3 Correlation coefficients between physiological indices and germination parameters across all mosses and treatments

Variables Chlorophyll Sugar Protein MDA Germination Increment

Sugar 0762lowastlowast

Protein 0747lowastlowast 0781lowastlowast

MDA 0220 0402lowastlowast 0510lowastlowast

Germination 0473lowastlowast 0414lowastlowast 0313lowast minus0022Increment minus0239 minus0187 minus0249 minus0344lowast 0388lowastlowast

Vigor index minus0158 minus0122 minus0191 minus0328lowast 0441lowastlowast 0995lowastlowast

Chlorophyll chlorophyll content sugar soluble sugar content protein soluble protein content MDA MDA contentgermination gametophyte germination increment gametophyte increment vigor index gametophyte vigor indexThe lowast symbol indicates a significant correlation at P lt 005 lowastlowast indicates a significant correlation at P lt 001

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804 Y Guo and Y Zhao Effects of storage temperature on desiccation-tolerant mosses

Table 4 Gray incidence degree between physiological indices and the gametophyte vigor index across all treatments

Reference sequences B unguiculata D vinealis D tectorum

Chlorophyll content (X1) 060plusmn 020 055plusmn 027 066plusmn 021Soluble sugar content (X2) 057plusmn 020 062plusmn 023 062plusmn 017Soluble protein content (X3) 057plusmn 022 074plusmn 028 070plusmn 025MDA content (X4) 077plusmn 020 073plusmn 022 076plusmn 027

4 Discussion

41 Effects of storage temperature on the vegetativepropagation of mosses

For more than a century researchers have studied many as-pects of mosses such as inocula pretreatment (eg stor-age and sterilization) culture methods and culture condi-tions (Duckett et al 2004 Hoffman 1966) Some of thesestudies have implied that the physiological characteristics ofmoss gametophytes are closely related to the success of ar-tificial cultivation for example pretreatment with sucroseandor abscisic acid can improve the viability of mosses byincreasing DT (Burch and Wilkinson 2002) In line with pre-vious studies this study found that gametophyte regenera-tion within the same species after desiccation varied amongdifferent temperatures (Fig 1a and b Table 2) which islikely related to species-specific DT The regenerative capac-ity of mosses can be summarily described by the gameto-phyte vigor index on the basis of Eqs (1)ndash(3) and Table 3The gametophyte vigor index most sensitive to storage tem-perature was that of D tectorum whereas that of D vinealisvaried little with storage temperature with no significant dif-ferences among temperatures (Table 2) Thus the effect ofstorage temperature on regenerative capacity was strongestin D tectorum and weakest in D vinealis

The 40-day storage period adversely affected regenera-tion in most gametophytes (Fig 1a and b Table 1) how-ever some gametophytes of D tectorum stored at 17 and25 C produced more new shoots than before It is not clearwhether this enhanced regeneration was associated with thelow-temperature tolerance of D tectorum D tectorum possi-bly suffered low-temperature stress in early winter Further-more higher temperatures (eg 30 C) injured the gameto-phytes of D tectorum as did the lower temperatures of 0and 4 C These findings suggest that extreme temperaturesare unsuitable for storing this moss species Further stud-ies are warranted on the impact of the storage environmenton desiccation-tolerant mosses For example Burch (2003)found that the survival and regeneration of dehydrated pro-tonemata were reduced after cryopreservation due to dam-age caused by intracellular ice crystals The desiccationtime can also affect the restorability of vegetative propaga-tion in desiccation-tolerant mosses and their physiologicalcharacteristics (Keever 1957 Proctor 2001) Environmental

changes or variation in the dormancy period of cells mightinfluence the restoration results after rehydration

42 Effects of storage temperature on the physiologicalcharacteristics of mosses

MDA an important product of membrane lipid peroxidationincreased in all mosses over the storage period This find-ing indicated that the 40-day storage period caused cell dam-age (Fig 2d Table 1) Accordingly the soluble sugar con-tent increased to protect the membranes and proteins in thedried gametophytes (Fig 2b Table 1) Sugars are the mainsubstance used to stabilize protein structures in desiccation-tolerant cells (Hoekstra et al 2001) However the solublesugar content of B unguiculata stored at 0 and 4 C was de-creased relative to the initial value This result might havebeen due to the low temperatures preventing the conversionfrom starch to soluble sugar (Pressel et al 2006) Whenmosses suffered oxidative damage the increases in chloro-phyll content and soluble protein content in some gameto-phytes were related to the recovery ability of desiccation-tolerant cells (Fig 2a and c Table 1) In previous studiesthe chlorophyll content of mosses increased during desicca-tion and their photosynthetic capacity recovered rapidly af-ter rewetting (Alpert 1988 Csintalan et al 1999) Similarlyprotein synthesis recovered after rehydration (Oliver 1991)since cellular recovery is an important part of DT (Proctoret al 2007)

The recovery of photosynthesis and protein synthesis inB unguiculata was facilitated by higher temperatures (notmore than 30 C Fig 2a and c) This finding is inconsis-tent with the pattern in other mosses in which viabilitytends to be lower at increased temperatures (Hearnshaw andProctor 1982) However the increasing trend of MDA con-tent from 17 to 30 C suggests that more extensive mem-brane damage may be caused by storage temperatures above30 C (Fig 2d) The adverse effects of the higher tempera-tures in D vinealis and D tectorum were clearly reflectedby the slower recovery of photosynthesis and protein syn-thesis (Fig 2a and c) The changes in the MDA content inD vinealis suggested more rapid repair of cell membranewith increasing temperature however the species possiblyhad stronger tolerance under the protection of abundant sug-ars when the recovery of photosynthesis and protein synthe-sis was slower (Fig 2andashd)

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Y Guo and Y Zhao Effects of storage temperature on desiccation-tolerant mosses 805

The responses of the physiological characteristics of thethree species to temperature reflected species variation inrestoration ability over a short rehydration time Because therewetting periods were longer than 30 days in the cultiva-tion the vegetative propagation results can be considered asreflecting the long-term recovery of mosses Thus the long-term effect of cell recovery during short-term rehydration canbe explained by the relationships between the physiologi-cal characteristics and vegetative propagation of desiccation-tolerant mosses

43 Relationships between physiological characteristicsand the vegetative propagation of mosses

Before storage the four physiological indices of gameto-phytes showed significant differences between D vinealisand D tectorum However no significant differences be-tween the two species were observed in regard to the threegermination parameters (Table 1) Mosses of similar fertilityshowed significant differences in physiological characteris-tics Species differences in DT led to larger differences invegetative propagation among species than before as evi-denced by the values of the gametophyte vigor indices withinthe same treatment (Tables 1 and 2) Therefore the recoveryability of dried mosses with respect to development and re-generation might be more informative for screening suitableinocula than using fresh mosses in dry habitats Many stud-ies have indicated that desiccation-tolerant mosses can re-cover from drying once they are rehydrated (Csintalan et al1999 Pressel et al 2006) However long periods of desic-cation would impede the reuse of moss specimens and therestoration of dried biocrusts This study showed that cellswere subjected to oxidative damage after the 40-day desicca-tion period (Fig 2d Table 1) Over this period the regener-ative capacity of the three species declined (Table 2) whichsuggested that membrane integrity andor other factors af-fected the vegetative propagation of the desiccation-tolerantmosses

Based on the correlation coefficients among the physio-logical indices and germination parameters of desiccation-tolerant mosses (Table 3) gametophyte germination was sig-nificantly and positively correlated with chlorophyll contentsoluble sugar content and soluble protein content In addi-tion gametophyte increment and gametophyte vigor indexwere significantly and negatively correlated with MDA con-tent These findings are in accordance with the observationsthat metabolic repair is favorable to the germination of newgametophytes and that long-term recovery is more dependenton cell integrity than metabolic repair Therefore to quan-titatively compare the effects of the four physiological in-dices on vegetative propagation the gray incidence degreebetween the physiological indices and the gametophyte vigorindex for each of the three moss species was calculated byusing Eqs (4)ndash(6) As shown in Table 4 the effect of MDAcontent on the gametophyte vigor index was the strongest

in B unguiculata and D tectorum and the incidence degreeof MDA (073) in D vinealis was similar to the maximum(074) In all three mosses MDA content increased as stor-age temperature decreased from 17 to 0 C Smaller gameto-phyte vigor index values were observed for D vinealis andD tectorum at 0 and 4 C than at 25 and 30 C (Fig 2d Ta-ble 2) This result indicated that the greater membrane dam-age incurred at low temperatures caused the decline in regen-erative capacity In addition the higher gametophyte vigorindex values of D tectorum at 17 and 25 C than before stor-age were possibly related to the reduced formation of intra-cellular ice crystals at these temperatures during the storageperiod (Burch 2003) which facilitated more rapid recoveryupon rehydration (Table 2) However the number of nega-tive effects on physiological characteristics increased withincreasing temperature (Fig 2andashc) The high temperatureswere unfavorable to the recovery of the mosses (Hearnshawand Proctor 1982) When cells suffered damage under desic-cation and temperature stress the protection provided by ad-ditional sugars was important for maintaining cell integrityin the dry state (Fig 2d Table 1) D vinealis showed no sig-nificant difference in regenerative capacity among tempera-tures potentially because the level of cellular protection wasequivalent among the different temperatures

Researchers have summarized the recovery mechanisms ofmosses upon rehydration such as the rapid recovery of pho-tosynthesis respiration and protein synthesis within min-utes to hours (Proctor et al 2007) However recovery ofthe carbon balance cell cycle and the cytoskeleton requiremore than 24 h (Alpert and Oechel 1985 Mansour and Hal-let 1981 Pressel et al 2006) Based on these results it hasbeen speculated that cell integrity is more difficult to recoverthan physiological reactions and that cell integrity greatlylimits the recovery and regenerative capacity of desiccation-tolerant mosses Over long-term desiccation the cumulativedamage affects cell function and integrity (Proctor 2001)different temperatures might enhance or suppress such celldamage Thus the effects of temperature on the ecology ofDT in bryophytes warrant investigation especially during thedry season in semiarid and arid areas The greater sensitiv-ity of D tectorum observed here might provide insight intowhy this species is not a widely distributed species such asD vinealis in the study region Furthermore the ecologi-cal niche requirements of different mosses in both dry andwet periods will influence the choice of moss inocula for ar-tificial cultivation and biocrust restoration Field studies areneeded to better understand the ecological requirements ofdried mosses Furthermore a precise description of micro-climates and the application of quantitative methods wouldbe helpful

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806 Y Guo and Y Zhao Effects of storage temperature on desiccation-tolerant mosses

5 Conclusions

The conducted experiment explored the effect of storage tem-perature on the vegetative propagation of desiccation-tolerantmosses and influencing factors The results indicated that thedecline in regenerative capacity in mosses observed follow-ing storage was related to cell damage caused by dehydra-tion during storage The storage temperature during dehydra-tion influenced the vegetative propagation of mosses throughchanges in moss cell activity Further analysis showed thatthe factor with the strongest effect on vegetative propagationwas membrane damage During storage soluble sugars in-creased to protect the cells highlighting the important role ofcell integrity in influencing the physiological characteristicsand vegetative propagation of desiccation-tolerant mosses Inthis study the optimal storage temperature of D vinealis andD tectorum was 17 C whereas the optimal temperature forB unguiculata was 4 C Different responses to temperatureamong the three moss species were associated with speciesdifferences in DT These findings can potentially guide futureresearch on suitable storage methods for inoculation materialto improve the artificial cultivation of moss biocrusts

In general the properties of inoculation material are keyfactors affecting the development and recovery of mossbiocrusts such as species physiological features andorother factors The results provide insight into the factors thatinfluence the vegetative propagation of desiccation-tolerantmosses and highlight the potential applicability of a rapidexperimental approach for screening suitable inocula

Data availability Currently data can only be accessed in the formof Excel sheets via contact with the corresponding author

Competing interests The authors declare that they have no conflictof interest

Special issue statement This article is part of the special issue ldquoBi-ological soil crusts and their role in biogeochemical processes andcyclingrdquo It is a result of the BIOCRUST3 conference Moab USA26 to 30 September 2016

Acknowledgements The research was supported by the NationalNatural Science Foundation of China (grant nos 4157126841271298) We also express our gratitude to the anonymousreviewers and editors for their constructive comments and sugges-tions

Edited by Bettina WeberReviewed by three anonymous referees

References

Abdul-baki A A and Anderson J D Relation-ship between decarboxylation of glutamic-acid andvigor in soybean seed Crop Sci 13 227ndash232httpsdoiorg102135cropsci19730011183X001300020023x1973

Alpert P Survival of a desiccation-tolerant moss Grimmia laevi-gata beyond its observed microdistributional limits J Bryol15 219ndash227 httpsdoiorg101179jbr1988151219 1988

Alpert P and Oechel W C Carbon balance limits microdistribu-tion of Grimmia laevigata a desiccation-tolerant plant Ecology66 660ndash669 httpsdoiorg1023071940527 1985

Antoninka A Bowker M A Reed S C and Doherty K Pro-duction of greenhouse-grown biocrust mosses and associatedcyanobacteria to rehabilitate dryland soil function Restor Ecol24 324ndash335 httpsdoiorg101111rec12311 2016

Belnap J and Eldridge D Disturbance and recovery of biologicalsoil crusts in Biological Soil Crusts Structure Function andManagement edited by Belnap J and Lange O L SpringerBerlin Germany 363ndash383 2003

Belnap J and Lange O L Structure and functioning of biolog-ical soil crusts a synthesis in Biological Soil Crusts Struc-ture Function and Management edited by Belnap J andLange O L Springer Berlin Germany 471ndash479 2003

Belnap J Weber B and Buumldel B Biological soil crusts as an or-ganizing principle in drylands in Biological Soil Crusts An Or-ganizing Principle in Drylands edited by Weber B Buumldel Band Belnap J Springer Berlin Germany 3ndash13 2016

Bradford M M A rapid and sensitive method for the quantifi-cation of microgram quantities of protein utilizing the prin-ciple of protein dye binding Anal Biochem 72 248ndash254httpsdoiorg1010160003-2697(76)90527-3 1976

Burch J Some mosses survive cryopreserva-tion without prior pretreatment Bryologist106 270ndash277 httpsdoiorg1016390007-2745(2003)106[0270SMSCWP]20CO2 2003

Burch J and Wilkinson T Cryopreservation of protonemataof Ditrichum cornubicum (Paton) comparing the effectivenessof four cryoprotectant pretreatments Cryoletters 23 197ndash2082002

Chinese Central Meteorological Station httpwwwnmccnpublishforecastASNansaihtml last access 2 August 2017

Cleavitt N L Stress tolerance of rare and common moss speciesin relation to their occupied environments and asexual dispersalpotential J Ecol 90 785ndash795 httpsdoiorg101046j1365-2745200200713x 2002

Csintalan Z Proctor M C F and Tuba Z Chlorophyll fluo-rescence during drying and rehydration in the mosses Rhytidi-adelphus loreus (Hedw) Warnst Anomodon viticulosus (Hedw)Hook amp Tayl and Grimmia pulvinata (Hedw) Sm Ann Bot-London 84 235ndash244 httpsdoiorg101006anbo199909191999

Deng J L Control problems of grey systems Syst Control Lett1 288ndash294 httpsdoiorg101016S0167-6911(82)80025-X1982

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Y Guo and Y Zhao Effects of storage temperature on desiccation-tolerant mosses 799

22 Experimental design

Some of the three moss crusts were used to measure initialvalues of physiological indices (chlorophyll content solublesugar content soluble protein content and MDA content)and germination parameters (gametophyte germination ga-metophyte increment and gametophyte vigor index) imme-diately following their transport to the laboratory The rest ofthe moss crusts were stored at one of five temperature levelsie 0 4 17 25 and 30 C Each temperature was controlledwithin plusmn1 C around the target On the 41st day of storagethe moss crusts were removed and the physiological indicesand germination parameters described above were measured

23 Moss crusts storage and mosses collection

The crusts of three species of mosses were collected frommany colonies and then air-dried in the shade for 24ndash48 hmost of crust samples were dried in the field Then the sam-ples were transported to the laboratory of the State Key Lab-oratory of Soil Erosion and Dry-land Farming on the LoessPlateau in Yangling Shaanxi Province Samples were storedin one of two refrigerators (at 0 or 4 C) or one of threegrowth chambers (at 17 25 and 30 C) Before storage themoss crusts had been placed in resealable plastic bags to pre-vent changes in water content The samples were stored in thedark under light-blocking fabric The water content measure-ments of the moss gametophytes were all less than 10 andthe equilibrating relative humidity during storage was 55 After the 40-day dry period subsamples of desiccated game-tophytes were collected to measure the physiological indicesand germination parameters

24 Measurement of the physiological indices andgermination parameters

241 Physiological indices

Living mature gametophytes of B unguiculata D vinealisand D tectorum were collected from the moss crusts Shortlyafter being rehydrated and washed with deionized water thegametophytes were measured for the contents of chlorophyllsoluble sugar soluble protein and MDA Approximately01 g fresh mass of gametophytes was used to measure thecontents of soluble sugar soluble protein and MDA in eachreplicate whereas the measurements of chlorophyll contentused approximately 005 g fresh mass per replicate The fourindicators were measured by using the following protocolswith three replications

The chlorophyll was extracted by 95 (vv) ethanol andthe solution was boiled at 85 C for 5 min After being cen-trifuged at 4000 rpm for 10 min the chlorophyll in the super-natant was measured at absorbances of 665 and 649 nm witha spectrophotometer (UV-2300 Techcomp Shanghai ChinaWellburn and Lichtenthaler 1984)

After the soluble protein was extracted in ice-cold50 mmolLminus1 phosphate buffer (pH 78) the suspension wascentrifuged at 8000 rpm for 30 min at 4 C and the super-natant was collected The soluble protein was stained withCoomassie brilliant blue G-250 and the absorbance was readat 595 nm (Bradford 1976)

MDA and soluble protein were extracted and centrifugedThen the supernatant was homogenized with 06 (WV )thiobarbituric acid dissolved by 1 molLminus1 NaOH and 10 (WV ) trichloroacetic acid The mixed solution was heatedat 100 C for 20 min and then the absorbance was read at450 523 and 600 nm (Hodges et al 1999) The TechcompUV-2300 spectrophotometer was used to measure the ab-sorbance of the MDA and soluble protein

Soluble sugar was extracted by distilled water at 100 Cfor 30 min After being filtered and diluted the extract wasadded to an anthronendashsulfuric acid solution The mixed so-lution was used to measure the absorbance at 620 nm witha spectrophotometer (UV-1601 Shimadzu Kyoto JapanMorris 1948)

The fresh weight of gametophytes was measured shortlyafter rehydration and dry weight was measured after ovendrying to a constant weight at 70 C (Schonfeld et al 1988)The fresh and dry weights were used to calculate the fourphysiological indices on a dry basis

242 Germination parameters

At the same time as the physiological indices was measuredsome gametophytes of each of the three moss species werecollected to measure the germination parameters The loes-sial soil (uniform soil texture of Calciustepts) collected fromthe study region was used to culture the mosses The soilwas sieved through a 025 mm mesh and placed in each poreof a six-well plate each pore had a diameter of 35 mm anda depth of 12 mm Then the soil water content was adjustedto 23 (WW ) (the field water-holding capacity of the soil)by adding deionized water and the surface was flattened be-fore inoculation Five inocula representing the top 2 mm ofliving mature gametophytes of the mosses were cut rehy-drated washed and placed in each well Thirty inocula wereplaced in each six-well plate as one replication Three six-well plates were established for each moss species In total90 experimental inoculations were established for the mea-surement of germination parameters before and after storageat each of the five temperature levels for each moss speciesMeanwhile three six-well plates without inoculated mosseswere set up as experimental controls for the effect of otherpropagules such as spores in the experimental soil Thesix-well plates were wrapped tightly with transparent plasticfilm to retain the soil moisture Next they were placed intoa growth chamber (AGC-D003N Qiushi Hangzhou China)to incubate The parameters of the growth chamber were setto a 12 h photoperiod (4500ndash5500 Lux) a constant temper-ature of 17 C (plusmn1 C) and a relative humidity of 60ndash70

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800 Y Guo and Y Zhao Effects of storage temperature on desiccation-tolerant mosses

During the incubation period deionized water was suppliedto maintain the soil moisture at 23 The new gametophyteswere counted every 5 days beginning on the day they werefound Five observations were made over the subsequent25 days This paper reports the results of cultivation at thefifth observation No new gametophytes were found in theblank six-well plates during the entire incubation period Itwas difficult to distinguish protonemal germination betweenthe underside of original inocula and the soil substrate there-fore protonemal growth was not quantified

By analogy with seed germination the vegetative propa-gation of moss gametophytes was described by three germi-nation parameters gametophyte germination gametophyteincrement and the gametophyte vigor index In this papergametophyte germination is defined as the percent of mossinocula that germinated Gametophyte increment is the aver-age number of new gametophytes per six-well plate The ga-metophyte vigor index is analogous to the seed vigor indexwhich is calculated by multiplying the seed germination per-centage by the length of the hypocotyl (Abdul-baki and An-derson 1973) Here the seed germination percentage and thelength of hypocotyl were replaced by the gametophyte ger-mination and gametophyte increment respectively and usedto calculate the gametophyte vigor index Thus the germina-tion parameters were calculated by using Eqs (1)ndash(3)

gametophyte germination

=number of germinated inocula

number of total inoculatimes 100 (1)

gametophyte increment=number of new gametophyte

number of total inocula(2)

gametophyte vigor index= gametophyte germinationtimes gametophyte increment (3)

According to Eqs (1)ndash(3) the gametophyte vigor index sum-marizes the vegetative propagation of the mosses

25 Statistical analyses

The differences in physiological indices and germination pa-rameters among treatments and mosses were tested usingone-way analysis of variance (ANOVA) with Fisherrsquos leastsignificant difference post hoc test (LSD) at P lt 005 Therelationships between the physiological indices and germi-nation parameters of the three moss species were quantifiedby calculating Pearson correlation coefficients These statis-tical analyses were completed using SPSS 220

The effects of physiological characteristics on vegetativepropagation were analyzed by a gray incidence analysis inMicrosoft Excel 2010 (Deng 1982 Lin et al 2009) Thegray incidence degree between each of the reference se-quences (physiological indices) and the compared sequence(gametophyte vigor index) was calculated by using Eqs (4)ndash

(6)

1i(k)= |y(k)minus xi(k)| k = 12 n i = 1234 (4)

ξi (XiY )=minimink1i(k)+ ρmaximaxk1i(k)

1i (k)+ ρmaximaxk1i(k)

k = 12 n i = 1234 (5)

ri =1n

sumn

k=1ξi(k) k = 12 n i = 1234 (6)

where 1i (k) and ξi (XiY ) are the absolute difference andthe gray relational coefficient respectively between Xi(physiological index i) and Y (gametophyte vigor index) atpoint k The gray relational coefficient (ri) is between the ithphysiological index and its gametophyte vigor index whenthe distinguishing coefficient (ρ) is 05

The gray incidence degree is the sum of the gray relationalcoefficients

3 Results

31 The initial measurement values of the mosses

The three moss species began to germinate new gameto-phytes from the original inocula at different times whereasno gametophyte germinated in the control groups as of thefinal (fifth) observation B unguiculata germinated on the11th day of inoculation and the entire length of its cultiva-tion period was 35 days D vinealis and D tectorum eachgerminated on the sixth day with a 30-day cultivation pe-riod The initial values of the physiological indices and ger-mination parameters of the three mosses are shown in Ta-ble 1 It can be seen that the four physiological indices andgametophyte germination of D vinealis were significantlyhigher than those of the other two species The largest val-ues of gametophyte increment and gametophyte vigor indexwere found in D tectorum and the lowest germination pa-rameter values were found in B unguiculata However nosignificant differences in the contents of chlorophyll solubleprotein and MDA between D tectorum and B unguiculatawere found

32 Effect of storage temperature on the vegetativepropagation of mosses

The germination times of each of the three mosses after stor-age at each temperature did not differ significantly from theinitial values whereas controls still had no gametophyte Atthe fifth observation the gametophyte germination of eachof the three species had changed from the initial value byno more than 20 (Fig 1a Table 1) The highest gameto-phyte germination of B unguiculata was 9444 at 17 CNo significant difference was found between the maximumvalue and minimum value (7556 at 0 C) In D vinealisgametophyte germination did not significantly differ amongthe storage temperatures and ranged from 9556 (0 C) to

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Y Guo and Y Zhao Effects of storage temperature on desiccation-tolerant mosses 801

Figure 1 Data (averageplusmn1 SE) for the three moss species on (a) gametophyte germination and (b) gametophyte increment after the 40-daystorage period at each of the five temperatures Different letters indicate significant differences (P lt 005) among the five temperatureswithin the same species Dotted lines represent the approximate values of the two germination parameters before storage for each species(the true values are shown in Table 1)

Table 1 Initial values of physiological indices and germination parameters in the three mosses

Index B unguiculata D vinealis D tectorum

Chlorophyll content (mggminus1) 153plusmn 013a 333plusmn 018b 219plusmn 044aSoluble sugar content (mggminus1) 3002plusmn 367a 4413plusmn 341b 1419plusmn 177cSoluble protein content (mggminus1) 628plusmn 140a 1224plusmn 026b 792plusmn 046aMDA content (micromolgminus1) 2402plusmn 047a 3507plusmn 312b 2368plusmn 050aGametophyte germination () 8293plusmn 1000a 10000plusmn 000a 9833plusmn 236aGametophyte increment 154plusmn 018a 182plusmn 040ab 237plusmn 005bGametophyte vigor index 128plusmn 015a 182plusmn 040ab 233plusmn 005b

Data are averageplusmn1 SE and different letters indicate significant differences (P lt 005) among the three species

9889 (17 C) The only significant difference in gameto-phyte germination was observed in D tectorum and was be-tween 8192 and 100 after storage at 0 and 25 C respec-tively

The changes in gametophyte increment were all more than20 after storage except in D tectorum at 30 C for whicha slight decrease of 657 was observed (Fig 1b Table 1)After storage the largest gametophyte increment of B un-guiculata was 111 at 4 C whereas the smallest gameto-phyte increment was 081 at 25 C Except for a significantdifference between 4 and 25 C no significant difference ingametophyte increment was found among the storage tem-peratures in B unguiculata Similarly no significant differ-ence in the gametophyte increment of D vinealis was ob-served among the storage temperatures The maximum andminimum gametophyte increments after storage were 103and 123 at 0 and 17 C respectively for D vinealis Largerdifferences in gametophyte increment among the storagetemperatures were observed in D tectorum except for the dif-ference in gametophyte increment between 0 and 4 C The

maximum gametophyte increment of D tectorum was 374at 17 C after storage and the minimum value was 132 at0 C

The gametophyte vigor index of the three moss speciesshowed significant changes over the 40-day storage period(Table 2) The largest changes in gametophyte vigor indexafter storage were observed in D tectorum with the indexranging from a 5336 decrease (0 C) from the initial valueto a 5732 increase (17 C) No significant difference inthe gametophyte vigor index among the five temperatureswas observed in D vinealis However the index values wereall significantly lower than the initial value (before storage)representing decreases of 3286 (17 C) to 4565 (0 C)After storage the gametophyte vigor index values of B un-guiculata decreased the least by 1881 at 4 C and the mostby 4920 at 25 C representing changes between those ofD vinealis and D tectorum

After the 40-day storage at the five temperatures the high-est gametophyte germination percentages of B unguiculataand D vinealis were at 17 C whereas the highest percent-

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802 Y Guo and Y Zhao Effects of storage temperature on desiccation-tolerant mosses

Table 2 Gametophyte vigor index of the three mosses under treat-ments

Treatment B unguiculata D vinealis D tectorum

Initial value 128plusmn 015a 182plusmn 040a 233plusmn 005a0 C 068plusmn 022b 099plusmn 017b 109plusmn 010b4 C 104plusmn 002ac 111plusmn 013b 126plusmn 003b17 C 095plusmn 005c 122plusmn 010b 366plusmn 035c25 C 065plusmn 006b 117plusmn 013b 290plusmn 046a30 C 086plusmn 018bc 115plusmn 017b 204plusmn 033a

Data are averageplusmn 1 SE and different letters indicate significant differences(P lt 005) among treatments within the same species

age in D tectorum was at 25 C The highest gametophyteincrement of B unguiculata was at 4 C The highest game-tophyte increment values in D vinealis and D tectorum wereboth at 17 C as observed for the gametophyte vigor indexvalues of these two species

33 Effects of storage temperature on the physiologicalindices of mosses

As shown in Table 1 and Fig 2a the chlorophyll content ofB unguiculata increased after storage at four of the five tem-peratures ie all but 0 C The chlorophyll content of B un-guiculata showed an increasing trend with increasing stor-age temperature with the maximum increase of 7308 ob-served at 30 C The smallest change in chlorophyll contentwas observed in D vinealis which showed a maximum de-crease of 1789 at 4 C and a minimum decrease of 239 at 17 C The chlorophyll content of D tectorum after storagewas decreased by 3151 at 17 C and increased by 1850 at 25 C yielding the highest and lowest content values re-spectively

A similar increasing trend with temperature was found forsoluble sugar content (Fig 2b) The soluble sugar contentwas consistently higher after storage than before except inB unguiculata in which sugar content was decreased by5652 and 4047 at 0 and 4 C respectively (Fig 2b Ta-ble 1) The soluble sugar content of D vinealis showed lessvariation than the other species No significant difference wasfound between the minimum and maximum increases whichwere 992 at 0 C and 2314 at 25 C respectively Thegreatest changes in soluble sugar content with greater than65 increases at all storage temperatures occurred in D tec-torum

MDA content showed greater variation than sugar contentincreasing by more than 50 in all stored gametophytes(Fig 2d Table 1) The MDA content of both B unguicu-lata and D tectorum decreased as the temperature increasedfrom 0 to 17 C the minimum value of MDA content (at17 C) was 170 times and 206 times the initial value respec-tively However the MDA content of D vinealis was 154 to

298 times the initial value after storage and continuously de-creased with increasing temperature

Some temperatures caused the soluble protein content tochange significantly (Fig 2c Table 1) The soluble proteincontent of B unguiculata increased abruptly from a 3179 decrease from the initial value to a 4006 increase with in-creasing temperature In contrast soluble protein showed theopposite trend in D vinealis and D tectorum Both speciespresented a maximum increase at 0 C which was 1664 inD vinealis and 2365 in D tectorum The lowest solubleprotein content of D vinealis and D tectorum representeda decrease of 1600 at 25 C and a decrease of 2138 at30 C respectively

Our results indicated that the sharpest changes in chloro-phyll content and soluble protein content with increasingtemperature were observed in B unguiculata furthermoresoluble sugar content and MDA content changed morerapidly with increasing temperature in this species than inD vinealis and D tectorum (Fig 2andashd Table 1) D vinealisshowed slower changes in chlorophyll soluble sugar andsoluble protein contents with increasing temperature thanthe other two species MDA content however varied widelywith temperature The largest increases in soluble sugar con-tent and MDA content after 40 days of storage were observedin D tectorum In all three moss species the greatest changeswere observed in MDA content followed by soluble sugarcontent (Fig 2b and d Table 1)

34 Relationships between physiological characteristicsand the vegetative propagation of mosses

After analyzing the correlations between the physiologi-cal indices and germination parameters of the desiccation-tolerant mosses a significant correlation (P lt 001) wasfound between each physiological index except for chloro-phyll content and MDA content (Table 3) Gametophytegermination was significantly correlated (P lt 005) withsoluble protein content and highly significantly correlated(P lt 001) with both chlorophyll content and soluble sugarcontent MDA content was significantly negatively correlated(P lt 005) with both gametophyte increment and gameto-phyte vigor index

At a distinguishing coefficient of 05 the gray incidencedegrees between the physiological indices (X1 chlorophyllcontent X2 soluble sugar content X3 soluble protein con-tent X4 MDA content) and the gametophyte vigor indexin the three moss species were (1) X4gtX1gtX2=X3 inB unguiculata (2) X3gtX4gtX2gtX1 in D vinealis and(3) X4gtX3gtX1gtX2 in D tectorum (Table 4)

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Y Guo and Y Zhao Effects of storage temperature on desiccation-tolerant mosses 803

Figure 2 (andashd) Data (averageplusmn 1 SE) for the three moss species on (a) chlorophyll content (b) soluble sugar content (c) soluble proteincontent and (d) MDA content after the 40-day storage period at each of the five temperatures Different letters indicate significant differences(P lt 005) among the five temperatures within the same species Dotted lines represent the approximate values of the two germinationparameters before storage for each species (the true values are shown in Table 1)

Table 3 Correlation coefficients between physiological indices and germination parameters across all mosses and treatments

Variables Chlorophyll Sugar Protein MDA Germination Increment

Sugar 0762lowastlowast

Protein 0747lowastlowast 0781lowastlowast

MDA 0220 0402lowastlowast 0510lowastlowast

Germination 0473lowastlowast 0414lowastlowast 0313lowast minus0022Increment minus0239 minus0187 minus0249 minus0344lowast 0388lowastlowast

Vigor index minus0158 minus0122 minus0191 minus0328lowast 0441lowastlowast 0995lowastlowast

Chlorophyll chlorophyll content sugar soluble sugar content protein soluble protein content MDA MDA contentgermination gametophyte germination increment gametophyte increment vigor index gametophyte vigor indexThe lowast symbol indicates a significant correlation at P lt 005 lowastlowast indicates a significant correlation at P lt 001

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804 Y Guo and Y Zhao Effects of storage temperature on desiccation-tolerant mosses

Table 4 Gray incidence degree between physiological indices and the gametophyte vigor index across all treatments

Reference sequences B unguiculata D vinealis D tectorum

Chlorophyll content (X1) 060plusmn 020 055plusmn 027 066plusmn 021Soluble sugar content (X2) 057plusmn 020 062plusmn 023 062plusmn 017Soluble protein content (X3) 057plusmn 022 074plusmn 028 070plusmn 025MDA content (X4) 077plusmn 020 073plusmn 022 076plusmn 027

4 Discussion

41 Effects of storage temperature on the vegetativepropagation of mosses

For more than a century researchers have studied many as-pects of mosses such as inocula pretreatment (eg stor-age and sterilization) culture methods and culture condi-tions (Duckett et al 2004 Hoffman 1966) Some of thesestudies have implied that the physiological characteristics ofmoss gametophytes are closely related to the success of ar-tificial cultivation for example pretreatment with sucroseandor abscisic acid can improve the viability of mosses byincreasing DT (Burch and Wilkinson 2002) In line with pre-vious studies this study found that gametophyte regenera-tion within the same species after desiccation varied amongdifferent temperatures (Fig 1a and b Table 2) which islikely related to species-specific DT The regenerative capac-ity of mosses can be summarily described by the gameto-phyte vigor index on the basis of Eqs (1)ndash(3) and Table 3The gametophyte vigor index most sensitive to storage tem-perature was that of D tectorum whereas that of D vinealisvaried little with storage temperature with no significant dif-ferences among temperatures (Table 2) Thus the effect ofstorage temperature on regenerative capacity was strongestin D tectorum and weakest in D vinealis

The 40-day storage period adversely affected regenera-tion in most gametophytes (Fig 1a and b Table 1) how-ever some gametophytes of D tectorum stored at 17 and25 C produced more new shoots than before It is not clearwhether this enhanced regeneration was associated with thelow-temperature tolerance of D tectorum D tectorum possi-bly suffered low-temperature stress in early winter Further-more higher temperatures (eg 30 C) injured the gameto-phytes of D tectorum as did the lower temperatures of 0and 4 C These findings suggest that extreme temperaturesare unsuitable for storing this moss species Further stud-ies are warranted on the impact of the storage environmenton desiccation-tolerant mosses For example Burch (2003)found that the survival and regeneration of dehydrated pro-tonemata were reduced after cryopreservation due to dam-age caused by intracellular ice crystals The desiccationtime can also affect the restorability of vegetative propaga-tion in desiccation-tolerant mosses and their physiologicalcharacteristics (Keever 1957 Proctor 2001) Environmental

changes or variation in the dormancy period of cells mightinfluence the restoration results after rehydration

42 Effects of storage temperature on the physiologicalcharacteristics of mosses

MDA an important product of membrane lipid peroxidationincreased in all mosses over the storage period This find-ing indicated that the 40-day storage period caused cell dam-age (Fig 2d Table 1) Accordingly the soluble sugar con-tent increased to protect the membranes and proteins in thedried gametophytes (Fig 2b Table 1) Sugars are the mainsubstance used to stabilize protein structures in desiccation-tolerant cells (Hoekstra et al 2001) However the solublesugar content of B unguiculata stored at 0 and 4 C was de-creased relative to the initial value This result might havebeen due to the low temperatures preventing the conversionfrom starch to soluble sugar (Pressel et al 2006) Whenmosses suffered oxidative damage the increases in chloro-phyll content and soluble protein content in some gameto-phytes were related to the recovery ability of desiccation-tolerant cells (Fig 2a and c Table 1) In previous studiesthe chlorophyll content of mosses increased during desicca-tion and their photosynthetic capacity recovered rapidly af-ter rewetting (Alpert 1988 Csintalan et al 1999) Similarlyprotein synthesis recovered after rehydration (Oliver 1991)since cellular recovery is an important part of DT (Proctoret al 2007)

The recovery of photosynthesis and protein synthesis inB unguiculata was facilitated by higher temperatures (notmore than 30 C Fig 2a and c) This finding is inconsis-tent with the pattern in other mosses in which viabilitytends to be lower at increased temperatures (Hearnshaw andProctor 1982) However the increasing trend of MDA con-tent from 17 to 30 C suggests that more extensive mem-brane damage may be caused by storage temperatures above30 C (Fig 2d) The adverse effects of the higher tempera-tures in D vinealis and D tectorum were clearly reflectedby the slower recovery of photosynthesis and protein syn-thesis (Fig 2a and c) The changes in the MDA content inD vinealis suggested more rapid repair of cell membranewith increasing temperature however the species possiblyhad stronger tolerance under the protection of abundant sug-ars when the recovery of photosynthesis and protein synthe-sis was slower (Fig 2andashd)

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Y Guo and Y Zhao Effects of storage temperature on desiccation-tolerant mosses 805

The responses of the physiological characteristics of thethree species to temperature reflected species variation inrestoration ability over a short rehydration time Because therewetting periods were longer than 30 days in the cultiva-tion the vegetative propagation results can be considered asreflecting the long-term recovery of mosses Thus the long-term effect of cell recovery during short-term rehydration canbe explained by the relationships between the physiologi-cal characteristics and vegetative propagation of desiccation-tolerant mosses

43 Relationships between physiological characteristicsand the vegetative propagation of mosses

Before storage the four physiological indices of gameto-phytes showed significant differences between D vinealisand D tectorum However no significant differences be-tween the two species were observed in regard to the threegermination parameters (Table 1) Mosses of similar fertilityshowed significant differences in physiological characteris-tics Species differences in DT led to larger differences invegetative propagation among species than before as evi-denced by the values of the gametophyte vigor indices withinthe same treatment (Tables 1 and 2) Therefore the recoveryability of dried mosses with respect to development and re-generation might be more informative for screening suitableinocula than using fresh mosses in dry habitats Many stud-ies have indicated that desiccation-tolerant mosses can re-cover from drying once they are rehydrated (Csintalan et al1999 Pressel et al 2006) However long periods of desic-cation would impede the reuse of moss specimens and therestoration of dried biocrusts This study showed that cellswere subjected to oxidative damage after the 40-day desicca-tion period (Fig 2d Table 1) Over this period the regener-ative capacity of the three species declined (Table 2) whichsuggested that membrane integrity andor other factors af-fected the vegetative propagation of the desiccation-tolerantmosses

Based on the correlation coefficients among the physio-logical indices and germination parameters of desiccation-tolerant mosses (Table 3) gametophyte germination was sig-nificantly and positively correlated with chlorophyll contentsoluble sugar content and soluble protein content In addi-tion gametophyte increment and gametophyte vigor indexwere significantly and negatively correlated with MDA con-tent These findings are in accordance with the observationsthat metabolic repair is favorable to the germination of newgametophytes and that long-term recovery is more dependenton cell integrity than metabolic repair Therefore to quan-titatively compare the effects of the four physiological in-dices on vegetative propagation the gray incidence degreebetween the physiological indices and the gametophyte vigorindex for each of the three moss species was calculated byusing Eqs (4)ndash(6) As shown in Table 4 the effect of MDAcontent on the gametophyte vigor index was the strongest

in B unguiculata and D tectorum and the incidence degreeof MDA (073) in D vinealis was similar to the maximum(074) In all three mosses MDA content increased as stor-age temperature decreased from 17 to 0 C Smaller gameto-phyte vigor index values were observed for D vinealis andD tectorum at 0 and 4 C than at 25 and 30 C (Fig 2d Ta-ble 2) This result indicated that the greater membrane dam-age incurred at low temperatures caused the decline in regen-erative capacity In addition the higher gametophyte vigorindex values of D tectorum at 17 and 25 C than before stor-age were possibly related to the reduced formation of intra-cellular ice crystals at these temperatures during the storageperiod (Burch 2003) which facilitated more rapid recoveryupon rehydration (Table 2) However the number of nega-tive effects on physiological characteristics increased withincreasing temperature (Fig 2andashc) The high temperatureswere unfavorable to the recovery of the mosses (Hearnshawand Proctor 1982) When cells suffered damage under desic-cation and temperature stress the protection provided by ad-ditional sugars was important for maintaining cell integrityin the dry state (Fig 2d Table 1) D vinealis showed no sig-nificant difference in regenerative capacity among tempera-tures potentially because the level of cellular protection wasequivalent among the different temperatures

Researchers have summarized the recovery mechanisms ofmosses upon rehydration such as the rapid recovery of pho-tosynthesis respiration and protein synthesis within min-utes to hours (Proctor et al 2007) However recovery ofthe carbon balance cell cycle and the cytoskeleton requiremore than 24 h (Alpert and Oechel 1985 Mansour and Hal-let 1981 Pressel et al 2006) Based on these results it hasbeen speculated that cell integrity is more difficult to recoverthan physiological reactions and that cell integrity greatlylimits the recovery and regenerative capacity of desiccation-tolerant mosses Over long-term desiccation the cumulativedamage affects cell function and integrity (Proctor 2001)different temperatures might enhance or suppress such celldamage Thus the effects of temperature on the ecology ofDT in bryophytes warrant investigation especially during thedry season in semiarid and arid areas The greater sensitiv-ity of D tectorum observed here might provide insight intowhy this species is not a widely distributed species such asD vinealis in the study region Furthermore the ecologi-cal niche requirements of different mosses in both dry andwet periods will influence the choice of moss inocula for ar-tificial cultivation and biocrust restoration Field studies areneeded to better understand the ecological requirements ofdried mosses Furthermore a precise description of micro-climates and the application of quantitative methods wouldbe helpful

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806 Y Guo and Y Zhao Effects of storage temperature on desiccation-tolerant mosses

5 Conclusions

The conducted experiment explored the effect of storage tem-perature on the vegetative propagation of desiccation-tolerantmosses and influencing factors The results indicated that thedecline in regenerative capacity in mosses observed follow-ing storage was related to cell damage caused by dehydra-tion during storage The storage temperature during dehydra-tion influenced the vegetative propagation of mosses throughchanges in moss cell activity Further analysis showed thatthe factor with the strongest effect on vegetative propagationwas membrane damage During storage soluble sugars in-creased to protect the cells highlighting the important role ofcell integrity in influencing the physiological characteristicsand vegetative propagation of desiccation-tolerant mosses Inthis study the optimal storage temperature of D vinealis andD tectorum was 17 C whereas the optimal temperature forB unguiculata was 4 C Different responses to temperatureamong the three moss species were associated with speciesdifferences in DT These findings can potentially guide futureresearch on suitable storage methods for inoculation materialto improve the artificial cultivation of moss biocrusts

In general the properties of inoculation material are keyfactors affecting the development and recovery of mossbiocrusts such as species physiological features andorother factors The results provide insight into the factors thatinfluence the vegetative propagation of desiccation-tolerantmosses and highlight the potential applicability of a rapidexperimental approach for screening suitable inocula

Data availability Currently data can only be accessed in the formof Excel sheets via contact with the corresponding author

Competing interests The authors declare that they have no conflictof interest

Special issue statement This article is part of the special issue ldquoBi-ological soil crusts and their role in biogeochemical processes andcyclingrdquo It is a result of the BIOCRUST3 conference Moab USA26 to 30 September 2016

Acknowledgements The research was supported by the NationalNatural Science Foundation of China (grant nos 4157126841271298) We also express our gratitude to the anonymousreviewers and editors for their constructive comments and sugges-tions

Edited by Bettina WeberReviewed by three anonymous referees

References

Abdul-baki A A and Anderson J D Relation-ship between decarboxylation of glutamic-acid andvigor in soybean seed Crop Sci 13 227ndash232httpsdoiorg102135cropsci19730011183X001300020023x1973

Alpert P Survival of a desiccation-tolerant moss Grimmia laevi-gata beyond its observed microdistributional limits J Bryol15 219ndash227 httpsdoiorg101179jbr1988151219 1988

Alpert P and Oechel W C Carbon balance limits microdistribu-tion of Grimmia laevigata a desiccation-tolerant plant Ecology66 660ndash669 httpsdoiorg1023071940527 1985

Antoninka A Bowker M A Reed S C and Doherty K Pro-duction of greenhouse-grown biocrust mosses and associatedcyanobacteria to rehabilitate dryland soil function Restor Ecol24 324ndash335 httpsdoiorg101111rec12311 2016

Belnap J and Eldridge D Disturbance and recovery of biologicalsoil crusts in Biological Soil Crusts Structure Function andManagement edited by Belnap J and Lange O L SpringerBerlin Germany 363ndash383 2003

Belnap J and Lange O L Structure and functioning of biolog-ical soil crusts a synthesis in Biological Soil Crusts Struc-ture Function and Management edited by Belnap J andLange O L Springer Berlin Germany 471ndash479 2003

Belnap J Weber B and Buumldel B Biological soil crusts as an or-ganizing principle in drylands in Biological Soil Crusts An Or-ganizing Principle in Drylands edited by Weber B Buumldel Band Belnap J Springer Berlin Germany 3ndash13 2016

Bradford M M A rapid and sensitive method for the quantifi-cation of microgram quantities of protein utilizing the prin-ciple of protein dye binding Anal Biochem 72 248ndash254httpsdoiorg1010160003-2697(76)90527-3 1976

Burch J Some mosses survive cryopreserva-tion without prior pretreatment Bryologist106 270ndash277 httpsdoiorg1016390007-2745(2003)106[0270SMSCWP]20CO2 2003

Burch J and Wilkinson T Cryopreservation of protonemataof Ditrichum cornubicum (Paton) comparing the effectivenessof four cryoprotectant pretreatments Cryoletters 23 197ndash2082002

Chinese Central Meteorological Station httpwwwnmccnpublishforecastASNansaihtml last access 2 August 2017

Cleavitt N L Stress tolerance of rare and common moss speciesin relation to their occupied environments and asexual dispersalpotential J Ecol 90 785ndash795 httpsdoiorg101046j1365-2745200200713x 2002

Csintalan Z Proctor M C F and Tuba Z Chlorophyll fluo-rescence during drying and rehydration in the mosses Rhytidi-adelphus loreus (Hedw) Warnst Anomodon viticulosus (Hedw)Hook amp Tayl and Grimmia pulvinata (Hedw) Sm Ann Bot-London 84 235ndash244 httpsdoiorg101006anbo199909191999

Deng J L Control problems of grey systems Syst Control Lett1 288ndash294 httpsdoiorg101016S0167-6911(82)80025-X1982

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800 Y Guo and Y Zhao Effects of storage temperature on desiccation-tolerant mosses

During the incubation period deionized water was suppliedto maintain the soil moisture at 23 The new gametophyteswere counted every 5 days beginning on the day they werefound Five observations were made over the subsequent25 days This paper reports the results of cultivation at thefifth observation No new gametophytes were found in theblank six-well plates during the entire incubation period Itwas difficult to distinguish protonemal germination betweenthe underside of original inocula and the soil substrate there-fore protonemal growth was not quantified

By analogy with seed germination the vegetative propa-gation of moss gametophytes was described by three germi-nation parameters gametophyte germination gametophyteincrement and the gametophyte vigor index In this papergametophyte germination is defined as the percent of mossinocula that germinated Gametophyte increment is the aver-age number of new gametophytes per six-well plate The ga-metophyte vigor index is analogous to the seed vigor indexwhich is calculated by multiplying the seed germination per-centage by the length of the hypocotyl (Abdul-baki and An-derson 1973) Here the seed germination percentage and thelength of hypocotyl were replaced by the gametophyte ger-mination and gametophyte increment respectively and usedto calculate the gametophyte vigor index Thus the germina-tion parameters were calculated by using Eqs (1)ndash(3)

gametophyte germination

=number of germinated inocula

number of total inoculatimes 100 (1)

gametophyte increment=number of new gametophyte

number of total inocula(2)

gametophyte vigor index= gametophyte germinationtimes gametophyte increment (3)

According to Eqs (1)ndash(3) the gametophyte vigor index sum-marizes the vegetative propagation of the mosses

25 Statistical analyses

The differences in physiological indices and germination pa-rameters among treatments and mosses were tested usingone-way analysis of variance (ANOVA) with Fisherrsquos leastsignificant difference post hoc test (LSD) at P lt 005 Therelationships between the physiological indices and germi-nation parameters of the three moss species were quantifiedby calculating Pearson correlation coefficients These statis-tical analyses were completed using SPSS 220

The effects of physiological characteristics on vegetativepropagation were analyzed by a gray incidence analysis inMicrosoft Excel 2010 (Deng 1982 Lin et al 2009) Thegray incidence degree between each of the reference se-quences (physiological indices) and the compared sequence(gametophyte vigor index) was calculated by using Eqs (4)ndash

(6)

1i(k)= |y(k)minus xi(k)| k = 12 n i = 1234 (4)

ξi (XiY )=minimink1i(k)+ ρmaximaxk1i(k)

1i (k)+ ρmaximaxk1i(k)

k = 12 n i = 1234 (5)

ri =1n

sumn

k=1ξi(k) k = 12 n i = 1234 (6)

where 1i (k) and ξi (XiY ) are the absolute difference andthe gray relational coefficient respectively between Xi(physiological index i) and Y (gametophyte vigor index) atpoint k The gray relational coefficient (ri) is between the ithphysiological index and its gametophyte vigor index whenthe distinguishing coefficient (ρ) is 05

The gray incidence degree is the sum of the gray relationalcoefficients

3 Results

31 The initial measurement values of the mosses

The three moss species began to germinate new gameto-phytes from the original inocula at different times whereasno gametophyte germinated in the control groups as of thefinal (fifth) observation B unguiculata germinated on the11th day of inoculation and the entire length of its cultiva-tion period was 35 days D vinealis and D tectorum eachgerminated on the sixth day with a 30-day cultivation pe-riod The initial values of the physiological indices and ger-mination parameters of the three mosses are shown in Ta-ble 1 It can be seen that the four physiological indices andgametophyte germination of D vinealis were significantlyhigher than those of the other two species The largest val-ues of gametophyte increment and gametophyte vigor indexwere found in D tectorum and the lowest germination pa-rameter values were found in B unguiculata However nosignificant differences in the contents of chlorophyll solubleprotein and MDA between D tectorum and B unguiculatawere found

32 Effect of storage temperature on the vegetativepropagation of mosses

The germination times of each of the three mosses after stor-age at each temperature did not differ significantly from theinitial values whereas controls still had no gametophyte Atthe fifth observation the gametophyte germination of eachof the three species had changed from the initial value byno more than 20 (Fig 1a Table 1) The highest gameto-phyte germination of B unguiculata was 9444 at 17 CNo significant difference was found between the maximumvalue and minimum value (7556 at 0 C) In D vinealisgametophyte germination did not significantly differ amongthe storage temperatures and ranged from 9556 (0 C) to

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Y Guo and Y Zhao Effects of storage temperature on desiccation-tolerant mosses 801

Figure 1 Data (averageplusmn1 SE) for the three moss species on (a) gametophyte germination and (b) gametophyte increment after the 40-daystorage period at each of the five temperatures Different letters indicate significant differences (P lt 005) among the five temperatureswithin the same species Dotted lines represent the approximate values of the two germination parameters before storage for each species(the true values are shown in Table 1)

Table 1 Initial values of physiological indices and germination parameters in the three mosses

Index B unguiculata D vinealis D tectorum

Chlorophyll content (mggminus1) 153plusmn 013a 333plusmn 018b 219plusmn 044aSoluble sugar content (mggminus1) 3002plusmn 367a 4413plusmn 341b 1419plusmn 177cSoluble protein content (mggminus1) 628plusmn 140a 1224plusmn 026b 792plusmn 046aMDA content (micromolgminus1) 2402plusmn 047a 3507plusmn 312b 2368plusmn 050aGametophyte germination () 8293plusmn 1000a 10000plusmn 000a 9833plusmn 236aGametophyte increment 154plusmn 018a 182plusmn 040ab 237plusmn 005bGametophyte vigor index 128plusmn 015a 182plusmn 040ab 233plusmn 005b

Data are averageplusmn1 SE and different letters indicate significant differences (P lt 005) among the three species

9889 (17 C) The only significant difference in gameto-phyte germination was observed in D tectorum and was be-tween 8192 and 100 after storage at 0 and 25 C respec-tively

The changes in gametophyte increment were all more than20 after storage except in D tectorum at 30 C for whicha slight decrease of 657 was observed (Fig 1b Table 1)After storage the largest gametophyte increment of B un-guiculata was 111 at 4 C whereas the smallest gameto-phyte increment was 081 at 25 C Except for a significantdifference between 4 and 25 C no significant difference ingametophyte increment was found among the storage tem-peratures in B unguiculata Similarly no significant differ-ence in the gametophyte increment of D vinealis was ob-served among the storage temperatures The maximum andminimum gametophyte increments after storage were 103and 123 at 0 and 17 C respectively for D vinealis Largerdifferences in gametophyte increment among the storagetemperatures were observed in D tectorum except for the dif-ference in gametophyte increment between 0 and 4 C The

maximum gametophyte increment of D tectorum was 374at 17 C after storage and the minimum value was 132 at0 C

The gametophyte vigor index of the three moss speciesshowed significant changes over the 40-day storage period(Table 2) The largest changes in gametophyte vigor indexafter storage were observed in D tectorum with the indexranging from a 5336 decrease (0 C) from the initial valueto a 5732 increase (17 C) No significant difference inthe gametophyte vigor index among the five temperatureswas observed in D vinealis However the index values wereall significantly lower than the initial value (before storage)representing decreases of 3286 (17 C) to 4565 (0 C)After storage the gametophyte vigor index values of B un-guiculata decreased the least by 1881 at 4 C and the mostby 4920 at 25 C representing changes between those ofD vinealis and D tectorum

After the 40-day storage at the five temperatures the high-est gametophyte germination percentages of B unguiculataand D vinealis were at 17 C whereas the highest percent-

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802 Y Guo and Y Zhao Effects of storage temperature on desiccation-tolerant mosses

Table 2 Gametophyte vigor index of the three mosses under treat-ments

Treatment B unguiculata D vinealis D tectorum

Initial value 128plusmn 015a 182plusmn 040a 233plusmn 005a0 C 068plusmn 022b 099plusmn 017b 109plusmn 010b4 C 104plusmn 002ac 111plusmn 013b 126plusmn 003b17 C 095plusmn 005c 122plusmn 010b 366plusmn 035c25 C 065plusmn 006b 117plusmn 013b 290plusmn 046a30 C 086plusmn 018bc 115plusmn 017b 204plusmn 033a

Data are averageplusmn 1 SE and different letters indicate significant differences(P lt 005) among treatments within the same species

age in D tectorum was at 25 C The highest gametophyteincrement of B unguiculata was at 4 C The highest game-tophyte increment values in D vinealis and D tectorum wereboth at 17 C as observed for the gametophyte vigor indexvalues of these two species

33 Effects of storage temperature on the physiologicalindices of mosses

As shown in Table 1 and Fig 2a the chlorophyll content ofB unguiculata increased after storage at four of the five tem-peratures ie all but 0 C The chlorophyll content of B un-guiculata showed an increasing trend with increasing stor-age temperature with the maximum increase of 7308 ob-served at 30 C The smallest change in chlorophyll contentwas observed in D vinealis which showed a maximum de-crease of 1789 at 4 C and a minimum decrease of 239 at 17 C The chlorophyll content of D tectorum after storagewas decreased by 3151 at 17 C and increased by 1850 at 25 C yielding the highest and lowest content values re-spectively

A similar increasing trend with temperature was found forsoluble sugar content (Fig 2b) The soluble sugar contentwas consistently higher after storage than before except inB unguiculata in which sugar content was decreased by5652 and 4047 at 0 and 4 C respectively (Fig 2b Ta-ble 1) The soluble sugar content of D vinealis showed lessvariation than the other species No significant difference wasfound between the minimum and maximum increases whichwere 992 at 0 C and 2314 at 25 C respectively Thegreatest changes in soluble sugar content with greater than65 increases at all storage temperatures occurred in D tec-torum

MDA content showed greater variation than sugar contentincreasing by more than 50 in all stored gametophytes(Fig 2d Table 1) The MDA content of both B unguicu-lata and D tectorum decreased as the temperature increasedfrom 0 to 17 C the minimum value of MDA content (at17 C) was 170 times and 206 times the initial value respec-tively However the MDA content of D vinealis was 154 to

298 times the initial value after storage and continuously de-creased with increasing temperature

Some temperatures caused the soluble protein content tochange significantly (Fig 2c Table 1) The soluble proteincontent of B unguiculata increased abruptly from a 3179 decrease from the initial value to a 4006 increase with in-creasing temperature In contrast soluble protein showed theopposite trend in D vinealis and D tectorum Both speciespresented a maximum increase at 0 C which was 1664 inD vinealis and 2365 in D tectorum The lowest solubleprotein content of D vinealis and D tectorum representeda decrease of 1600 at 25 C and a decrease of 2138 at30 C respectively

Our results indicated that the sharpest changes in chloro-phyll content and soluble protein content with increasingtemperature were observed in B unguiculata furthermoresoluble sugar content and MDA content changed morerapidly with increasing temperature in this species than inD vinealis and D tectorum (Fig 2andashd Table 1) D vinealisshowed slower changes in chlorophyll soluble sugar andsoluble protein contents with increasing temperature thanthe other two species MDA content however varied widelywith temperature The largest increases in soluble sugar con-tent and MDA content after 40 days of storage were observedin D tectorum In all three moss species the greatest changeswere observed in MDA content followed by soluble sugarcontent (Fig 2b and d Table 1)

34 Relationships between physiological characteristicsand the vegetative propagation of mosses

After analyzing the correlations between the physiologi-cal indices and germination parameters of the desiccation-tolerant mosses a significant correlation (P lt 001) wasfound between each physiological index except for chloro-phyll content and MDA content (Table 3) Gametophytegermination was significantly correlated (P lt 005) withsoluble protein content and highly significantly correlated(P lt 001) with both chlorophyll content and soluble sugarcontent MDA content was significantly negatively correlated(P lt 005) with both gametophyte increment and gameto-phyte vigor index

At a distinguishing coefficient of 05 the gray incidencedegrees between the physiological indices (X1 chlorophyllcontent X2 soluble sugar content X3 soluble protein con-tent X4 MDA content) and the gametophyte vigor indexin the three moss species were (1) X4gtX1gtX2=X3 inB unguiculata (2) X3gtX4gtX2gtX1 in D vinealis and(3) X4gtX3gtX1gtX2 in D tectorum (Table 4)

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Y Guo and Y Zhao Effects of storage temperature on desiccation-tolerant mosses 803

Figure 2 (andashd) Data (averageplusmn 1 SE) for the three moss species on (a) chlorophyll content (b) soluble sugar content (c) soluble proteincontent and (d) MDA content after the 40-day storage period at each of the five temperatures Different letters indicate significant differences(P lt 005) among the five temperatures within the same species Dotted lines represent the approximate values of the two germinationparameters before storage for each species (the true values are shown in Table 1)

Table 3 Correlation coefficients between physiological indices and germination parameters across all mosses and treatments

Variables Chlorophyll Sugar Protein MDA Germination Increment

Sugar 0762lowastlowast

Protein 0747lowastlowast 0781lowastlowast

MDA 0220 0402lowastlowast 0510lowastlowast

Germination 0473lowastlowast 0414lowastlowast 0313lowast minus0022Increment minus0239 minus0187 minus0249 minus0344lowast 0388lowastlowast

Vigor index minus0158 minus0122 minus0191 minus0328lowast 0441lowastlowast 0995lowastlowast

Chlorophyll chlorophyll content sugar soluble sugar content protein soluble protein content MDA MDA contentgermination gametophyte germination increment gametophyte increment vigor index gametophyte vigor indexThe lowast symbol indicates a significant correlation at P lt 005 lowastlowast indicates a significant correlation at P lt 001

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804 Y Guo and Y Zhao Effects of storage temperature on desiccation-tolerant mosses

Table 4 Gray incidence degree between physiological indices and the gametophyte vigor index across all treatments

Reference sequences B unguiculata D vinealis D tectorum

Chlorophyll content (X1) 060plusmn 020 055plusmn 027 066plusmn 021Soluble sugar content (X2) 057plusmn 020 062plusmn 023 062plusmn 017Soluble protein content (X3) 057plusmn 022 074plusmn 028 070plusmn 025MDA content (X4) 077plusmn 020 073plusmn 022 076plusmn 027

4 Discussion

41 Effects of storage temperature on the vegetativepropagation of mosses

For more than a century researchers have studied many as-pects of mosses such as inocula pretreatment (eg stor-age and sterilization) culture methods and culture condi-tions (Duckett et al 2004 Hoffman 1966) Some of thesestudies have implied that the physiological characteristics ofmoss gametophytes are closely related to the success of ar-tificial cultivation for example pretreatment with sucroseandor abscisic acid can improve the viability of mosses byincreasing DT (Burch and Wilkinson 2002) In line with pre-vious studies this study found that gametophyte regenera-tion within the same species after desiccation varied amongdifferent temperatures (Fig 1a and b Table 2) which islikely related to species-specific DT The regenerative capac-ity of mosses can be summarily described by the gameto-phyte vigor index on the basis of Eqs (1)ndash(3) and Table 3The gametophyte vigor index most sensitive to storage tem-perature was that of D tectorum whereas that of D vinealisvaried little with storage temperature with no significant dif-ferences among temperatures (Table 2) Thus the effect ofstorage temperature on regenerative capacity was strongestin D tectorum and weakest in D vinealis

The 40-day storage period adversely affected regenera-tion in most gametophytes (Fig 1a and b Table 1) how-ever some gametophytes of D tectorum stored at 17 and25 C produced more new shoots than before It is not clearwhether this enhanced regeneration was associated with thelow-temperature tolerance of D tectorum D tectorum possi-bly suffered low-temperature stress in early winter Further-more higher temperatures (eg 30 C) injured the gameto-phytes of D tectorum as did the lower temperatures of 0and 4 C These findings suggest that extreme temperaturesare unsuitable for storing this moss species Further stud-ies are warranted on the impact of the storage environmenton desiccation-tolerant mosses For example Burch (2003)found that the survival and regeneration of dehydrated pro-tonemata were reduced after cryopreservation due to dam-age caused by intracellular ice crystals The desiccationtime can also affect the restorability of vegetative propaga-tion in desiccation-tolerant mosses and their physiologicalcharacteristics (Keever 1957 Proctor 2001) Environmental

changes or variation in the dormancy period of cells mightinfluence the restoration results after rehydration

42 Effects of storage temperature on the physiologicalcharacteristics of mosses

MDA an important product of membrane lipid peroxidationincreased in all mosses over the storage period This find-ing indicated that the 40-day storage period caused cell dam-age (Fig 2d Table 1) Accordingly the soluble sugar con-tent increased to protect the membranes and proteins in thedried gametophytes (Fig 2b Table 1) Sugars are the mainsubstance used to stabilize protein structures in desiccation-tolerant cells (Hoekstra et al 2001) However the solublesugar content of B unguiculata stored at 0 and 4 C was de-creased relative to the initial value This result might havebeen due to the low temperatures preventing the conversionfrom starch to soluble sugar (Pressel et al 2006) Whenmosses suffered oxidative damage the increases in chloro-phyll content and soluble protein content in some gameto-phytes were related to the recovery ability of desiccation-tolerant cells (Fig 2a and c Table 1) In previous studiesthe chlorophyll content of mosses increased during desicca-tion and their photosynthetic capacity recovered rapidly af-ter rewetting (Alpert 1988 Csintalan et al 1999) Similarlyprotein synthesis recovered after rehydration (Oliver 1991)since cellular recovery is an important part of DT (Proctoret al 2007)

The recovery of photosynthesis and protein synthesis inB unguiculata was facilitated by higher temperatures (notmore than 30 C Fig 2a and c) This finding is inconsis-tent with the pattern in other mosses in which viabilitytends to be lower at increased temperatures (Hearnshaw andProctor 1982) However the increasing trend of MDA con-tent from 17 to 30 C suggests that more extensive mem-brane damage may be caused by storage temperatures above30 C (Fig 2d) The adverse effects of the higher tempera-tures in D vinealis and D tectorum were clearly reflectedby the slower recovery of photosynthesis and protein syn-thesis (Fig 2a and c) The changes in the MDA content inD vinealis suggested more rapid repair of cell membranewith increasing temperature however the species possiblyhad stronger tolerance under the protection of abundant sug-ars when the recovery of photosynthesis and protein synthe-sis was slower (Fig 2andashd)

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Y Guo and Y Zhao Effects of storage temperature on desiccation-tolerant mosses 805

The responses of the physiological characteristics of thethree species to temperature reflected species variation inrestoration ability over a short rehydration time Because therewetting periods were longer than 30 days in the cultiva-tion the vegetative propagation results can be considered asreflecting the long-term recovery of mosses Thus the long-term effect of cell recovery during short-term rehydration canbe explained by the relationships between the physiologi-cal characteristics and vegetative propagation of desiccation-tolerant mosses

43 Relationships between physiological characteristicsand the vegetative propagation of mosses

Before storage the four physiological indices of gameto-phytes showed significant differences between D vinealisand D tectorum However no significant differences be-tween the two species were observed in regard to the threegermination parameters (Table 1) Mosses of similar fertilityshowed significant differences in physiological characteris-tics Species differences in DT led to larger differences invegetative propagation among species than before as evi-denced by the values of the gametophyte vigor indices withinthe same treatment (Tables 1 and 2) Therefore the recoveryability of dried mosses with respect to development and re-generation might be more informative for screening suitableinocula than using fresh mosses in dry habitats Many stud-ies have indicated that desiccation-tolerant mosses can re-cover from drying once they are rehydrated (Csintalan et al1999 Pressel et al 2006) However long periods of desic-cation would impede the reuse of moss specimens and therestoration of dried biocrusts This study showed that cellswere subjected to oxidative damage after the 40-day desicca-tion period (Fig 2d Table 1) Over this period the regener-ative capacity of the three species declined (Table 2) whichsuggested that membrane integrity andor other factors af-fected the vegetative propagation of the desiccation-tolerantmosses

Based on the correlation coefficients among the physio-logical indices and germination parameters of desiccation-tolerant mosses (Table 3) gametophyte germination was sig-nificantly and positively correlated with chlorophyll contentsoluble sugar content and soluble protein content In addi-tion gametophyte increment and gametophyte vigor indexwere significantly and negatively correlated with MDA con-tent These findings are in accordance with the observationsthat metabolic repair is favorable to the germination of newgametophytes and that long-term recovery is more dependenton cell integrity than metabolic repair Therefore to quan-titatively compare the effects of the four physiological in-dices on vegetative propagation the gray incidence degreebetween the physiological indices and the gametophyte vigorindex for each of the three moss species was calculated byusing Eqs (4)ndash(6) As shown in Table 4 the effect of MDAcontent on the gametophyte vigor index was the strongest

in B unguiculata and D tectorum and the incidence degreeof MDA (073) in D vinealis was similar to the maximum(074) In all three mosses MDA content increased as stor-age temperature decreased from 17 to 0 C Smaller gameto-phyte vigor index values were observed for D vinealis andD tectorum at 0 and 4 C than at 25 and 30 C (Fig 2d Ta-ble 2) This result indicated that the greater membrane dam-age incurred at low temperatures caused the decline in regen-erative capacity In addition the higher gametophyte vigorindex values of D tectorum at 17 and 25 C than before stor-age were possibly related to the reduced formation of intra-cellular ice crystals at these temperatures during the storageperiod (Burch 2003) which facilitated more rapid recoveryupon rehydration (Table 2) However the number of nega-tive effects on physiological characteristics increased withincreasing temperature (Fig 2andashc) The high temperatureswere unfavorable to the recovery of the mosses (Hearnshawand Proctor 1982) When cells suffered damage under desic-cation and temperature stress the protection provided by ad-ditional sugars was important for maintaining cell integrityin the dry state (Fig 2d Table 1) D vinealis showed no sig-nificant difference in regenerative capacity among tempera-tures potentially because the level of cellular protection wasequivalent among the different temperatures

Researchers have summarized the recovery mechanisms ofmosses upon rehydration such as the rapid recovery of pho-tosynthesis respiration and protein synthesis within min-utes to hours (Proctor et al 2007) However recovery ofthe carbon balance cell cycle and the cytoskeleton requiremore than 24 h (Alpert and Oechel 1985 Mansour and Hal-let 1981 Pressel et al 2006) Based on these results it hasbeen speculated that cell integrity is more difficult to recoverthan physiological reactions and that cell integrity greatlylimits the recovery and regenerative capacity of desiccation-tolerant mosses Over long-term desiccation the cumulativedamage affects cell function and integrity (Proctor 2001)different temperatures might enhance or suppress such celldamage Thus the effects of temperature on the ecology ofDT in bryophytes warrant investigation especially during thedry season in semiarid and arid areas The greater sensitiv-ity of D tectorum observed here might provide insight intowhy this species is not a widely distributed species such asD vinealis in the study region Furthermore the ecologi-cal niche requirements of different mosses in both dry andwet periods will influence the choice of moss inocula for ar-tificial cultivation and biocrust restoration Field studies areneeded to better understand the ecological requirements ofdried mosses Furthermore a precise description of micro-climates and the application of quantitative methods wouldbe helpful

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806 Y Guo and Y Zhao Effects of storage temperature on desiccation-tolerant mosses

5 Conclusions

The conducted experiment explored the effect of storage tem-perature on the vegetative propagation of desiccation-tolerantmosses and influencing factors The results indicated that thedecline in regenerative capacity in mosses observed follow-ing storage was related to cell damage caused by dehydra-tion during storage The storage temperature during dehydra-tion influenced the vegetative propagation of mosses throughchanges in moss cell activity Further analysis showed thatthe factor with the strongest effect on vegetative propagationwas membrane damage During storage soluble sugars in-creased to protect the cells highlighting the important role ofcell integrity in influencing the physiological characteristicsand vegetative propagation of desiccation-tolerant mosses Inthis study the optimal storage temperature of D vinealis andD tectorum was 17 C whereas the optimal temperature forB unguiculata was 4 C Different responses to temperatureamong the three moss species were associated with speciesdifferences in DT These findings can potentially guide futureresearch on suitable storage methods for inoculation materialto improve the artificial cultivation of moss biocrusts

In general the properties of inoculation material are keyfactors affecting the development and recovery of mossbiocrusts such as species physiological features andorother factors The results provide insight into the factors thatinfluence the vegetative propagation of desiccation-tolerantmosses and highlight the potential applicability of a rapidexperimental approach for screening suitable inocula

Data availability Currently data can only be accessed in the formof Excel sheets via contact with the corresponding author

Competing interests The authors declare that they have no conflictof interest

Special issue statement This article is part of the special issue ldquoBi-ological soil crusts and their role in biogeochemical processes andcyclingrdquo It is a result of the BIOCRUST3 conference Moab USA26 to 30 September 2016

Acknowledgements The research was supported by the NationalNatural Science Foundation of China (grant nos 4157126841271298) We also express our gratitude to the anonymousreviewers and editors for their constructive comments and sugges-tions

Edited by Bettina WeberReviewed by three anonymous referees

References

Abdul-baki A A and Anderson J D Relation-ship between decarboxylation of glutamic-acid andvigor in soybean seed Crop Sci 13 227ndash232httpsdoiorg102135cropsci19730011183X001300020023x1973

Alpert P Survival of a desiccation-tolerant moss Grimmia laevi-gata beyond its observed microdistributional limits J Bryol15 219ndash227 httpsdoiorg101179jbr1988151219 1988

Alpert P and Oechel W C Carbon balance limits microdistribu-tion of Grimmia laevigata a desiccation-tolerant plant Ecology66 660ndash669 httpsdoiorg1023071940527 1985

Antoninka A Bowker M A Reed S C and Doherty K Pro-duction of greenhouse-grown biocrust mosses and associatedcyanobacteria to rehabilitate dryland soil function Restor Ecol24 324ndash335 httpsdoiorg101111rec12311 2016

Belnap J and Eldridge D Disturbance and recovery of biologicalsoil crusts in Biological Soil Crusts Structure Function andManagement edited by Belnap J and Lange O L SpringerBerlin Germany 363ndash383 2003

Belnap J and Lange O L Structure and functioning of biolog-ical soil crusts a synthesis in Biological Soil Crusts Struc-ture Function and Management edited by Belnap J andLange O L Springer Berlin Germany 471ndash479 2003

Belnap J Weber B and Buumldel B Biological soil crusts as an or-ganizing principle in drylands in Biological Soil Crusts An Or-ganizing Principle in Drylands edited by Weber B Buumldel Band Belnap J Springer Berlin Germany 3ndash13 2016

Bradford M M A rapid and sensitive method for the quantifi-cation of microgram quantities of protein utilizing the prin-ciple of protein dye binding Anal Biochem 72 248ndash254httpsdoiorg1010160003-2697(76)90527-3 1976

Burch J Some mosses survive cryopreserva-tion without prior pretreatment Bryologist106 270ndash277 httpsdoiorg1016390007-2745(2003)106[0270SMSCWP]20CO2 2003

Burch J and Wilkinson T Cryopreservation of protonemataof Ditrichum cornubicum (Paton) comparing the effectivenessof four cryoprotectant pretreatments Cryoletters 23 197ndash2082002

Chinese Central Meteorological Station httpwwwnmccnpublishforecastASNansaihtml last access 2 August 2017

Cleavitt N L Stress tolerance of rare and common moss speciesin relation to their occupied environments and asexual dispersalpotential J Ecol 90 785ndash795 httpsdoiorg101046j1365-2745200200713x 2002

Csintalan Z Proctor M C F and Tuba Z Chlorophyll fluo-rescence during drying and rehydration in the mosses Rhytidi-adelphus loreus (Hedw) Warnst Anomodon viticulosus (Hedw)Hook amp Tayl and Grimmia pulvinata (Hedw) Sm Ann Bot-London 84 235ndash244 httpsdoiorg101006anbo199909191999

Deng J L Control problems of grey systems Syst Control Lett1 288ndash294 httpsdoiorg101016S0167-6911(82)80025-X1982

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Y Guo and Y Zhao Effects of storage temperature on desiccation-tolerant mosses 801

Figure 1 Data (averageplusmn1 SE) for the three moss species on (a) gametophyte germination and (b) gametophyte increment after the 40-daystorage period at each of the five temperatures Different letters indicate significant differences (P lt 005) among the five temperatureswithin the same species Dotted lines represent the approximate values of the two germination parameters before storage for each species(the true values are shown in Table 1)

Table 1 Initial values of physiological indices and germination parameters in the three mosses

Index B unguiculata D vinealis D tectorum

Chlorophyll content (mggminus1) 153plusmn 013a 333plusmn 018b 219plusmn 044aSoluble sugar content (mggminus1) 3002plusmn 367a 4413plusmn 341b 1419plusmn 177cSoluble protein content (mggminus1) 628plusmn 140a 1224plusmn 026b 792plusmn 046aMDA content (micromolgminus1) 2402plusmn 047a 3507plusmn 312b 2368plusmn 050aGametophyte germination () 8293plusmn 1000a 10000plusmn 000a 9833plusmn 236aGametophyte increment 154plusmn 018a 182plusmn 040ab 237plusmn 005bGametophyte vigor index 128plusmn 015a 182plusmn 040ab 233plusmn 005b

Data are averageplusmn1 SE and different letters indicate significant differences (P lt 005) among the three species

9889 (17 C) The only significant difference in gameto-phyte germination was observed in D tectorum and was be-tween 8192 and 100 after storage at 0 and 25 C respec-tively

The changes in gametophyte increment were all more than20 after storage except in D tectorum at 30 C for whicha slight decrease of 657 was observed (Fig 1b Table 1)After storage the largest gametophyte increment of B un-guiculata was 111 at 4 C whereas the smallest gameto-phyte increment was 081 at 25 C Except for a significantdifference between 4 and 25 C no significant difference ingametophyte increment was found among the storage tem-peratures in B unguiculata Similarly no significant differ-ence in the gametophyte increment of D vinealis was ob-served among the storage temperatures The maximum andminimum gametophyte increments after storage were 103and 123 at 0 and 17 C respectively for D vinealis Largerdifferences in gametophyte increment among the storagetemperatures were observed in D tectorum except for the dif-ference in gametophyte increment between 0 and 4 C The

maximum gametophyte increment of D tectorum was 374at 17 C after storage and the minimum value was 132 at0 C

The gametophyte vigor index of the three moss speciesshowed significant changes over the 40-day storage period(Table 2) The largest changes in gametophyte vigor indexafter storage were observed in D tectorum with the indexranging from a 5336 decrease (0 C) from the initial valueto a 5732 increase (17 C) No significant difference inthe gametophyte vigor index among the five temperatureswas observed in D vinealis However the index values wereall significantly lower than the initial value (before storage)representing decreases of 3286 (17 C) to 4565 (0 C)After storage the gametophyte vigor index values of B un-guiculata decreased the least by 1881 at 4 C and the mostby 4920 at 25 C representing changes between those ofD vinealis and D tectorum

After the 40-day storage at the five temperatures the high-est gametophyte germination percentages of B unguiculataand D vinealis were at 17 C whereas the highest percent-

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802 Y Guo and Y Zhao Effects of storage temperature on desiccation-tolerant mosses

Table 2 Gametophyte vigor index of the three mosses under treat-ments

Treatment B unguiculata D vinealis D tectorum

Initial value 128plusmn 015a 182plusmn 040a 233plusmn 005a0 C 068plusmn 022b 099plusmn 017b 109plusmn 010b4 C 104plusmn 002ac 111plusmn 013b 126plusmn 003b17 C 095plusmn 005c 122plusmn 010b 366plusmn 035c25 C 065plusmn 006b 117plusmn 013b 290plusmn 046a30 C 086plusmn 018bc 115plusmn 017b 204plusmn 033a

Data are averageplusmn 1 SE and different letters indicate significant differences(P lt 005) among treatments within the same species

age in D tectorum was at 25 C The highest gametophyteincrement of B unguiculata was at 4 C The highest game-tophyte increment values in D vinealis and D tectorum wereboth at 17 C as observed for the gametophyte vigor indexvalues of these two species

33 Effects of storage temperature on the physiologicalindices of mosses

As shown in Table 1 and Fig 2a the chlorophyll content ofB unguiculata increased after storage at four of the five tem-peratures ie all but 0 C The chlorophyll content of B un-guiculata showed an increasing trend with increasing stor-age temperature with the maximum increase of 7308 ob-served at 30 C The smallest change in chlorophyll contentwas observed in D vinealis which showed a maximum de-crease of 1789 at 4 C and a minimum decrease of 239 at 17 C The chlorophyll content of D tectorum after storagewas decreased by 3151 at 17 C and increased by 1850 at 25 C yielding the highest and lowest content values re-spectively

A similar increasing trend with temperature was found forsoluble sugar content (Fig 2b) The soluble sugar contentwas consistently higher after storage than before except inB unguiculata in which sugar content was decreased by5652 and 4047 at 0 and 4 C respectively (Fig 2b Ta-ble 1) The soluble sugar content of D vinealis showed lessvariation than the other species No significant difference wasfound between the minimum and maximum increases whichwere 992 at 0 C and 2314 at 25 C respectively Thegreatest changes in soluble sugar content with greater than65 increases at all storage temperatures occurred in D tec-torum

MDA content showed greater variation than sugar contentincreasing by more than 50 in all stored gametophytes(Fig 2d Table 1) The MDA content of both B unguicu-lata and D tectorum decreased as the temperature increasedfrom 0 to 17 C the minimum value of MDA content (at17 C) was 170 times and 206 times the initial value respec-tively However the MDA content of D vinealis was 154 to

298 times the initial value after storage and continuously de-creased with increasing temperature

Some temperatures caused the soluble protein content tochange significantly (Fig 2c Table 1) The soluble proteincontent of B unguiculata increased abruptly from a 3179 decrease from the initial value to a 4006 increase with in-creasing temperature In contrast soluble protein showed theopposite trend in D vinealis and D tectorum Both speciespresented a maximum increase at 0 C which was 1664 inD vinealis and 2365 in D tectorum The lowest solubleprotein content of D vinealis and D tectorum representeda decrease of 1600 at 25 C and a decrease of 2138 at30 C respectively

Our results indicated that the sharpest changes in chloro-phyll content and soluble protein content with increasingtemperature were observed in B unguiculata furthermoresoluble sugar content and MDA content changed morerapidly with increasing temperature in this species than inD vinealis and D tectorum (Fig 2andashd Table 1) D vinealisshowed slower changes in chlorophyll soluble sugar andsoluble protein contents with increasing temperature thanthe other two species MDA content however varied widelywith temperature The largest increases in soluble sugar con-tent and MDA content after 40 days of storage were observedin D tectorum In all three moss species the greatest changeswere observed in MDA content followed by soluble sugarcontent (Fig 2b and d Table 1)

34 Relationships between physiological characteristicsand the vegetative propagation of mosses

After analyzing the correlations between the physiologi-cal indices and germination parameters of the desiccation-tolerant mosses a significant correlation (P lt 001) wasfound between each physiological index except for chloro-phyll content and MDA content (Table 3) Gametophytegermination was significantly correlated (P lt 005) withsoluble protein content and highly significantly correlated(P lt 001) with both chlorophyll content and soluble sugarcontent MDA content was significantly negatively correlated(P lt 005) with both gametophyte increment and gameto-phyte vigor index

At a distinguishing coefficient of 05 the gray incidencedegrees between the physiological indices (X1 chlorophyllcontent X2 soluble sugar content X3 soluble protein con-tent X4 MDA content) and the gametophyte vigor indexin the three moss species were (1) X4gtX1gtX2=X3 inB unguiculata (2) X3gtX4gtX2gtX1 in D vinealis and(3) X4gtX3gtX1gtX2 in D tectorum (Table 4)

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Y Guo and Y Zhao Effects of storage temperature on desiccation-tolerant mosses 803

Figure 2 (andashd) Data (averageplusmn 1 SE) for the three moss species on (a) chlorophyll content (b) soluble sugar content (c) soluble proteincontent and (d) MDA content after the 40-day storage period at each of the five temperatures Different letters indicate significant differences(P lt 005) among the five temperatures within the same species Dotted lines represent the approximate values of the two germinationparameters before storage for each species (the true values are shown in Table 1)

Table 3 Correlation coefficients between physiological indices and germination parameters across all mosses and treatments

Variables Chlorophyll Sugar Protein MDA Germination Increment

Sugar 0762lowastlowast

Protein 0747lowastlowast 0781lowastlowast

MDA 0220 0402lowastlowast 0510lowastlowast

Germination 0473lowastlowast 0414lowastlowast 0313lowast minus0022Increment minus0239 minus0187 minus0249 minus0344lowast 0388lowastlowast

Vigor index minus0158 minus0122 minus0191 minus0328lowast 0441lowastlowast 0995lowastlowast

Chlorophyll chlorophyll content sugar soluble sugar content protein soluble protein content MDA MDA contentgermination gametophyte germination increment gametophyte increment vigor index gametophyte vigor indexThe lowast symbol indicates a significant correlation at P lt 005 lowastlowast indicates a significant correlation at P lt 001

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804 Y Guo and Y Zhao Effects of storage temperature on desiccation-tolerant mosses

Table 4 Gray incidence degree between physiological indices and the gametophyte vigor index across all treatments

Reference sequences B unguiculata D vinealis D tectorum

Chlorophyll content (X1) 060plusmn 020 055plusmn 027 066plusmn 021Soluble sugar content (X2) 057plusmn 020 062plusmn 023 062plusmn 017Soluble protein content (X3) 057plusmn 022 074plusmn 028 070plusmn 025MDA content (X4) 077plusmn 020 073plusmn 022 076plusmn 027

4 Discussion

41 Effects of storage temperature on the vegetativepropagation of mosses

For more than a century researchers have studied many as-pects of mosses such as inocula pretreatment (eg stor-age and sterilization) culture methods and culture condi-tions (Duckett et al 2004 Hoffman 1966) Some of thesestudies have implied that the physiological characteristics ofmoss gametophytes are closely related to the success of ar-tificial cultivation for example pretreatment with sucroseandor abscisic acid can improve the viability of mosses byincreasing DT (Burch and Wilkinson 2002) In line with pre-vious studies this study found that gametophyte regenera-tion within the same species after desiccation varied amongdifferent temperatures (Fig 1a and b Table 2) which islikely related to species-specific DT The regenerative capac-ity of mosses can be summarily described by the gameto-phyte vigor index on the basis of Eqs (1)ndash(3) and Table 3The gametophyte vigor index most sensitive to storage tem-perature was that of D tectorum whereas that of D vinealisvaried little with storage temperature with no significant dif-ferences among temperatures (Table 2) Thus the effect ofstorage temperature on regenerative capacity was strongestin D tectorum and weakest in D vinealis

The 40-day storage period adversely affected regenera-tion in most gametophytes (Fig 1a and b Table 1) how-ever some gametophytes of D tectorum stored at 17 and25 C produced more new shoots than before It is not clearwhether this enhanced regeneration was associated with thelow-temperature tolerance of D tectorum D tectorum possi-bly suffered low-temperature stress in early winter Further-more higher temperatures (eg 30 C) injured the gameto-phytes of D tectorum as did the lower temperatures of 0and 4 C These findings suggest that extreme temperaturesare unsuitable for storing this moss species Further stud-ies are warranted on the impact of the storage environmenton desiccation-tolerant mosses For example Burch (2003)found that the survival and regeneration of dehydrated pro-tonemata were reduced after cryopreservation due to dam-age caused by intracellular ice crystals The desiccationtime can also affect the restorability of vegetative propaga-tion in desiccation-tolerant mosses and their physiologicalcharacteristics (Keever 1957 Proctor 2001) Environmental

changes or variation in the dormancy period of cells mightinfluence the restoration results after rehydration

42 Effects of storage temperature on the physiologicalcharacteristics of mosses

MDA an important product of membrane lipid peroxidationincreased in all mosses over the storage period This find-ing indicated that the 40-day storage period caused cell dam-age (Fig 2d Table 1) Accordingly the soluble sugar con-tent increased to protect the membranes and proteins in thedried gametophytes (Fig 2b Table 1) Sugars are the mainsubstance used to stabilize protein structures in desiccation-tolerant cells (Hoekstra et al 2001) However the solublesugar content of B unguiculata stored at 0 and 4 C was de-creased relative to the initial value This result might havebeen due to the low temperatures preventing the conversionfrom starch to soluble sugar (Pressel et al 2006) Whenmosses suffered oxidative damage the increases in chloro-phyll content and soluble protein content in some gameto-phytes were related to the recovery ability of desiccation-tolerant cells (Fig 2a and c Table 1) In previous studiesthe chlorophyll content of mosses increased during desicca-tion and their photosynthetic capacity recovered rapidly af-ter rewetting (Alpert 1988 Csintalan et al 1999) Similarlyprotein synthesis recovered after rehydration (Oliver 1991)since cellular recovery is an important part of DT (Proctoret al 2007)

The recovery of photosynthesis and protein synthesis inB unguiculata was facilitated by higher temperatures (notmore than 30 C Fig 2a and c) This finding is inconsis-tent with the pattern in other mosses in which viabilitytends to be lower at increased temperatures (Hearnshaw andProctor 1982) However the increasing trend of MDA con-tent from 17 to 30 C suggests that more extensive mem-brane damage may be caused by storage temperatures above30 C (Fig 2d) The adverse effects of the higher tempera-tures in D vinealis and D tectorum were clearly reflectedby the slower recovery of photosynthesis and protein syn-thesis (Fig 2a and c) The changes in the MDA content inD vinealis suggested more rapid repair of cell membranewith increasing temperature however the species possiblyhad stronger tolerance under the protection of abundant sug-ars when the recovery of photosynthesis and protein synthe-sis was slower (Fig 2andashd)

Biogeosciences 15 797ndash808 2018 wwwbiogeosciencesnet157972018

Y Guo and Y Zhao Effects of storage temperature on desiccation-tolerant mosses 805

The responses of the physiological characteristics of thethree species to temperature reflected species variation inrestoration ability over a short rehydration time Because therewetting periods were longer than 30 days in the cultiva-tion the vegetative propagation results can be considered asreflecting the long-term recovery of mosses Thus the long-term effect of cell recovery during short-term rehydration canbe explained by the relationships between the physiologi-cal characteristics and vegetative propagation of desiccation-tolerant mosses

43 Relationships between physiological characteristicsand the vegetative propagation of mosses

Before storage the four physiological indices of gameto-phytes showed significant differences between D vinealisand D tectorum However no significant differences be-tween the two species were observed in regard to the threegermination parameters (Table 1) Mosses of similar fertilityshowed significant differences in physiological characteris-tics Species differences in DT led to larger differences invegetative propagation among species than before as evi-denced by the values of the gametophyte vigor indices withinthe same treatment (Tables 1 and 2) Therefore the recoveryability of dried mosses with respect to development and re-generation might be more informative for screening suitableinocula than using fresh mosses in dry habitats Many stud-ies have indicated that desiccation-tolerant mosses can re-cover from drying once they are rehydrated (Csintalan et al1999 Pressel et al 2006) However long periods of desic-cation would impede the reuse of moss specimens and therestoration of dried biocrusts This study showed that cellswere subjected to oxidative damage after the 40-day desicca-tion period (Fig 2d Table 1) Over this period the regener-ative capacity of the three species declined (Table 2) whichsuggested that membrane integrity andor other factors af-fected the vegetative propagation of the desiccation-tolerantmosses

Based on the correlation coefficients among the physio-logical indices and germination parameters of desiccation-tolerant mosses (Table 3) gametophyte germination was sig-nificantly and positively correlated with chlorophyll contentsoluble sugar content and soluble protein content In addi-tion gametophyte increment and gametophyte vigor indexwere significantly and negatively correlated with MDA con-tent These findings are in accordance with the observationsthat metabolic repair is favorable to the germination of newgametophytes and that long-term recovery is more dependenton cell integrity than metabolic repair Therefore to quan-titatively compare the effects of the four physiological in-dices on vegetative propagation the gray incidence degreebetween the physiological indices and the gametophyte vigorindex for each of the three moss species was calculated byusing Eqs (4)ndash(6) As shown in Table 4 the effect of MDAcontent on the gametophyte vigor index was the strongest

in B unguiculata and D tectorum and the incidence degreeof MDA (073) in D vinealis was similar to the maximum(074) In all three mosses MDA content increased as stor-age temperature decreased from 17 to 0 C Smaller gameto-phyte vigor index values were observed for D vinealis andD tectorum at 0 and 4 C than at 25 and 30 C (Fig 2d Ta-ble 2) This result indicated that the greater membrane dam-age incurred at low temperatures caused the decline in regen-erative capacity In addition the higher gametophyte vigorindex values of D tectorum at 17 and 25 C than before stor-age were possibly related to the reduced formation of intra-cellular ice crystals at these temperatures during the storageperiod (Burch 2003) which facilitated more rapid recoveryupon rehydration (Table 2) However the number of nega-tive effects on physiological characteristics increased withincreasing temperature (Fig 2andashc) The high temperatureswere unfavorable to the recovery of the mosses (Hearnshawand Proctor 1982) When cells suffered damage under desic-cation and temperature stress the protection provided by ad-ditional sugars was important for maintaining cell integrityin the dry state (Fig 2d Table 1) D vinealis showed no sig-nificant difference in regenerative capacity among tempera-tures potentially because the level of cellular protection wasequivalent among the different temperatures

Researchers have summarized the recovery mechanisms ofmosses upon rehydration such as the rapid recovery of pho-tosynthesis respiration and protein synthesis within min-utes to hours (Proctor et al 2007) However recovery ofthe carbon balance cell cycle and the cytoskeleton requiremore than 24 h (Alpert and Oechel 1985 Mansour and Hal-let 1981 Pressel et al 2006) Based on these results it hasbeen speculated that cell integrity is more difficult to recoverthan physiological reactions and that cell integrity greatlylimits the recovery and regenerative capacity of desiccation-tolerant mosses Over long-term desiccation the cumulativedamage affects cell function and integrity (Proctor 2001)different temperatures might enhance or suppress such celldamage Thus the effects of temperature on the ecology ofDT in bryophytes warrant investigation especially during thedry season in semiarid and arid areas The greater sensitiv-ity of D tectorum observed here might provide insight intowhy this species is not a widely distributed species such asD vinealis in the study region Furthermore the ecologi-cal niche requirements of different mosses in both dry andwet periods will influence the choice of moss inocula for ar-tificial cultivation and biocrust restoration Field studies areneeded to better understand the ecological requirements ofdried mosses Furthermore a precise description of micro-climates and the application of quantitative methods wouldbe helpful

wwwbiogeosciencesnet157972018 Biogeosciences 15 797ndash808 2018

806 Y Guo and Y Zhao Effects of storage temperature on desiccation-tolerant mosses

5 Conclusions

The conducted experiment explored the effect of storage tem-perature on the vegetative propagation of desiccation-tolerantmosses and influencing factors The results indicated that thedecline in regenerative capacity in mosses observed follow-ing storage was related to cell damage caused by dehydra-tion during storage The storage temperature during dehydra-tion influenced the vegetative propagation of mosses throughchanges in moss cell activity Further analysis showed thatthe factor with the strongest effect on vegetative propagationwas membrane damage During storage soluble sugars in-creased to protect the cells highlighting the important role ofcell integrity in influencing the physiological characteristicsand vegetative propagation of desiccation-tolerant mosses Inthis study the optimal storage temperature of D vinealis andD tectorum was 17 C whereas the optimal temperature forB unguiculata was 4 C Different responses to temperatureamong the three moss species were associated with speciesdifferences in DT These findings can potentially guide futureresearch on suitable storage methods for inoculation materialto improve the artificial cultivation of moss biocrusts

In general the properties of inoculation material are keyfactors affecting the development and recovery of mossbiocrusts such as species physiological features andorother factors The results provide insight into the factors thatinfluence the vegetative propagation of desiccation-tolerantmosses and highlight the potential applicability of a rapidexperimental approach for screening suitable inocula

Data availability Currently data can only be accessed in the formof Excel sheets via contact with the corresponding author

Competing interests The authors declare that they have no conflictof interest

Special issue statement This article is part of the special issue ldquoBi-ological soil crusts and their role in biogeochemical processes andcyclingrdquo It is a result of the BIOCRUST3 conference Moab USA26 to 30 September 2016

Acknowledgements The research was supported by the NationalNatural Science Foundation of China (grant nos 4157126841271298) We also express our gratitude to the anonymousreviewers and editors for their constructive comments and sugges-tions

Edited by Bettina WeberReviewed by three anonymous referees

References

Abdul-baki A A and Anderson J D Relation-ship between decarboxylation of glutamic-acid andvigor in soybean seed Crop Sci 13 227ndash232httpsdoiorg102135cropsci19730011183X001300020023x1973

Alpert P Survival of a desiccation-tolerant moss Grimmia laevi-gata beyond its observed microdistributional limits J Bryol15 219ndash227 httpsdoiorg101179jbr1988151219 1988

Alpert P and Oechel W C Carbon balance limits microdistribu-tion of Grimmia laevigata a desiccation-tolerant plant Ecology66 660ndash669 httpsdoiorg1023071940527 1985

Antoninka A Bowker M A Reed S C and Doherty K Pro-duction of greenhouse-grown biocrust mosses and associatedcyanobacteria to rehabilitate dryland soil function Restor Ecol24 324ndash335 httpsdoiorg101111rec12311 2016

Belnap J and Eldridge D Disturbance and recovery of biologicalsoil crusts in Biological Soil Crusts Structure Function andManagement edited by Belnap J and Lange O L SpringerBerlin Germany 363ndash383 2003

Belnap J and Lange O L Structure and functioning of biolog-ical soil crusts a synthesis in Biological Soil Crusts Struc-ture Function and Management edited by Belnap J andLange O L Springer Berlin Germany 471ndash479 2003

Belnap J Weber B and Buumldel B Biological soil crusts as an or-ganizing principle in drylands in Biological Soil Crusts An Or-ganizing Principle in Drylands edited by Weber B Buumldel Band Belnap J Springer Berlin Germany 3ndash13 2016

Bradford M M A rapid and sensitive method for the quantifi-cation of microgram quantities of protein utilizing the prin-ciple of protein dye binding Anal Biochem 72 248ndash254httpsdoiorg1010160003-2697(76)90527-3 1976

Burch J Some mosses survive cryopreserva-tion without prior pretreatment Bryologist106 270ndash277 httpsdoiorg1016390007-2745(2003)106[0270SMSCWP]20CO2 2003

Burch J and Wilkinson T Cryopreservation of protonemataof Ditrichum cornubicum (Paton) comparing the effectivenessof four cryoprotectant pretreatments Cryoletters 23 197ndash2082002

Chinese Central Meteorological Station httpwwwnmccnpublishforecastASNansaihtml last access 2 August 2017

Cleavitt N L Stress tolerance of rare and common moss speciesin relation to their occupied environments and asexual dispersalpotential J Ecol 90 785ndash795 httpsdoiorg101046j1365-2745200200713x 2002

Csintalan Z Proctor M C F and Tuba Z Chlorophyll fluo-rescence during drying and rehydration in the mosses Rhytidi-adelphus loreus (Hedw) Warnst Anomodon viticulosus (Hedw)Hook amp Tayl and Grimmia pulvinata (Hedw) Sm Ann Bot-London 84 235ndash244 httpsdoiorg101006anbo199909191999

Deng J L Control problems of grey systems Syst Control Lett1 288ndash294 httpsdoiorg101016S0167-6911(82)80025-X1982

Biogeosciences 15 797ndash808 2018 wwwbiogeosciencesnet157972018

802 Y Guo and Y Zhao Effects of storage temperature on desiccation-tolerant mosses

Table 2 Gametophyte vigor index of the three mosses under treat-ments

Treatment B unguiculata D vinealis D tectorum

Initial value 128plusmn 015a 182plusmn 040a 233plusmn 005a0 C 068plusmn 022b 099plusmn 017b 109plusmn 010b4 C 104plusmn 002ac 111plusmn 013b 126plusmn 003b17 C 095plusmn 005c 122plusmn 010b 366plusmn 035c25 C 065plusmn 006b 117plusmn 013b 290plusmn 046a30 C 086plusmn 018bc 115plusmn 017b 204plusmn 033a

Data are averageplusmn 1 SE and different letters indicate significant differences(P lt 005) among treatments within the same species

age in D tectorum was at 25 C The highest gametophyteincrement of B unguiculata was at 4 C The highest game-tophyte increment values in D vinealis and D tectorum wereboth at 17 C as observed for the gametophyte vigor indexvalues of these two species

33 Effects of storage temperature on the physiologicalindices of mosses

As shown in Table 1 and Fig 2a the chlorophyll content ofB unguiculata increased after storage at four of the five tem-peratures ie all but 0 C The chlorophyll content of B un-guiculata showed an increasing trend with increasing stor-age temperature with the maximum increase of 7308 ob-served at 30 C The smallest change in chlorophyll contentwas observed in D vinealis which showed a maximum de-crease of 1789 at 4 C and a minimum decrease of 239 at 17 C The chlorophyll content of D tectorum after storagewas decreased by 3151 at 17 C and increased by 1850 at 25 C yielding the highest and lowest content values re-spectively

A similar increasing trend with temperature was found forsoluble sugar content (Fig 2b) The soluble sugar contentwas consistently higher after storage than before except inB unguiculata in which sugar content was decreased by5652 and 4047 at 0 and 4 C respectively (Fig 2b Ta-ble 1) The soluble sugar content of D vinealis showed lessvariation than the other species No significant difference wasfound between the minimum and maximum increases whichwere 992 at 0 C and 2314 at 25 C respectively Thegreatest changes in soluble sugar content with greater than65 increases at all storage temperatures occurred in D tec-torum

MDA content showed greater variation than sugar contentincreasing by more than 50 in all stored gametophytes(Fig 2d Table 1) The MDA content of both B unguicu-lata and D tectorum decreased as the temperature increasedfrom 0 to 17 C the minimum value of MDA content (at17 C) was 170 times and 206 times the initial value respec-tively However the MDA content of D vinealis was 154 to

298 times the initial value after storage and continuously de-creased with increasing temperature

Some temperatures caused the soluble protein content tochange significantly (Fig 2c Table 1) The soluble proteincontent of B unguiculata increased abruptly from a 3179 decrease from the initial value to a 4006 increase with in-creasing temperature In contrast soluble protein showed theopposite trend in D vinealis and D tectorum Both speciespresented a maximum increase at 0 C which was 1664 inD vinealis and 2365 in D tectorum The lowest solubleprotein content of D vinealis and D tectorum representeda decrease of 1600 at 25 C and a decrease of 2138 at30 C respectively

Our results indicated that the sharpest changes in chloro-phyll content and soluble protein content with increasingtemperature were observed in B unguiculata furthermoresoluble sugar content and MDA content changed morerapidly with increasing temperature in this species than inD vinealis and D tectorum (Fig 2andashd Table 1) D vinealisshowed slower changes in chlorophyll soluble sugar andsoluble protein contents with increasing temperature thanthe other two species MDA content however varied widelywith temperature The largest increases in soluble sugar con-tent and MDA content after 40 days of storage were observedin D tectorum In all three moss species the greatest changeswere observed in MDA content followed by soluble sugarcontent (Fig 2b and d Table 1)

34 Relationships between physiological characteristicsand the vegetative propagation of mosses

After analyzing the correlations between the physiologi-cal indices and germination parameters of the desiccation-tolerant mosses a significant correlation (P lt 001) wasfound between each physiological index except for chloro-phyll content and MDA content (Table 3) Gametophytegermination was significantly correlated (P lt 005) withsoluble protein content and highly significantly correlated(P lt 001) with both chlorophyll content and soluble sugarcontent MDA content was significantly negatively correlated(P lt 005) with both gametophyte increment and gameto-phyte vigor index

At a distinguishing coefficient of 05 the gray incidencedegrees between the physiological indices (X1 chlorophyllcontent X2 soluble sugar content X3 soluble protein con-tent X4 MDA content) and the gametophyte vigor indexin the three moss species were (1) X4gtX1gtX2=X3 inB unguiculata (2) X3gtX4gtX2gtX1 in D vinealis and(3) X4gtX3gtX1gtX2 in D tectorum (Table 4)

Biogeosciences 15 797ndash808 2018 wwwbiogeosciencesnet157972018

Y Guo and Y Zhao Effects of storage temperature on desiccation-tolerant mosses 803

Figure 2 (andashd) Data (averageplusmn 1 SE) for the three moss species on (a) chlorophyll content (b) soluble sugar content (c) soluble proteincontent and (d) MDA content after the 40-day storage period at each of the five temperatures Different letters indicate significant differences(P lt 005) among the five temperatures within the same species Dotted lines represent the approximate values of the two germinationparameters before storage for each species (the true values are shown in Table 1)

Table 3 Correlation coefficients between physiological indices and germination parameters across all mosses and treatments

Variables Chlorophyll Sugar Protein MDA Germination Increment

Sugar 0762lowastlowast

Protein 0747lowastlowast 0781lowastlowast

MDA 0220 0402lowastlowast 0510lowastlowast

Germination 0473lowastlowast 0414lowastlowast 0313lowast minus0022Increment minus0239 minus0187 minus0249 minus0344lowast 0388lowastlowast

Vigor index minus0158 minus0122 minus0191 minus0328lowast 0441lowastlowast 0995lowastlowast

Chlorophyll chlorophyll content sugar soluble sugar content protein soluble protein content MDA MDA contentgermination gametophyte germination increment gametophyte increment vigor index gametophyte vigor indexThe lowast symbol indicates a significant correlation at P lt 005 lowastlowast indicates a significant correlation at P lt 001

wwwbiogeosciencesnet157972018 Biogeosciences 15 797ndash808 2018

804 Y Guo and Y Zhao Effects of storage temperature on desiccation-tolerant mosses

Table 4 Gray incidence degree between physiological indices and the gametophyte vigor index across all treatments

Reference sequences B unguiculata D vinealis D tectorum

Chlorophyll content (X1) 060plusmn 020 055plusmn 027 066plusmn 021Soluble sugar content (X2) 057plusmn 020 062plusmn 023 062plusmn 017Soluble protein content (X3) 057plusmn 022 074plusmn 028 070plusmn 025MDA content (X4) 077plusmn 020 073plusmn 022 076plusmn 027

4 Discussion

41 Effects of storage temperature on the vegetativepropagation of mosses

For more than a century researchers have studied many as-pects of mosses such as inocula pretreatment (eg stor-age and sterilization) culture methods and culture condi-tions (Duckett et al 2004 Hoffman 1966) Some of thesestudies have implied that the physiological characteristics ofmoss gametophytes are closely related to the success of ar-tificial cultivation for example pretreatment with sucroseandor abscisic acid can improve the viability of mosses byincreasing DT (Burch and Wilkinson 2002) In line with pre-vious studies this study found that gametophyte regenera-tion within the same species after desiccation varied amongdifferent temperatures (Fig 1a and b Table 2) which islikely related to species-specific DT The regenerative capac-ity of mosses can be summarily described by the gameto-phyte vigor index on the basis of Eqs (1)ndash(3) and Table 3The gametophyte vigor index most sensitive to storage tem-perature was that of D tectorum whereas that of D vinealisvaried little with storage temperature with no significant dif-ferences among temperatures (Table 2) Thus the effect ofstorage temperature on regenerative capacity was strongestin D tectorum and weakest in D vinealis

The 40-day storage period adversely affected regenera-tion in most gametophytes (Fig 1a and b Table 1) how-ever some gametophytes of D tectorum stored at 17 and25 C produced more new shoots than before It is not clearwhether this enhanced regeneration was associated with thelow-temperature tolerance of D tectorum D tectorum possi-bly suffered low-temperature stress in early winter Further-more higher temperatures (eg 30 C) injured the gameto-phytes of D tectorum as did the lower temperatures of 0and 4 C These findings suggest that extreme temperaturesare unsuitable for storing this moss species Further stud-ies are warranted on the impact of the storage environmenton desiccation-tolerant mosses For example Burch (2003)found that the survival and regeneration of dehydrated pro-tonemata were reduced after cryopreservation due to dam-age caused by intracellular ice crystals The desiccationtime can also affect the restorability of vegetative propaga-tion in desiccation-tolerant mosses and their physiologicalcharacteristics (Keever 1957 Proctor 2001) Environmental

changes or variation in the dormancy period of cells mightinfluence the restoration results after rehydration

42 Effects of storage temperature on the physiologicalcharacteristics of mosses

MDA an important product of membrane lipid peroxidationincreased in all mosses over the storage period This find-ing indicated that the 40-day storage period caused cell dam-age (Fig 2d Table 1) Accordingly the soluble sugar con-tent increased to protect the membranes and proteins in thedried gametophytes (Fig 2b Table 1) Sugars are the mainsubstance used to stabilize protein structures in desiccation-tolerant cells (Hoekstra et al 2001) However the solublesugar content of B unguiculata stored at 0 and 4 C was de-creased relative to the initial value This result might havebeen due to the low temperatures preventing the conversionfrom starch to soluble sugar (Pressel et al 2006) Whenmosses suffered oxidative damage the increases in chloro-phyll content and soluble protein content in some gameto-phytes were related to the recovery ability of desiccation-tolerant cells (Fig 2a and c Table 1) In previous studiesthe chlorophyll content of mosses increased during desicca-tion and their photosynthetic capacity recovered rapidly af-ter rewetting (Alpert 1988 Csintalan et al 1999) Similarlyprotein synthesis recovered after rehydration (Oliver 1991)since cellular recovery is an important part of DT (Proctoret al 2007)

The recovery of photosynthesis and protein synthesis inB unguiculata was facilitated by higher temperatures (notmore than 30 C Fig 2a and c) This finding is inconsis-tent with the pattern in other mosses in which viabilitytends to be lower at increased temperatures (Hearnshaw andProctor 1982) However the increasing trend of MDA con-tent from 17 to 30 C suggests that more extensive mem-brane damage may be caused by storage temperatures above30 C (Fig 2d) The adverse effects of the higher tempera-tures in D vinealis and D tectorum were clearly reflectedby the slower recovery of photosynthesis and protein syn-thesis (Fig 2a and c) The changes in the MDA content inD vinealis suggested more rapid repair of cell membranewith increasing temperature however the species possiblyhad stronger tolerance under the protection of abundant sug-ars when the recovery of photosynthesis and protein synthe-sis was slower (Fig 2andashd)

Biogeosciences 15 797ndash808 2018 wwwbiogeosciencesnet157972018

Y Guo and Y Zhao Effects of storage temperature on desiccation-tolerant mosses 805

The responses of the physiological characteristics of thethree species to temperature reflected species variation inrestoration ability over a short rehydration time Because therewetting periods were longer than 30 days in the cultiva-tion the vegetative propagation results can be considered asreflecting the long-term recovery of mosses Thus the long-term effect of cell recovery during short-term rehydration canbe explained by the relationships between the physiologi-cal characteristics and vegetative propagation of desiccation-tolerant mosses

43 Relationships between physiological characteristicsand the vegetative propagation of mosses

Before storage the four physiological indices of gameto-phytes showed significant differences between D vinealisand D tectorum However no significant differences be-tween the two species were observed in regard to the threegermination parameters (Table 1) Mosses of similar fertilityshowed significant differences in physiological characteris-tics Species differences in DT led to larger differences invegetative propagation among species than before as evi-denced by the values of the gametophyte vigor indices withinthe same treatment (Tables 1 and 2) Therefore the recoveryability of dried mosses with respect to development and re-generation might be more informative for screening suitableinocula than using fresh mosses in dry habitats Many stud-ies have indicated that desiccation-tolerant mosses can re-cover from drying once they are rehydrated (Csintalan et al1999 Pressel et al 2006) However long periods of desic-cation would impede the reuse of moss specimens and therestoration of dried biocrusts This study showed that cellswere subjected to oxidative damage after the 40-day desicca-tion period (Fig 2d Table 1) Over this period the regener-ative capacity of the three species declined (Table 2) whichsuggested that membrane integrity andor other factors af-fected the vegetative propagation of the desiccation-tolerantmosses

Based on the correlation coefficients among the physio-logical indices and germination parameters of desiccation-tolerant mosses (Table 3) gametophyte germination was sig-nificantly and positively correlated with chlorophyll contentsoluble sugar content and soluble protein content In addi-tion gametophyte increment and gametophyte vigor indexwere significantly and negatively correlated with MDA con-tent These findings are in accordance with the observationsthat metabolic repair is favorable to the germination of newgametophytes and that long-term recovery is more dependenton cell integrity than metabolic repair Therefore to quan-titatively compare the effects of the four physiological in-dices on vegetative propagation the gray incidence degreebetween the physiological indices and the gametophyte vigorindex for each of the three moss species was calculated byusing Eqs (4)ndash(6) As shown in Table 4 the effect of MDAcontent on the gametophyte vigor index was the strongest

in B unguiculata and D tectorum and the incidence degreeof MDA (073) in D vinealis was similar to the maximum(074) In all three mosses MDA content increased as stor-age temperature decreased from 17 to 0 C Smaller gameto-phyte vigor index values were observed for D vinealis andD tectorum at 0 and 4 C than at 25 and 30 C (Fig 2d Ta-ble 2) This result indicated that the greater membrane dam-age incurred at low temperatures caused the decline in regen-erative capacity In addition the higher gametophyte vigorindex values of D tectorum at 17 and 25 C than before stor-age were possibly related to the reduced formation of intra-cellular ice crystals at these temperatures during the storageperiod (Burch 2003) which facilitated more rapid recoveryupon rehydration (Table 2) However the number of nega-tive effects on physiological characteristics increased withincreasing temperature (Fig 2andashc) The high temperatureswere unfavorable to the recovery of the mosses (Hearnshawand Proctor 1982) When cells suffered damage under desic-cation and temperature stress the protection provided by ad-ditional sugars was important for maintaining cell integrityin the dry state (Fig 2d Table 1) D vinealis showed no sig-nificant difference in regenerative capacity among tempera-tures potentially because the level of cellular protection wasequivalent among the different temperatures

Researchers have summarized the recovery mechanisms ofmosses upon rehydration such as the rapid recovery of pho-tosynthesis respiration and protein synthesis within min-utes to hours (Proctor et al 2007) However recovery ofthe carbon balance cell cycle and the cytoskeleton requiremore than 24 h (Alpert and Oechel 1985 Mansour and Hal-let 1981 Pressel et al 2006) Based on these results it hasbeen speculated that cell integrity is more difficult to recoverthan physiological reactions and that cell integrity greatlylimits the recovery and regenerative capacity of desiccation-tolerant mosses Over long-term desiccation the cumulativedamage affects cell function and integrity (Proctor 2001)different temperatures might enhance or suppress such celldamage Thus the effects of temperature on the ecology ofDT in bryophytes warrant investigation especially during thedry season in semiarid and arid areas The greater sensitiv-ity of D tectorum observed here might provide insight intowhy this species is not a widely distributed species such asD vinealis in the study region Furthermore the ecologi-cal niche requirements of different mosses in both dry andwet periods will influence the choice of moss inocula for ar-tificial cultivation and biocrust restoration Field studies areneeded to better understand the ecological requirements ofdried mosses Furthermore a precise description of micro-climates and the application of quantitative methods wouldbe helpful

wwwbiogeosciencesnet157972018 Biogeosciences 15 797ndash808 2018

806 Y Guo and Y Zhao Effects of storage temperature on desiccation-tolerant mosses

5 Conclusions

The conducted experiment explored the effect of storage tem-perature on the vegetative propagation of desiccation-tolerantmosses and influencing factors The results indicated that thedecline in regenerative capacity in mosses observed follow-ing storage was related to cell damage caused by dehydra-tion during storage The storage temperature during dehydra-tion influenced the vegetative propagation of mosses throughchanges in moss cell activity Further analysis showed thatthe factor with the strongest effect on vegetative propagationwas membrane damage During storage soluble sugars in-creased to protect the cells highlighting the important role ofcell integrity in influencing the physiological characteristicsand vegetative propagation of desiccation-tolerant mosses Inthis study the optimal storage temperature of D vinealis andD tectorum was 17 C whereas the optimal temperature forB unguiculata was 4 C Different responses to temperatureamong the three moss species were associated with speciesdifferences in DT These findings can potentially guide futureresearch on suitable storage methods for inoculation materialto improve the artificial cultivation of moss biocrusts

In general the properties of inoculation material are keyfactors affecting the development and recovery of mossbiocrusts such as species physiological features andorother factors The results provide insight into the factors thatinfluence the vegetative propagation of desiccation-tolerantmosses and highlight the potential applicability of a rapidexperimental approach for screening suitable inocula

Data availability Currently data can only be accessed in the formof Excel sheets via contact with the corresponding author

Competing interests The authors declare that they have no conflictof interest

Special issue statement This article is part of the special issue ldquoBi-ological soil crusts and their role in biogeochemical processes andcyclingrdquo It is a result of the BIOCRUST3 conference Moab USA26 to 30 September 2016

Acknowledgements The research was supported by the NationalNatural Science Foundation of China (grant nos 4157126841271298) We also express our gratitude to the anonymousreviewers and editors for their constructive comments and sugges-tions

Edited by Bettina WeberReviewed by three anonymous referees

References

Abdul-baki A A and Anderson J D Relation-ship between decarboxylation of glutamic-acid andvigor in soybean seed Crop Sci 13 227ndash232httpsdoiorg102135cropsci19730011183X001300020023x1973

Alpert P Survival of a desiccation-tolerant moss Grimmia laevi-gata beyond its observed microdistributional limits J Bryol15 219ndash227 httpsdoiorg101179jbr1988151219 1988

Alpert P and Oechel W C Carbon balance limits microdistribu-tion of Grimmia laevigata a desiccation-tolerant plant Ecology66 660ndash669 httpsdoiorg1023071940527 1985

Antoninka A Bowker M A Reed S C and Doherty K Pro-duction of greenhouse-grown biocrust mosses and associatedcyanobacteria to rehabilitate dryland soil function Restor Ecol24 324ndash335 httpsdoiorg101111rec12311 2016

Belnap J and Eldridge D Disturbance and recovery of biologicalsoil crusts in Biological Soil Crusts Structure Function andManagement edited by Belnap J and Lange O L SpringerBerlin Germany 363ndash383 2003

Belnap J and Lange O L Structure and functioning of biolog-ical soil crusts a synthesis in Biological Soil Crusts Struc-ture Function and Management edited by Belnap J andLange O L Springer Berlin Germany 471ndash479 2003

Belnap J Weber B and Buumldel B Biological soil crusts as an or-ganizing principle in drylands in Biological Soil Crusts An Or-ganizing Principle in Drylands edited by Weber B Buumldel Band Belnap J Springer Berlin Germany 3ndash13 2016

Bradford M M A rapid and sensitive method for the quantifi-cation of microgram quantities of protein utilizing the prin-ciple of protein dye binding Anal Biochem 72 248ndash254httpsdoiorg1010160003-2697(76)90527-3 1976

Burch J Some mosses survive cryopreserva-tion without prior pretreatment Bryologist106 270ndash277 httpsdoiorg1016390007-2745(2003)106[0270SMSCWP]20CO2 2003

Burch J and Wilkinson T Cryopreservation of protonemataof Ditrichum cornubicum (Paton) comparing the effectivenessof four cryoprotectant pretreatments Cryoletters 23 197ndash2082002

Chinese Central Meteorological Station httpwwwnmccnpublishforecastASNansaihtml last access 2 August 2017

Cleavitt N L Stress tolerance of rare and common moss speciesin relation to their occupied environments and asexual dispersalpotential J Ecol 90 785ndash795 httpsdoiorg101046j1365-2745200200713x 2002

Csintalan Z Proctor M C F and Tuba Z Chlorophyll fluo-rescence during drying and rehydration in the mosses Rhytidi-adelphus loreus (Hedw) Warnst Anomodon viticulosus (Hedw)Hook amp Tayl and Grimmia pulvinata (Hedw) Sm Ann Bot-London 84 235ndash244 httpsdoiorg101006anbo199909191999

Deng J L Control problems of grey systems Syst Control Lett1 288ndash294 httpsdoiorg101016S0167-6911(82)80025-X1982

Biogeosciences 15 797ndash808 2018 wwwbiogeosciencesnet157972018

Y Guo and Y Zhao Effects of storage temperature on desiccation-tolerant mosses 803

Figure 2 (andashd) Data (averageplusmn 1 SE) for the three moss species on (a) chlorophyll content (b) soluble sugar content (c) soluble proteincontent and (d) MDA content after the 40-day storage period at each of the five temperatures Different letters indicate significant differences(P lt 005) among the five temperatures within the same species Dotted lines represent the approximate values of the two germinationparameters before storage for each species (the true values are shown in Table 1)

Table 3 Correlation coefficients between physiological indices and germination parameters across all mosses and treatments

Variables Chlorophyll Sugar Protein MDA Germination Increment

Sugar 0762lowastlowast

Protein 0747lowastlowast 0781lowastlowast

MDA 0220 0402lowastlowast 0510lowastlowast

Germination 0473lowastlowast 0414lowastlowast 0313lowast minus0022Increment minus0239 minus0187 minus0249 minus0344lowast 0388lowastlowast

Vigor index minus0158 minus0122 minus0191 minus0328lowast 0441lowastlowast 0995lowastlowast

Chlorophyll chlorophyll content sugar soluble sugar content protein soluble protein content MDA MDA contentgermination gametophyte germination increment gametophyte increment vigor index gametophyte vigor indexThe lowast symbol indicates a significant correlation at P lt 005 lowastlowast indicates a significant correlation at P lt 001

wwwbiogeosciencesnet157972018 Biogeosciences 15 797ndash808 2018

804 Y Guo and Y Zhao Effects of storage temperature on desiccation-tolerant mosses

Table 4 Gray incidence degree between physiological indices and the gametophyte vigor index across all treatments

Reference sequences B unguiculata D vinealis D tectorum

Chlorophyll content (X1) 060plusmn 020 055plusmn 027 066plusmn 021Soluble sugar content (X2) 057plusmn 020 062plusmn 023 062plusmn 017Soluble protein content (X3) 057plusmn 022 074plusmn 028 070plusmn 025MDA content (X4) 077plusmn 020 073plusmn 022 076plusmn 027

4 Discussion

41 Effects of storage temperature on the vegetativepropagation of mosses

For more than a century researchers have studied many as-pects of mosses such as inocula pretreatment (eg stor-age and sterilization) culture methods and culture condi-tions (Duckett et al 2004 Hoffman 1966) Some of thesestudies have implied that the physiological characteristics ofmoss gametophytes are closely related to the success of ar-tificial cultivation for example pretreatment with sucroseandor abscisic acid can improve the viability of mosses byincreasing DT (Burch and Wilkinson 2002) In line with pre-vious studies this study found that gametophyte regenera-tion within the same species after desiccation varied amongdifferent temperatures (Fig 1a and b Table 2) which islikely related to species-specific DT The regenerative capac-ity of mosses can be summarily described by the gameto-phyte vigor index on the basis of Eqs (1)ndash(3) and Table 3The gametophyte vigor index most sensitive to storage tem-perature was that of D tectorum whereas that of D vinealisvaried little with storage temperature with no significant dif-ferences among temperatures (Table 2) Thus the effect ofstorage temperature on regenerative capacity was strongestin D tectorum and weakest in D vinealis

The 40-day storage period adversely affected regenera-tion in most gametophytes (Fig 1a and b Table 1) how-ever some gametophytes of D tectorum stored at 17 and25 C produced more new shoots than before It is not clearwhether this enhanced regeneration was associated with thelow-temperature tolerance of D tectorum D tectorum possi-bly suffered low-temperature stress in early winter Further-more higher temperatures (eg 30 C) injured the gameto-phytes of D tectorum as did the lower temperatures of 0and 4 C These findings suggest that extreme temperaturesare unsuitable for storing this moss species Further stud-ies are warranted on the impact of the storage environmenton desiccation-tolerant mosses For example Burch (2003)found that the survival and regeneration of dehydrated pro-tonemata were reduced after cryopreservation due to dam-age caused by intracellular ice crystals The desiccationtime can also affect the restorability of vegetative propaga-tion in desiccation-tolerant mosses and their physiologicalcharacteristics (Keever 1957 Proctor 2001) Environmental

changes or variation in the dormancy period of cells mightinfluence the restoration results after rehydration

42 Effects of storage temperature on the physiologicalcharacteristics of mosses

MDA an important product of membrane lipid peroxidationincreased in all mosses over the storage period This find-ing indicated that the 40-day storage period caused cell dam-age (Fig 2d Table 1) Accordingly the soluble sugar con-tent increased to protect the membranes and proteins in thedried gametophytes (Fig 2b Table 1) Sugars are the mainsubstance used to stabilize protein structures in desiccation-tolerant cells (Hoekstra et al 2001) However the solublesugar content of B unguiculata stored at 0 and 4 C was de-creased relative to the initial value This result might havebeen due to the low temperatures preventing the conversionfrom starch to soluble sugar (Pressel et al 2006) Whenmosses suffered oxidative damage the increases in chloro-phyll content and soluble protein content in some gameto-phytes were related to the recovery ability of desiccation-tolerant cells (Fig 2a and c Table 1) In previous studiesthe chlorophyll content of mosses increased during desicca-tion and their photosynthetic capacity recovered rapidly af-ter rewetting (Alpert 1988 Csintalan et al 1999) Similarlyprotein synthesis recovered after rehydration (Oliver 1991)since cellular recovery is an important part of DT (Proctoret al 2007)

The recovery of photosynthesis and protein synthesis inB unguiculata was facilitated by higher temperatures (notmore than 30 C Fig 2a and c) This finding is inconsis-tent with the pattern in other mosses in which viabilitytends to be lower at increased temperatures (Hearnshaw andProctor 1982) However the increasing trend of MDA con-tent from 17 to 30 C suggests that more extensive mem-brane damage may be caused by storage temperatures above30 C (Fig 2d) The adverse effects of the higher tempera-tures in D vinealis and D tectorum were clearly reflectedby the slower recovery of photosynthesis and protein syn-thesis (Fig 2a and c) The changes in the MDA content inD vinealis suggested more rapid repair of cell membranewith increasing temperature however the species possiblyhad stronger tolerance under the protection of abundant sug-ars when the recovery of photosynthesis and protein synthe-sis was slower (Fig 2andashd)

Biogeosciences 15 797ndash808 2018 wwwbiogeosciencesnet157972018

Y Guo and Y Zhao Effects of storage temperature on desiccation-tolerant mosses 805

The responses of the physiological characteristics of thethree species to temperature reflected species variation inrestoration ability over a short rehydration time Because therewetting periods were longer than 30 days in the cultiva-tion the vegetative propagation results can be considered asreflecting the long-term recovery of mosses Thus the long-term effect of cell recovery during short-term rehydration canbe explained by the relationships between the physiologi-cal characteristics and vegetative propagation of desiccation-tolerant mosses

43 Relationships between physiological characteristicsand the vegetative propagation of mosses

Before storage the four physiological indices of gameto-phytes showed significant differences between D vinealisand D tectorum However no significant differences be-tween the two species were observed in regard to the threegermination parameters (Table 1) Mosses of similar fertilityshowed significant differences in physiological characteris-tics Species differences in DT led to larger differences invegetative propagation among species than before as evi-denced by the values of the gametophyte vigor indices withinthe same treatment (Tables 1 and 2) Therefore the recoveryability of dried mosses with respect to development and re-generation might be more informative for screening suitableinocula than using fresh mosses in dry habitats Many stud-ies have indicated that desiccation-tolerant mosses can re-cover from drying once they are rehydrated (Csintalan et al1999 Pressel et al 2006) However long periods of desic-cation would impede the reuse of moss specimens and therestoration of dried biocrusts This study showed that cellswere subjected to oxidative damage after the 40-day desicca-tion period (Fig 2d Table 1) Over this period the regener-ative capacity of the three species declined (Table 2) whichsuggested that membrane integrity andor other factors af-fected the vegetative propagation of the desiccation-tolerantmosses

Based on the correlation coefficients among the physio-logical indices and germination parameters of desiccation-tolerant mosses (Table 3) gametophyte germination was sig-nificantly and positively correlated with chlorophyll contentsoluble sugar content and soluble protein content In addi-tion gametophyte increment and gametophyte vigor indexwere significantly and negatively correlated with MDA con-tent These findings are in accordance with the observationsthat metabolic repair is favorable to the germination of newgametophytes and that long-term recovery is more dependenton cell integrity than metabolic repair Therefore to quan-titatively compare the effects of the four physiological in-dices on vegetative propagation the gray incidence degreebetween the physiological indices and the gametophyte vigorindex for each of the three moss species was calculated byusing Eqs (4)ndash(6) As shown in Table 4 the effect of MDAcontent on the gametophyte vigor index was the strongest

in B unguiculata and D tectorum and the incidence degreeof MDA (073) in D vinealis was similar to the maximum(074) In all three mosses MDA content increased as stor-age temperature decreased from 17 to 0 C Smaller gameto-phyte vigor index values were observed for D vinealis andD tectorum at 0 and 4 C than at 25 and 30 C (Fig 2d Ta-ble 2) This result indicated that the greater membrane dam-age incurred at low temperatures caused the decline in regen-erative capacity In addition the higher gametophyte vigorindex values of D tectorum at 17 and 25 C than before stor-age were possibly related to the reduced formation of intra-cellular ice crystals at these temperatures during the storageperiod (Burch 2003) which facilitated more rapid recoveryupon rehydration (Table 2) However the number of nega-tive effects on physiological characteristics increased withincreasing temperature (Fig 2andashc) The high temperatureswere unfavorable to the recovery of the mosses (Hearnshawand Proctor 1982) When cells suffered damage under desic-cation and temperature stress the protection provided by ad-ditional sugars was important for maintaining cell integrityin the dry state (Fig 2d Table 1) D vinealis showed no sig-nificant difference in regenerative capacity among tempera-tures potentially because the level of cellular protection wasequivalent among the different temperatures

Researchers have summarized the recovery mechanisms ofmosses upon rehydration such as the rapid recovery of pho-tosynthesis respiration and protein synthesis within min-utes to hours (Proctor et al 2007) However recovery ofthe carbon balance cell cycle and the cytoskeleton requiremore than 24 h (Alpert and Oechel 1985 Mansour and Hal-let 1981 Pressel et al 2006) Based on these results it hasbeen speculated that cell integrity is more difficult to recoverthan physiological reactions and that cell integrity greatlylimits the recovery and regenerative capacity of desiccation-tolerant mosses Over long-term desiccation the cumulativedamage affects cell function and integrity (Proctor 2001)different temperatures might enhance or suppress such celldamage Thus the effects of temperature on the ecology ofDT in bryophytes warrant investigation especially during thedry season in semiarid and arid areas The greater sensitiv-ity of D tectorum observed here might provide insight intowhy this species is not a widely distributed species such asD vinealis in the study region Furthermore the ecologi-cal niche requirements of different mosses in both dry andwet periods will influence the choice of moss inocula for ar-tificial cultivation and biocrust restoration Field studies areneeded to better understand the ecological requirements ofdried mosses Furthermore a precise description of micro-climates and the application of quantitative methods wouldbe helpful

wwwbiogeosciencesnet157972018 Biogeosciences 15 797ndash808 2018

806 Y Guo and Y Zhao Effects of storage temperature on desiccation-tolerant mosses

5 Conclusions

The conducted experiment explored the effect of storage tem-perature on the vegetative propagation of desiccation-tolerantmosses and influencing factors The results indicated that thedecline in regenerative capacity in mosses observed follow-ing storage was related to cell damage caused by dehydra-tion during storage The storage temperature during dehydra-tion influenced the vegetative propagation of mosses throughchanges in moss cell activity Further analysis showed thatthe factor with the strongest effect on vegetative propagationwas membrane damage During storage soluble sugars in-creased to protect the cells highlighting the important role ofcell integrity in influencing the physiological characteristicsand vegetative propagation of desiccation-tolerant mosses Inthis study the optimal storage temperature of D vinealis andD tectorum was 17 C whereas the optimal temperature forB unguiculata was 4 C Different responses to temperatureamong the three moss species were associated with speciesdifferences in DT These findings can potentially guide futureresearch on suitable storage methods for inoculation materialto improve the artificial cultivation of moss biocrusts

In general the properties of inoculation material are keyfactors affecting the development and recovery of mossbiocrusts such as species physiological features andorother factors The results provide insight into the factors thatinfluence the vegetative propagation of desiccation-tolerantmosses and highlight the potential applicability of a rapidexperimental approach for screening suitable inocula

Data availability Currently data can only be accessed in the formof Excel sheets via contact with the corresponding author

Competing interests The authors declare that they have no conflictof interest

Special issue statement This article is part of the special issue ldquoBi-ological soil crusts and their role in biogeochemical processes andcyclingrdquo It is a result of the BIOCRUST3 conference Moab USA26 to 30 September 2016

Acknowledgements The research was supported by the NationalNatural Science Foundation of China (grant nos 4157126841271298) We also express our gratitude to the anonymousreviewers and editors for their constructive comments and sugges-tions

Edited by Bettina WeberReviewed by three anonymous referees

References

Abdul-baki A A and Anderson J D Relation-ship between decarboxylation of glutamic-acid andvigor in soybean seed Crop Sci 13 227ndash232httpsdoiorg102135cropsci19730011183X001300020023x1973

Alpert P Survival of a desiccation-tolerant moss Grimmia laevi-gata beyond its observed microdistributional limits J Bryol15 219ndash227 httpsdoiorg101179jbr1988151219 1988

Alpert P and Oechel W C Carbon balance limits microdistribu-tion of Grimmia laevigata a desiccation-tolerant plant Ecology66 660ndash669 httpsdoiorg1023071940527 1985

Antoninka A Bowker M A Reed S C and Doherty K Pro-duction of greenhouse-grown biocrust mosses and associatedcyanobacteria to rehabilitate dryland soil function Restor Ecol24 324ndash335 httpsdoiorg101111rec12311 2016

Belnap J and Eldridge D Disturbance and recovery of biologicalsoil crusts in Biological Soil Crusts Structure Function andManagement edited by Belnap J and Lange O L SpringerBerlin Germany 363ndash383 2003

Belnap J and Lange O L Structure and functioning of biolog-ical soil crusts a synthesis in Biological Soil Crusts Struc-ture Function and Management edited by Belnap J andLange O L Springer Berlin Germany 471ndash479 2003

Belnap J Weber B and Buumldel B Biological soil crusts as an or-ganizing principle in drylands in Biological Soil Crusts An Or-ganizing Principle in Drylands edited by Weber B Buumldel Band Belnap J Springer Berlin Germany 3ndash13 2016

Bradford M M A rapid and sensitive method for the quantifi-cation of microgram quantities of protein utilizing the prin-ciple of protein dye binding Anal Biochem 72 248ndash254httpsdoiorg1010160003-2697(76)90527-3 1976

Burch J Some mosses survive cryopreserva-tion without prior pretreatment Bryologist106 270ndash277 httpsdoiorg1016390007-2745(2003)106[0270SMSCWP]20CO2 2003

Burch J and Wilkinson T Cryopreservation of protonemataof Ditrichum cornubicum (Paton) comparing the effectivenessof four cryoprotectant pretreatments Cryoletters 23 197ndash2082002

Chinese Central Meteorological Station httpwwwnmccnpublishforecastASNansaihtml last access 2 August 2017

Cleavitt N L Stress tolerance of rare and common moss speciesin relation to their occupied environments and asexual dispersalpotential J Ecol 90 785ndash795 httpsdoiorg101046j1365-2745200200713x 2002

Csintalan Z Proctor M C F and Tuba Z Chlorophyll fluo-rescence during drying and rehydration in the mosses Rhytidi-adelphus loreus (Hedw) Warnst Anomodon viticulosus (Hedw)Hook amp Tayl and Grimmia pulvinata (Hedw) Sm Ann Bot-London 84 235ndash244 httpsdoiorg101006anbo199909191999

Deng J L Control problems of grey systems Syst Control Lett1 288ndash294 httpsdoiorg101016S0167-6911(82)80025-X1982

Biogeosciences 15 797ndash808 2018 wwwbiogeosciencesnet157972018

804 Y Guo and Y Zhao Effects of storage temperature on desiccation-tolerant mosses

Table 4 Gray incidence degree between physiological indices and the gametophyte vigor index across all treatments

Reference sequences B unguiculata D vinealis D tectorum

Chlorophyll content (X1) 060plusmn 020 055plusmn 027 066plusmn 021Soluble sugar content (X2) 057plusmn 020 062plusmn 023 062plusmn 017Soluble protein content (X3) 057plusmn 022 074plusmn 028 070plusmn 025MDA content (X4) 077plusmn 020 073plusmn 022 076plusmn 027

4 Discussion

41 Effects of storage temperature on the vegetativepropagation of mosses

For more than a century researchers have studied many as-pects of mosses such as inocula pretreatment (eg stor-age and sterilization) culture methods and culture condi-tions (Duckett et al 2004 Hoffman 1966) Some of thesestudies have implied that the physiological characteristics ofmoss gametophytes are closely related to the success of ar-tificial cultivation for example pretreatment with sucroseandor abscisic acid can improve the viability of mosses byincreasing DT (Burch and Wilkinson 2002) In line with pre-vious studies this study found that gametophyte regenera-tion within the same species after desiccation varied amongdifferent temperatures (Fig 1a and b Table 2) which islikely related to species-specific DT The regenerative capac-ity of mosses can be summarily described by the gameto-phyte vigor index on the basis of Eqs (1)ndash(3) and Table 3The gametophyte vigor index most sensitive to storage tem-perature was that of D tectorum whereas that of D vinealisvaried little with storage temperature with no significant dif-ferences among temperatures (Table 2) Thus the effect ofstorage temperature on regenerative capacity was strongestin D tectorum and weakest in D vinealis

The 40-day storage period adversely affected regenera-tion in most gametophytes (Fig 1a and b Table 1) how-ever some gametophytes of D tectorum stored at 17 and25 C produced more new shoots than before It is not clearwhether this enhanced regeneration was associated with thelow-temperature tolerance of D tectorum D tectorum possi-bly suffered low-temperature stress in early winter Further-more higher temperatures (eg 30 C) injured the gameto-phytes of D tectorum as did the lower temperatures of 0and 4 C These findings suggest that extreme temperaturesare unsuitable for storing this moss species Further stud-ies are warranted on the impact of the storage environmenton desiccation-tolerant mosses For example Burch (2003)found that the survival and regeneration of dehydrated pro-tonemata were reduced after cryopreservation due to dam-age caused by intracellular ice crystals The desiccationtime can also affect the restorability of vegetative propaga-tion in desiccation-tolerant mosses and their physiologicalcharacteristics (Keever 1957 Proctor 2001) Environmental

changes or variation in the dormancy period of cells mightinfluence the restoration results after rehydration

42 Effects of storage temperature on the physiologicalcharacteristics of mosses

MDA an important product of membrane lipid peroxidationincreased in all mosses over the storage period This find-ing indicated that the 40-day storage period caused cell dam-age (Fig 2d Table 1) Accordingly the soluble sugar con-tent increased to protect the membranes and proteins in thedried gametophytes (Fig 2b Table 1) Sugars are the mainsubstance used to stabilize protein structures in desiccation-tolerant cells (Hoekstra et al 2001) However the solublesugar content of B unguiculata stored at 0 and 4 C was de-creased relative to the initial value This result might havebeen due to the low temperatures preventing the conversionfrom starch to soluble sugar (Pressel et al 2006) Whenmosses suffered oxidative damage the increases in chloro-phyll content and soluble protein content in some gameto-phytes were related to the recovery ability of desiccation-tolerant cells (Fig 2a and c Table 1) In previous studiesthe chlorophyll content of mosses increased during desicca-tion and their photosynthetic capacity recovered rapidly af-ter rewetting (Alpert 1988 Csintalan et al 1999) Similarlyprotein synthesis recovered after rehydration (Oliver 1991)since cellular recovery is an important part of DT (Proctoret al 2007)

The recovery of photosynthesis and protein synthesis inB unguiculata was facilitated by higher temperatures (notmore than 30 C Fig 2a and c) This finding is inconsis-tent with the pattern in other mosses in which viabilitytends to be lower at increased temperatures (Hearnshaw andProctor 1982) However the increasing trend of MDA con-tent from 17 to 30 C suggests that more extensive mem-brane damage may be caused by storage temperatures above30 C (Fig 2d) The adverse effects of the higher tempera-tures in D vinealis and D tectorum were clearly reflectedby the slower recovery of photosynthesis and protein syn-thesis (Fig 2a and c) The changes in the MDA content inD vinealis suggested more rapid repair of cell membranewith increasing temperature however the species possiblyhad stronger tolerance under the protection of abundant sug-ars when the recovery of photosynthesis and protein synthe-sis was slower (Fig 2andashd)

Biogeosciences 15 797ndash808 2018 wwwbiogeosciencesnet157972018

Y Guo and Y Zhao Effects of storage temperature on desiccation-tolerant mosses 805

The responses of the physiological characteristics of thethree species to temperature reflected species variation inrestoration ability over a short rehydration time Because therewetting periods were longer than 30 days in the cultiva-tion the vegetative propagation results can be considered asreflecting the long-term recovery of mosses Thus the long-term effect of cell recovery during short-term rehydration canbe explained by the relationships between the physiologi-cal characteristics and vegetative propagation of desiccation-tolerant mosses

43 Relationships between physiological characteristicsand the vegetative propagation of mosses

Before storage the four physiological indices of gameto-phytes showed significant differences between D vinealisand D tectorum However no significant differences be-tween the two species were observed in regard to the threegermination parameters (Table 1) Mosses of similar fertilityshowed significant differences in physiological characteris-tics Species differences in DT led to larger differences invegetative propagation among species than before as evi-denced by the values of the gametophyte vigor indices withinthe same treatment (Tables 1 and 2) Therefore the recoveryability of dried mosses with respect to development and re-generation might be more informative for screening suitableinocula than using fresh mosses in dry habitats Many stud-ies have indicated that desiccation-tolerant mosses can re-cover from drying once they are rehydrated (Csintalan et al1999 Pressel et al 2006) However long periods of desic-cation would impede the reuse of moss specimens and therestoration of dried biocrusts This study showed that cellswere subjected to oxidative damage after the 40-day desicca-tion period (Fig 2d Table 1) Over this period the regener-ative capacity of the three species declined (Table 2) whichsuggested that membrane integrity andor other factors af-fected the vegetative propagation of the desiccation-tolerantmosses

Based on the correlation coefficients among the physio-logical indices and germination parameters of desiccation-tolerant mosses (Table 3) gametophyte germination was sig-nificantly and positively correlated with chlorophyll contentsoluble sugar content and soluble protein content In addi-tion gametophyte increment and gametophyte vigor indexwere significantly and negatively correlated with MDA con-tent These findings are in accordance with the observationsthat metabolic repair is favorable to the germination of newgametophytes and that long-term recovery is more dependenton cell integrity than metabolic repair Therefore to quan-titatively compare the effects of the four physiological in-dices on vegetative propagation the gray incidence degreebetween the physiological indices and the gametophyte vigorindex for each of the three moss species was calculated byusing Eqs (4)ndash(6) As shown in Table 4 the effect of MDAcontent on the gametophyte vigor index was the strongest

in B unguiculata and D tectorum and the incidence degreeof MDA (073) in D vinealis was similar to the maximum(074) In all three mosses MDA content increased as stor-age temperature decreased from 17 to 0 C Smaller gameto-phyte vigor index values were observed for D vinealis andD tectorum at 0 and 4 C than at 25 and 30 C (Fig 2d Ta-ble 2) This result indicated that the greater membrane dam-age incurred at low temperatures caused the decline in regen-erative capacity In addition the higher gametophyte vigorindex values of D tectorum at 17 and 25 C than before stor-age were possibly related to the reduced formation of intra-cellular ice crystals at these temperatures during the storageperiod (Burch 2003) which facilitated more rapid recoveryupon rehydration (Table 2) However the number of nega-tive effects on physiological characteristics increased withincreasing temperature (Fig 2andashc) The high temperatureswere unfavorable to the recovery of the mosses (Hearnshawand Proctor 1982) When cells suffered damage under desic-cation and temperature stress the protection provided by ad-ditional sugars was important for maintaining cell integrityin the dry state (Fig 2d Table 1) D vinealis showed no sig-nificant difference in regenerative capacity among tempera-tures potentially because the level of cellular protection wasequivalent among the different temperatures

Researchers have summarized the recovery mechanisms ofmosses upon rehydration such as the rapid recovery of pho-tosynthesis respiration and protein synthesis within min-utes to hours (Proctor et al 2007) However recovery ofthe carbon balance cell cycle and the cytoskeleton requiremore than 24 h (Alpert and Oechel 1985 Mansour and Hal-let 1981 Pressel et al 2006) Based on these results it hasbeen speculated that cell integrity is more difficult to recoverthan physiological reactions and that cell integrity greatlylimits the recovery and regenerative capacity of desiccation-tolerant mosses Over long-term desiccation the cumulativedamage affects cell function and integrity (Proctor 2001)different temperatures might enhance or suppress such celldamage Thus the effects of temperature on the ecology ofDT in bryophytes warrant investigation especially during thedry season in semiarid and arid areas The greater sensitiv-ity of D tectorum observed here might provide insight intowhy this species is not a widely distributed species such asD vinealis in the study region Furthermore the ecologi-cal niche requirements of different mosses in both dry andwet periods will influence the choice of moss inocula for ar-tificial cultivation and biocrust restoration Field studies areneeded to better understand the ecological requirements ofdried mosses Furthermore a precise description of micro-climates and the application of quantitative methods wouldbe helpful

wwwbiogeosciencesnet157972018 Biogeosciences 15 797ndash808 2018

806 Y Guo and Y Zhao Effects of storage temperature on desiccation-tolerant mosses

5 Conclusions

The conducted experiment explored the effect of storage tem-perature on the vegetative propagation of desiccation-tolerantmosses and influencing factors The results indicated that thedecline in regenerative capacity in mosses observed follow-ing storage was related to cell damage caused by dehydra-tion during storage The storage temperature during dehydra-tion influenced the vegetative propagation of mosses throughchanges in moss cell activity Further analysis showed thatthe factor with the strongest effect on vegetative propagationwas membrane damage During storage soluble sugars in-creased to protect the cells highlighting the important role ofcell integrity in influencing the physiological characteristicsand vegetative propagation of desiccation-tolerant mosses Inthis study the optimal storage temperature of D vinealis andD tectorum was 17 C whereas the optimal temperature forB unguiculata was 4 C Different responses to temperatureamong the three moss species were associated with speciesdifferences in DT These findings can potentially guide futureresearch on suitable storage methods for inoculation materialto improve the artificial cultivation of moss biocrusts

In general the properties of inoculation material are keyfactors affecting the development and recovery of mossbiocrusts such as species physiological features andorother factors The results provide insight into the factors thatinfluence the vegetative propagation of desiccation-tolerantmosses and highlight the potential applicability of a rapidexperimental approach for screening suitable inocula

Data availability Currently data can only be accessed in the formof Excel sheets via contact with the corresponding author

Competing interests The authors declare that they have no conflictof interest

Special issue statement This article is part of the special issue ldquoBi-ological soil crusts and their role in biogeochemical processes andcyclingrdquo It is a result of the BIOCRUST3 conference Moab USA26 to 30 September 2016

Acknowledgements The research was supported by the NationalNatural Science Foundation of China (grant nos 4157126841271298) We also express our gratitude to the anonymousreviewers and editors for their constructive comments and sugges-tions

Edited by Bettina WeberReviewed by three anonymous referees

References

Abdul-baki A A and Anderson J D Relation-ship between decarboxylation of glutamic-acid andvigor in soybean seed Crop Sci 13 227ndash232httpsdoiorg102135cropsci19730011183X001300020023x1973

Alpert P Survival of a desiccation-tolerant moss Grimmia laevi-gata beyond its observed microdistributional limits J Bryol15 219ndash227 httpsdoiorg101179jbr1988151219 1988

Alpert P and Oechel W C Carbon balance limits microdistribu-tion of Grimmia laevigata a desiccation-tolerant plant Ecology66 660ndash669 httpsdoiorg1023071940527 1985

Antoninka A Bowker M A Reed S C and Doherty K Pro-duction of greenhouse-grown biocrust mosses and associatedcyanobacteria to rehabilitate dryland soil function Restor Ecol24 324ndash335 httpsdoiorg101111rec12311 2016

Belnap J and Eldridge D Disturbance and recovery of biologicalsoil crusts in Biological Soil Crusts Structure Function andManagement edited by Belnap J and Lange O L SpringerBerlin Germany 363ndash383 2003

Belnap J and Lange O L Structure and functioning of biolog-ical soil crusts a synthesis in Biological Soil Crusts Struc-ture Function and Management edited by Belnap J andLange O L Springer Berlin Germany 471ndash479 2003

Belnap J Weber B and Buumldel B Biological soil crusts as an or-ganizing principle in drylands in Biological Soil Crusts An Or-ganizing Principle in Drylands edited by Weber B Buumldel Band Belnap J Springer Berlin Germany 3ndash13 2016

Bradford M M A rapid and sensitive method for the quantifi-cation of microgram quantities of protein utilizing the prin-ciple of protein dye binding Anal Biochem 72 248ndash254httpsdoiorg1010160003-2697(76)90527-3 1976

Burch J Some mosses survive cryopreserva-tion without prior pretreatment Bryologist106 270ndash277 httpsdoiorg1016390007-2745(2003)106[0270SMSCWP]20CO2 2003

Burch J and Wilkinson T Cryopreservation of protonemataof Ditrichum cornubicum (Paton) comparing the effectivenessof four cryoprotectant pretreatments Cryoletters 23 197ndash2082002

Chinese Central Meteorological Station httpwwwnmccnpublishforecastASNansaihtml last access 2 August 2017

Cleavitt N L Stress tolerance of rare and common moss speciesin relation to their occupied environments and asexual dispersalpotential J Ecol 90 785ndash795 httpsdoiorg101046j1365-2745200200713x 2002

Csintalan Z Proctor M C F and Tuba Z Chlorophyll fluo-rescence during drying and rehydration in the mosses Rhytidi-adelphus loreus (Hedw) Warnst Anomodon viticulosus (Hedw)Hook amp Tayl and Grimmia pulvinata (Hedw) Sm Ann Bot-London 84 235ndash244 httpsdoiorg101006anbo199909191999

Deng J L Control problems of grey systems Syst Control Lett1 288ndash294 httpsdoiorg101016S0167-6911(82)80025-X1982

Biogeosciences 15 797ndash808 2018 wwwbiogeosciencesnet157972018

Y Guo and Y Zhao Effects of storage temperature on desiccation-tolerant mosses 805

The responses of the physiological characteristics of thethree species to temperature reflected species variation inrestoration ability over a short rehydration time Because therewetting periods were longer than 30 days in the cultiva-tion the vegetative propagation results can be considered asreflecting the long-term recovery of mosses Thus the long-term effect of cell recovery during short-term rehydration canbe explained by the relationships between the physiologi-cal characteristics and vegetative propagation of desiccation-tolerant mosses

43 Relationships between physiological characteristicsand the vegetative propagation of mosses

Before storage the four physiological indices of gameto-phytes showed significant differences between D vinealisand D tectorum However no significant differences be-tween the two species were observed in regard to the threegermination parameters (Table 1) Mosses of similar fertilityshowed significant differences in physiological characteris-tics Species differences in DT led to larger differences invegetative propagation among species than before as evi-denced by the values of the gametophyte vigor indices withinthe same treatment (Tables 1 and 2) Therefore the recoveryability of dried mosses with respect to development and re-generation might be more informative for screening suitableinocula than using fresh mosses in dry habitats Many stud-ies have indicated that desiccation-tolerant mosses can re-cover from drying once they are rehydrated (Csintalan et al1999 Pressel et al 2006) However long periods of desic-cation would impede the reuse of moss specimens and therestoration of dried biocrusts This study showed that cellswere subjected to oxidative damage after the 40-day desicca-tion period (Fig 2d Table 1) Over this period the regener-ative capacity of the three species declined (Table 2) whichsuggested that membrane integrity andor other factors af-fected the vegetative propagation of the desiccation-tolerantmosses

Based on the correlation coefficients among the physio-logical indices and germination parameters of desiccation-tolerant mosses (Table 3) gametophyte germination was sig-nificantly and positively correlated with chlorophyll contentsoluble sugar content and soluble protein content In addi-tion gametophyte increment and gametophyte vigor indexwere significantly and negatively correlated with MDA con-tent These findings are in accordance with the observationsthat metabolic repair is favorable to the germination of newgametophytes and that long-term recovery is more dependenton cell integrity than metabolic repair Therefore to quan-titatively compare the effects of the four physiological in-dices on vegetative propagation the gray incidence degreebetween the physiological indices and the gametophyte vigorindex for each of the three moss species was calculated byusing Eqs (4)ndash(6) As shown in Table 4 the effect of MDAcontent on the gametophyte vigor index was the strongest

in B unguiculata and D tectorum and the incidence degreeof MDA (073) in D vinealis was similar to the maximum(074) In all three mosses MDA content increased as stor-age temperature decreased from 17 to 0 C Smaller gameto-phyte vigor index values were observed for D vinealis andD tectorum at 0 and 4 C than at 25 and 30 C (Fig 2d Ta-ble 2) This result indicated that the greater membrane dam-age incurred at low temperatures caused the decline in regen-erative capacity In addition the higher gametophyte vigorindex values of D tectorum at 17 and 25 C than before stor-age were possibly related to the reduced formation of intra-cellular ice crystals at these temperatures during the storageperiod (Burch 2003) which facilitated more rapid recoveryupon rehydration (Table 2) However the number of nega-tive effects on physiological characteristics increased withincreasing temperature (Fig 2andashc) The high temperatureswere unfavorable to the recovery of the mosses (Hearnshawand Proctor 1982) When cells suffered damage under desic-cation and temperature stress the protection provided by ad-ditional sugars was important for maintaining cell integrityin the dry state (Fig 2d Table 1) D vinealis showed no sig-nificant difference in regenerative capacity among tempera-tures potentially because the level of cellular protection wasequivalent among the different temperatures

Researchers have summarized the recovery mechanisms ofmosses upon rehydration such as the rapid recovery of pho-tosynthesis respiration and protein synthesis within min-utes to hours (Proctor et al 2007) However recovery ofthe carbon balance cell cycle and the cytoskeleton requiremore than 24 h (Alpert and Oechel 1985 Mansour and Hal-let 1981 Pressel et al 2006) Based on these results it hasbeen speculated that cell integrity is more difficult to recoverthan physiological reactions and that cell integrity greatlylimits the recovery and regenerative capacity of desiccation-tolerant mosses Over long-term desiccation the cumulativedamage affects cell function and integrity (Proctor 2001)different temperatures might enhance or suppress such celldamage Thus the effects of temperature on the ecology ofDT in bryophytes warrant investigation especially during thedry season in semiarid and arid areas The greater sensitiv-ity of D tectorum observed here might provide insight intowhy this species is not a widely distributed species such asD vinealis in the study region Furthermore the ecologi-cal niche requirements of different mosses in both dry andwet periods will influence the choice of moss inocula for ar-tificial cultivation and biocrust restoration Field studies areneeded to better understand the ecological requirements ofdried mosses Furthermore a precise description of micro-climates and the application of quantitative methods wouldbe helpful

wwwbiogeosciencesnet157972018 Biogeosciences 15 797ndash808 2018

806 Y Guo and Y Zhao Effects of storage temperature on desiccation-tolerant mosses

5 Conclusions

The conducted experiment explored the effect of storage tem-perature on the vegetative propagation of desiccation-tolerantmosses and influencing factors The results indicated that thedecline in regenerative capacity in mosses observed follow-ing storage was related to cell damage caused by dehydra-tion during storage The storage temperature during dehydra-tion influenced the vegetative propagation of mosses throughchanges in moss cell activity Further analysis showed thatthe factor with the strongest effect on vegetative propagationwas membrane damage During storage soluble sugars in-creased to protect the cells highlighting the important role ofcell integrity in influencing the physiological characteristicsand vegetative propagation of desiccation-tolerant mosses Inthis study the optimal storage temperature of D vinealis andD tectorum was 17 C whereas the optimal temperature forB unguiculata was 4 C Different responses to temperatureamong the three moss species were associated with speciesdifferences in DT These findings can potentially guide futureresearch on suitable storage methods for inoculation materialto improve the artificial cultivation of moss biocrusts

In general the properties of inoculation material are keyfactors affecting the development and recovery of mossbiocrusts such as species physiological features andorother factors The results provide insight into the factors thatinfluence the vegetative propagation of desiccation-tolerantmosses and highlight the potential applicability of a rapidexperimental approach for screening suitable inocula

Data availability Currently data can only be accessed in the formof Excel sheets via contact with the corresponding author

Competing interests The authors declare that they have no conflictof interest

Special issue statement This article is part of the special issue ldquoBi-ological soil crusts and their role in biogeochemical processes andcyclingrdquo It is a result of the BIOCRUST3 conference Moab USA26 to 30 September 2016

Acknowledgements The research was supported by the NationalNatural Science Foundation of China (grant nos 4157126841271298) We also express our gratitude to the anonymousreviewers and editors for their constructive comments and sugges-tions

Edited by Bettina WeberReviewed by three anonymous referees

References

Abdul-baki A A and Anderson J D Relation-ship between decarboxylation of glutamic-acid andvigor in soybean seed Crop Sci 13 227ndash232httpsdoiorg102135cropsci19730011183X001300020023x1973

Alpert P Survival of a desiccation-tolerant moss Grimmia laevi-gata beyond its observed microdistributional limits J Bryol15 219ndash227 httpsdoiorg101179jbr1988151219 1988

Alpert P and Oechel W C Carbon balance limits microdistribu-tion of Grimmia laevigata a desiccation-tolerant plant Ecology66 660ndash669 httpsdoiorg1023071940527 1985

Antoninka A Bowker M A Reed S C and Doherty K Pro-duction of greenhouse-grown biocrust mosses and associatedcyanobacteria to rehabilitate dryland soil function Restor Ecol24 324ndash335 httpsdoiorg101111rec12311 2016

Belnap J and Eldridge D Disturbance and recovery of biologicalsoil crusts in Biological Soil Crusts Structure Function andManagement edited by Belnap J and Lange O L SpringerBerlin Germany 363ndash383 2003

Belnap J and Lange O L Structure and functioning of biolog-ical soil crusts a synthesis in Biological Soil Crusts Struc-ture Function and Management edited by Belnap J andLange O L Springer Berlin Germany 471ndash479 2003

Belnap J Weber B and Buumldel B Biological soil crusts as an or-ganizing principle in drylands in Biological Soil Crusts An Or-ganizing Principle in Drylands edited by Weber B Buumldel Band Belnap J Springer Berlin Germany 3ndash13 2016

Bradford M M A rapid and sensitive method for the quantifi-cation of microgram quantities of protein utilizing the prin-ciple of protein dye binding Anal Biochem 72 248ndash254httpsdoiorg1010160003-2697(76)90527-3 1976

Burch J Some mosses survive cryopreserva-tion without prior pretreatment Bryologist106 270ndash277 httpsdoiorg1016390007-2745(2003)106[0270SMSCWP]20CO2 2003

Burch J and Wilkinson T Cryopreservation of protonemataof Ditrichum cornubicum (Paton) comparing the effectivenessof four cryoprotectant pretreatments Cryoletters 23 197ndash2082002

Chinese Central Meteorological Station httpwwwnmccnpublishforecastASNansaihtml last access 2 August 2017

Cleavitt N L Stress tolerance of rare and common moss speciesin relation to their occupied environments and asexual dispersalpotential J Ecol 90 785ndash795 httpsdoiorg101046j1365-2745200200713x 2002

Csintalan Z Proctor M C F and Tuba Z Chlorophyll fluo-rescence during drying and rehydration in the mosses Rhytidi-adelphus loreus (Hedw) Warnst Anomodon viticulosus (Hedw)Hook amp Tayl and Grimmia pulvinata (Hedw) Sm Ann Bot-London 84 235ndash244 httpsdoiorg101006anbo199909191999

Deng J L Control problems of grey systems Syst Control Lett1 288ndash294 httpsdoiorg101016S0167-6911(82)80025-X1982

Biogeosciences 15 797ndash808 2018 wwwbiogeosciencesnet157972018

806 Y Guo and Y Zhao Effects of storage temperature on desiccation-tolerant mosses

5 Conclusions

The conducted experiment explored the effect of storage tem-perature on the vegetative propagation of desiccation-tolerantmosses and influencing factors The results indicated that thedecline in regenerative capacity in mosses observed follow-ing storage was related to cell damage caused by dehydra-tion during storage The storage temperature during dehydra-tion influenced the vegetative propagation of mosses throughchanges in moss cell activity Further analysis showed thatthe factor with the strongest effect on vegetative propagationwas membrane damage During storage soluble sugars in-creased to protect the cells highlighting the important role ofcell integrity in influencing the physiological characteristicsand vegetative propagation of desiccation-tolerant mosses Inthis study the optimal storage temperature of D vinealis andD tectorum was 17 C whereas the optimal temperature forB unguiculata was 4 C Different responses to temperatureamong the three moss species were associated with speciesdifferences in DT These findings can potentially guide futureresearch on suitable storage methods for inoculation materialto improve the artificial cultivation of moss biocrusts

In general the properties of inoculation material are keyfactors affecting the development and recovery of mossbiocrusts such as species physiological features andorother factors The results provide insight into the factors thatinfluence the vegetative propagation of desiccation-tolerantmosses and highlight the potential applicability of a rapidexperimental approach for screening suitable inocula

Data availability Currently data can only be accessed in the formof Excel sheets via contact with the corresponding author

Competing interests The authors declare that they have no conflictof interest

Special issue statement This article is part of the special issue ldquoBi-ological soil crusts and their role in biogeochemical processes andcyclingrdquo It is a result of the BIOCRUST3 conference Moab USA26 to 30 September 2016

Acknowledgements The research was supported by the NationalNatural Science Foundation of China (grant nos 4157126841271298) We also express our gratitude to the anonymousreviewers and editors for their constructive comments and sugges-tions

Edited by Bettina WeberReviewed by three anonymous referees

References

Abdul-baki A A and Anderson J D Relation-ship between decarboxylation of glutamic-acid andvigor in soybean seed Crop Sci 13 227ndash232httpsdoiorg102135cropsci19730011183X001300020023x1973

Alpert P Survival of a desiccation-tolerant moss Grimmia laevi-gata beyond its observed microdistributional limits J Bryol15 219ndash227 httpsdoiorg101179jbr1988151219 1988

Alpert P and Oechel W C Carbon balance limits microdistribu-tion of Grimmia laevigata a desiccation-tolerant plant Ecology66 660ndash669 httpsdoiorg1023071940527 1985

Antoninka A Bowker M A Reed S C and Doherty K Pro-duction of greenhouse-grown biocrust mosses and associatedcyanobacteria to rehabilitate dryland soil function Restor Ecol24 324ndash335 httpsdoiorg101111rec12311 2016

Belnap J and Eldridge D Disturbance and recovery of biologicalsoil crusts in Biological Soil Crusts Structure Function andManagement edited by Belnap J and Lange O L SpringerBerlin Germany 363ndash383 2003

Belnap J and Lange O L Structure and functioning of biolog-ical soil crusts a synthesis in Biological Soil Crusts Struc-ture Function and Management edited by Belnap J andLange O L Springer Berlin Germany 471ndash479 2003

Belnap J Weber B and Buumldel B Biological soil crusts as an or-ganizing principle in drylands in Biological Soil Crusts An Or-ganizing Principle in Drylands edited by Weber B Buumldel Band Belnap J Springer Berlin Germany 3ndash13 2016

Bradford M M A rapid and sensitive method for the quantifi-cation of microgram quantities of protein utilizing the prin-ciple of protein dye binding Anal Biochem 72 248ndash254httpsdoiorg1010160003-2697(76)90527-3 1976

Burch J Some mosses survive cryopreserva-tion without prior pretreatment Bryologist106 270ndash277 httpsdoiorg1016390007-2745(2003)106[0270SMSCWP]20CO2 2003

Burch J and Wilkinson T Cryopreservation of protonemataof Ditrichum cornubicum (Paton) comparing the effectivenessof four cryoprotectant pretreatments Cryoletters 23 197ndash2082002

Chinese Central Meteorological Station httpwwwnmccnpublishforecastASNansaihtml last access 2 August 2017

Cleavitt N L Stress tolerance of rare and common moss speciesin relation to their occupied environments and asexual dispersalpotential J Ecol 90 785ndash795 httpsdoiorg101046j1365-2745200200713x 2002

Csintalan Z Proctor M C F and Tuba Z Chlorophyll fluo-rescence during drying and rehydration in the mosses Rhytidi-adelphus loreus (Hedw) Warnst Anomodon viticulosus (Hedw)Hook amp Tayl and Grimmia pulvinata (Hedw) Sm Ann Bot-London 84 235ndash244 httpsdoiorg101006anbo199909191999

Deng J L Control problems of grey systems Syst Control Lett1 288ndash294 httpsdoiorg101016S0167-6911(82)80025-X1982

Biogeosciences 15 797ndash808 2018 wwwbiogeosciencesnet157972018

Y Guo and Y Zhao Effects of storage temperature on desiccation-tolerant mosses 807

Duckett J G Burch J Fletcher P W Matcham H WRead D J Russell A J and Pressel S In vitrocultivation of bryophytes a review of practicalitiesproblems progress and promise J Bryol 26 3ndash20httpsdoiorg101179037366803235001742 2004

Gao L Q Bowker M A Xu M X Sun H Tuo D Fand Zhao Y G Biological soil crusts decrease erodi-bility by modifying inherent soil properties on theLoess Plateau China Soil Biol Biochem 105 49ndash58httpsdoiorg101016jsoilbio201611009 2017

Hearnshaw G F and Proctor M C F The effect of temperatureon the survival of dry bryophytes New Phytol 90 221ndash228httpsdoiorg101111j1469-81371982tb03254x 1982

Hodges D M DeLong J M Forney C F and Prange R KImproving the thiobarbituric acid-reactive-substances assay forestimating lipid peroxidation in plant tissues containing antho-cyanin and other interfering compounds Planta 207 604ndash611httpsdoiorg101007s004250050524 1999

Hoekstra F A Golovina E A and Buitink J Mechanismsof plant desiccation tolerance Trends Plant Sci 6 431ndash438httpsdoiorg101016S1360-1385(01)02052-0 2001

Hoffman G R Ecological studies of Funaria hygrometrica Hedwin Eastern Washington and Northern Idaho Ecol Monogr 36157ndash180 httpsdoiorg1023071942153 1966

Jones P R and Rosentreter R Gametophyte fragment growthof three common desert mosses on artificial and natural sub-strates Bryologist 109 166ndash172 httpsdoiorg1016390007-2745(2006)109[166GFGOTC]20CO2 2006

Keever C Establishment of Grimmia laevigata on bare graniteEcology 38 422ndash429 httpsdoiorg1023071929885 1957

Lan S B Wu L Zhang D L and Hu C X Successionalstages of biological soil crusts and their microstructure variabil-ity in Shapotou region (China) Environ Earth Sci 65 77ndash88httpsdoiorg101007s12665-011-1066-0 2012

Lin W Z Xiao X and Chou K C GPCR-GIA a web-serverfor identifying G-protein coupled receptors and their familieswith grey incidence analysis Protein Eng Des Sel 22 699ndash705 httpsdoiorg101093proteingzp057 2009

Mansour K S and Hallet J N Effect of desiccation on DNAsynthesis and the cell cycle of the moss Polytrichum formo-sum New Phytol 87 315ndash324 httpsdoiorg101111j1469-81371981tb03202x 1981

Mishler B D Reproductive ecology of bryophytes in PlantReproductive Ecology Patterns and Strategies edited byDoust J L and Doust L L Oxford University Press OxfordEngland 285ndash306 1988

Morris D L Quantitative determination of carbohydrateswith dreywoodrsquos anthrone reagent Science 107 254ndash255httpsdoiorg101126science1072775254 1948

Oliver M J Influence of protoplasmic water-loss on thecontrol of protein-synthesis in the desiccation-tolerant mossTortula ruralis ramifications for a repair-based mechanismof desiccation tolerance Plant Physiol 97 1501ndash1511httpsdoiorg101104pp9741501 1991

Oliver M J Tuba Z and Mishler B D The evolution of vege-tative desiccation tolerance in land plants Plant Ecol 151 85ndash100 httpsdoiorg101023A1026550808557 2000

Platt K A Oliver M J and Thomson W W Mem-branes and organelles of dehydrated Selaginella and Tor-tula retain their normal configuration and structural in-tegrity freeze fracture evidence Protoplasma 178 57ndash65httpsdoiorg101007BF01404121 1994

Pressel S Ligrone R and Duckett J G Effects of de- andrehydration on food-conducting cells in the moss Polytrichumformosum a cytological study Ann Bot-London 98 67ndash76httpsdoiorg101093aobmcl092 2006

Proctor M C F Patterns of desiccation tolerance and re-covery in bryophytes Plant Growth Regul 35 147ndash156httpsdoiorg101023A1014429720821 2001

Proctor M C F Oliver M J Wood A J Alpert PStark L R Cleavitt N L and Mishler B DDesiccation-tolerance in bryophytes a review Bry-ologist 110 595ndash621 httpsdoiorg1016390007-2745(2007)110[595DIBAR]20CO2 2007

Sabovljevic M Bijelovic A and Dragicevic I In vitro cultureof mosses Aloina aloides (K F Schultz) Kindb Brachytheciumvelutinum (Hedw) B S amp G Ceratodon purpureus (Hedw)Brid Eurhynchium praelongum (Hedw) B S amp G and Grim-mia pulvinata (Hedw) Sm Turk J Bot 27 441ndash446 2003

Schonfeld M A Johnson R C Carver B F andMornhinweg D W Water relations in winter-wheat asdrought resistance indicators Crop Sci 28 526ndash531httpsdoiorg102135cropsci19880011183X002800030021x1988

Seppelt R D Downing A J Deane-Coe K K Zhang Y Mand Zhang J Bryophytes within biological soil crusts in Bio-logical Soil Crusts An Organizing Principle in Drylands editedby Weber B Buumldel B and Belnap J Springer Berlin Ger-many 101ndash120 2016

Stark L R Greenwood J L and Brinda J CDesiccated Syntrichia ruralis shoots regenerate af-ter 20 years in the herbarium J Bryol 39 85ndash93httpsdoiorg1010800373668720161176307 2017

Tian G Q Bai X L Xu J and Wang X D Experimental stud-ies on natural regeneration and artificial cultures of moss crustson fixed dunes in the Tengger Desert Chinese Journal of PlantEcology 29 164ndash169 httpsdoiorg1017521cjpe200500212005 (in Chinese)

Wellburn A R and Lichtenthaler H Formulae and program to de-termine total carotenoids and chlorophylls a and b of leaf extractsin different solvents in Advances in Photosynthesis Researchedited by Sybesma C Springer Dordrecht the Netherlands9ndash12 1984

Xiao B Zhao Y G Wang Q H and Li C Devel-opment of artificial moss-dominated biological soil crustsand their effects on runoff and soil water content ina semi-arid environment J Arid Environ 117 75ndash83httpsdoiorg101016jjaridenv201502017 2015

Zhang G H Liu G B Wang G L and Wang Y X Ef-fects of vegetation cover and rainfall intensity on sediment-bound nutrient loss size composition and volume fractaldimension of sediment particles Pedosphere 21 676ndash684httpsdoiorg101016S1002-0160(11)60170-7 2011

Zhao Y G Qin N Q Weber B and Xu M X Response of bio-logical soil crusts to raindrop erosivity and underlying influences

wwwbiogeosciencesnet157972018 Biogeosciences 15 797ndash808 2018

808 Y Guo and Y Zhao Effects of storage temperature on desiccation-tolerant mosses

in the hilly Loess Plateau region China Biodivers Conserv 231669ndash1686 httpsdoiorg101007s10531-014-0680-z 2014

Zhao Y G Bowker M A Zhang Y M and Zaady E Enhancedrecovery of biological soil crusts after disturbance in Biologi-cal Soil Crusts An Organizing Principle in Drylands edited byWeber B Buumldel B and Belnap J Springer Berlin Germany499ndash523 2016

Biogeosciences 15 797ndash808 2018 wwwbiogeosciencesnet157972018