HORTSCIENCE 46(9):1333–1337. 2011.

Models to Describe Cycas micronesicaLeaf and Strobili DevelopmentThomas E. Marler1 and Nirmala DongolWestern Pacific Tropical Research Center, College of Natural and AppliedSciences, University of Guam, UOG Station, Mangilao, Guam 96923

Additional index words. Cycadaceae, Cycas micronesica, ontogeny, organ development,phenology

Abstract. Cycas micronesica leaf and strobili expansion patterns were measured in threelocations and seasons on Guam and then were fitted with non-linear models to evaluatethe use of the parameters for informing management decisions. All growth curves exceptfor microstrobili height conformed to a negative exponential function. Microstrobiliheight development could not be fitted with any traditional linear or non-linear function,so spline models were used to smooth the effect of elapsed days. Leaf and leaflet expansionpatterns were influenced by habitat and season, indicating development of the vegetativeorgan is plastic. In contrast, the models that described development of megasporophylllength, microstrobili height, and microstrobili diameter were not influenced by habitat orseason. Moreover, seed diameter developmental patterns were only minimally influencedby location. These results indicate developmental patterns of the reproductive structureswere primarily constitutive. We have demonstrated two empirical approaches to fittingmodels of Cycas micronesica organ growth and development and that both methods areuseful for determining the influence of spatial or temporal factors in the timing of organdevelopment. This approach may be used to inform horticultural or conservation questionsof other rare cycad species.

Understanding the developmental patternsand factors that control expansion of vegeta-tive and reproductive organs is of fundamentalimportance for refining conservation protocolsfor rare plants. For example, the period of vul-nerability to arthropod herbivory is ephemeralfor many plant organs, and prophylactic pro-tection activities can be restricted to thevulnerable phase. This requires a workable un-derstanding of the temporal dynamics of thatphase. In addition, understanding the patternand timing of pollen display and dispersalwithin male reproductive organs versus thatof ovule display and receptivity on femalereproductive organs is needed for managementof dioecious species to ensure successful pro-pagation operations. The vast majority of re-search in the literature that describes growthand development of Spermatophyte organshas been directed toward fleshy fruits (e.g.,Coombe, 1976), because these organs are ofspecial interest to horticulturists (Leopold andKriedemann, 1975).

The three families that comprise contem-porary cycad species are of interest for variousreasons, including striking morphological fea-tures and historical importance in vascularplant diversification (Norstog and Nicholls,1997). Because more than 60% of describedcycad species are threatened and some crit-ically endangered (IUCN, 2010), concern for

active conservation efforts in horticultural set-tings has increased. Despite this interest, em-pirical studies on issues that directly informmanagement decisions have been few (butsee Calonje et al. 2010). This deficiency is notrestricted to Cycadales, because most activemanagement programs for threatened and en-dangered plant species are based on generalfield observations and not on experimentalstudies (Given, 1994).

Field studies that tabulated frequency ofreproductive events have been reported fornumerous cycad species (Clark and Clark,1987, 1988; Keppel, 2001; Negron-Ortiz andGorchov, 2000; Ornduff, 1987, 1991, 1996;Tang, 1990; Terry et al., 2008; Vovides, 1990).Expansion of male cones during the few daysthat define the pollination stage has been de-scribed for Dioon edule (Tang, 1987), Ence-phalartos hildebrandtii (Tang, 1989), andZamia pumila (Tang, 1990). Some histolog-ical studies of organ development have beenreported for a range of species (Stevenson,1988. and citations therein; Vovides et al.,1993). Moreover, the influence of organ ageon physiological traits has been reported forone Cycas species (Marler, 2007; Marler et al.,2006). However, to our knowledge, no reportshave mathematically characterized organ ex-pansion patterns to establish a foundation forunderstanding levels of plasticity and improv-ing accuracy in management decisions.

Our foremost objective was to quantifyexpansion rates of various components ofleaves and strobili for the endangered Cycasmicronesica and then determine if the growthpatterns were amenable to being fitted withmathematical models. We then used the ap-propriate models to demonstrate if this ap-proach can be exploited to determine if these

developmental processes are constitutive orif temporal and spatial factors significantly in-fluence organ development traits.

Materials and Methods

Emerging C. micronesica leaves were se-lected in February and May 2004 from threeplants in each of three locations on the islandof Guam. Three leaves on each plant weretagged for a total of nine measurements perlocation on each date. The locations were sep-arated by 20 km and included an east coast lo-cation where abiotic stresses are most extreme,a west coast location where plants are pro-tected from the desiccating effects of chronictrade winds and aerosol oceanic salt spray,and a location in central Guam. The differencesin these combined stressors between the eastand west coast is enough to elicit a significantdifference in reproductive effort (Marler andNiklas, 2011). The lengths of each rachis andpetiole were measured every 3 to 4 d duringthe phase of rapid expansion and weeklythereafter. Additionally, leaflet length in themedian position of each rachis was measured.

Emerging microstrobili were selected onthree plants in each location in Feb., May, andNov. 2004. Microstrobili height was measureddirectly and strobili circumference was mea-sured with a flexible tape to ensure minor de-viations from radial symmetry did not confoundsequential measurements. Measurements weremade at 3- to 4-d intervals during the phases ofrapid expansion and weekly at other times.Strobilus diameter was calculated from thecircumference measurements.

The female Cycas strobilus is comprisedof a loose assemblage of individual mega-sporophylls. Plants with emerging megaspo-rophylls were selected within each of thelocations in Feb. 2004. The duration of growthof megastrobili is extensive, so we did not re-peat the selection of additional replications insubsequent seasons. Four plants in the eastcoast and central locations were selected, andfive plants in the west coast habitat were se-lected. Three sporophylls on each plant weretagged. Sporophyll length and seed diameterwere measured every 3 to 4 d during rapidexpansion phases and every 10 to 14 d there-after. Seeds of this species do not exhibit radialsymmetry but are isobilateral. Therefore, wepositioned the seed diameter measurementsto quantify the widest orientation at the mid-point of the seed.

The mean of the three tagged leaves, leaf-lets, microstrobili, megasporophylls, or seedsfrom each plant was used to represent each ofthese replications in the statistical analyses.Preliminary graphical analysis of each organexpansion data set revealed that the expectedrelationship between leaf, leaflet, or mega-sporophyll length expressed as a functionof elapsed days was the negative exponentialfunction Y = a� b*eð�kxÞ. Similarly, the re-lationship between male strobili and seed di-ameter as a function of elapsed days followedthe same model. After fitting several startingvalues in the SAS NLIN procedure (SAS In-stitute, Inc., Cary, NC), the final negative

Received for publication 6 Apr. 2011. Accepted forpublication 25 June 2011.Support provided by USDA CSREES #2003-05495.We thank George Fernandez, UNR Center for Re-search Design and Analysis, for statistical analyses.1To whom reprint requests should be addressed;e-mail tmarler@uguam.uog.edu.

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MISCELLANEOUS

exponential growth model was fitted for eachhabitat, season, and replication combination.The three negative exponential growth modelparameters, a, b, and k were calculated foreach replication and used to create a SAS dataset. Then a GLM model was fitted to deter-mine the significance of three habitats andtwo (leaf and megastrobili components) orthree (microstrobili) seasons on a, b, or k.

Microstrobili height growth pattern wasnot consistent with common linear or non-linear functions. Therefore, spline models weretried to model the non-linear growth patterns.We used the generalized mixed model withspline fit using the RSmooth option in theRandom statement in SAS GLIMMIX, whichuses a semiparametric approach to smoothcurves (Rupper et al., 2003). This analysistested the main sources of variation as season,habitat, and elapsed days with the effect ofelapsed days fitted as a spline effect.

Results

Leaves. Leaf length was comprised ex-clusively of rachis for the first 8 to 10 d afteremergence (Fig. 1A), after which the apicalportion of the petiole emerged and continuedleaf extension included both petiole and rachis(Fig. 1B). Petiole length reached a maximumby 18 to 20 d, and rachis length reached amaximum by 30 d (Fig. 2A). Total leaf lengthincreased slowly for the first 7 to 10 d afteremergence from the stem apex; then a lineargrowth phase with expansion rates of 8 to11 cm�d–1 was sustained until �30 d afteremergence.

Leaves emerged from the stem apex withcircinate leaflets (Fig. 1C), and leaflets re-mained so until�20 d after emergence. There-after, leaflets unfolded and exhibited rapidextension with increases of�1.5 mm�d–1 beingmaintained for 14 to 15 d (Fig. 2B).

After this 35-d expansion phase, the leavesranged in length from 101 to 185 cm andleaflets range in length from 169 to 275 mm.Percentage of total leaf length representedby petiole length ranged from 21% to 35%.Leaflet color continued to change as the leavesaged (Fig. 1D), until the new leaves portrayeda general appearance that was homogeneouswith subtending leaves by Days 80 to 90.

The main effect of season was significantfor a, b, and k non-linear model parametersthat described total leaf length and the maineffect of location was significant for k only(Table 1). Based on this outcome, six sepa-rate negative exponential growth modelswere fitted for three location and two seasoncombinations (Table 2). Season and locationdid not influence the non-linear model pa-rameter a for the function that described totalleaflet length (Table 1). However, the maineffects of season, location, and their interac-tion influenced b and k non-linear parametersfor leaflet expansion. Therefore, six separatenegative exponential growth models were fittedfor three location and two season combina-tions (Table 2).

Female sporophylls and seeds. Megaspo-rophylls emerged from the stem apex in a

linear growth phase with extension ratesof 8 mm�d–1 (Fig. 3A). Mature length wasachieved in �30 d when sporophylls werestill erect and ranged from 22.8 to 23.6 cm.Sporophyll length did not change thereafter,but orientation changed as sporophylls beganto radiate away from vertical. This change inorientation continued until sporophylls werepositively gravitropic. Diagnosis of the polli-nation stage by olfactory perception of thecharacteristic aromatic volatiles occurred at28.7 ± 0.5 d after megasporophyll emergencefrom the stem apex.

Ovules emerged on the sporophylls �11mm in diameter (Fig. 3B). Diameter did notchange while sporophyll extension was on-going, but a linear increase in seed diameterof 0.4 to 0.5 mm�d–1 was initiated thereafterin response to pollination events. The linearphase was retained until �130 d after sporo-phyll emergence. Thereafter, seed diametercontinued to increase at a progressively slowerpace until Day 300 when diameter ranged

from 59 to 69 mm. Finally, beginning Day550, the seed diameter decreased slightly asseed integument tissue lost hydration, anddiameter at Day 690 was 56 to 66 mm.

The GLM model determined that locationdid not influence sporophyll development.Therefore, sporophyll extension from allthree locations was described by one non-linear model, Length = 23:3731� 16:8980*eð�0:06793�daysÞ. The main effect of locationalso did not influence the non-linear modelparameter a, but it did influence model param-eters b (P # 0.0088) and k (P # 0.0300) forseed diameter developmental patterns. Basedon this outcome, three separate negative expo-nential growth models were fitted for the threelocations (Table 3). The differences were min-imal in comparison with differences in leafand leaflet development. The influence of dif-ferences in k (Table 3) was evident in a moregradual inflection of the growth curve for theeast coast location in comparison with the othertwo locations.

Fig. 1. Phenotype of Cycas micronesica organ expansion stages. (A) Newly emerging flush of leaves. (B)Erect leaves depict initial stages of expansion. (C) Cycas leaves express circinate leaflets initially. (D)Leaf color of young leaves is dissimilar to that of mature leaves until�3 months. (E) Newly emergingmegastrobili at Day 13. (F) Appearance of megastrobili at Day 35. (G) Bronze seed appearance atmaximum diameter, Day 420. (H) Dark brown seed appearance after shrinkage of seed integumenttissue, Day 590. (I) Newly emerging microstrobilus at Day 2. (J) Microstrobilus at Day 54 before finalstage of rapid height extension. (K) Microstrobilus at pollen dispersal and full height extension, Day 67.

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Male strobili. Microstrobili emerged witha rapid linear height growth phase that lasted20 to 25 d (Fig. 4A). Strobili continued toincrease in height at a continually slowingrate for another 30 d. At the end of this stage,the expansion rate was minimal. Then within7 to 9 d, these microstrobili doubled in height.This final rapid phase of extension was notevenly distributed vertically. Initially the cen-tral axis expanded at the base to loosen thebasal microsporophylls, and the loosening pro-gressed in an acropetal manner until all mi-crosporophylls were loosened. Ultimate matureheight was achieved in 60.4 ± 0.8 d and rangedfrom 35.3 to 56.5 cm. Microsporangia openedin a pattern that mirrored that of sporophyllloosening with basal microsporangia dispers-ing pollen initially at 62.4 ± 1.0 d and apicalmicrosporangia exhibiting the final pollen dis-persal at 67.8 ± 0.7 d.

Increase in microstrobili diameter did notfollow the two-phase pattern that character-ized increase in height. Instead, a smooth pat-tern occurred with a linear phase initiallyfollowed by a decrease in diameter increment(Fig. 4B). After pollen dispersal, these micro-strobili declined in diameter as microsporophylltissues began to become dehydrated. Maximumdiameter ranged from 10.3 to 14.5 cm and oc-curred at 54 to 71 d after emergence. Micro-strobili diameter decreased 5% to 18% duringthe 2 weeks after pollen shed.

The GLM model determined that locationdid not influence microstrobili diameter de-velopment. Therefore, microstrobili diameterfrom three locations and three seasons couldbe described by one non-linear model:

Diameter

= 116:8054 -115:1004*eð�0:088926�daysÞ:

Male strobili height exhibited the mostcomplex growth pattern observed in this study.Because of the non-parametric spline model fit,no model parameter estimates are available.However, the GLIMMIX procedure allowedthe test of significance for the main effect oflocation and season along with the spline ef-fect of elapsed days, so our objective of de-fining the influence of location and season onthe organ growth pattern was met. The TypeIII tests of location and season main effectsdid not influence the changes in male strobiliheight, but elapsed days was significant (P #0.0002).

Discussion

We have demonstrated the use of twostatistical approaches to describe cycad organdevelopment and have shown that both ap-proaches are amenable to quantifying the in-fluence of temporal and spatial factors on theprocesses. Whenever the data could be fittedwith a conventional non-linear model, we gen-erated model parameters for each replicationand then subjected each parameter to tradi-tional analysis of variance with season and lo-cation as sources of variation. When the growthcurves were atypical, we used elapsed days,season, and location as main sources of var-iation with elapsed days tested as a spline ef-fect. Both approaches enabled us to uncoverthe level of plasticity of Cycas micronesica

Fig. 2. (A) Length of Cycas micronesica petiole(h), rachis (n), and total leaf (s) as a functionof days after emergence. At least one of thethree non-linear model parameters was signif-icant for location and season effects. Therefore,data are exclusively from a west coast locationon Guam beginning Feb. 2004. (B) Length ofleaflets from the same leaves.

Table 1. Significance of two seasons and threelocations on the parameters a, b, and k thatdescribe the negative exponential function Y =a – b*e(–kx) for total Cycas micronesica leaf(cm) and leaflet (mm) extension with elapseddays as x-axis.

Source of variation a b kLeaf

Location 0.1435 0.1498 0.0452Season 0.0374 0.0387 0.0049Location · season 0.5441 0.5546 0.8571

LeafletLocation 0.1531 0.0015 <0.0001Season 0.1179 0.0065 0.0011Location · season 0.2977 0.0018 0.0012

Table 2. Model parameters for the negativeexponential function Y = a – b*e(–kx) for totalCycas micronesica leaf (cm) and leaflet (mm)extension fitted for six locations and seasons.

Location Season a b k

LeafEast coast Feb. 214.241 228.772 –0.03039East coast May 307.002 323.412 –0.01825Central Feb. 156.184 174.063 –0.04581Central May 208.248 227.242 –0.02934West

coastFeb. 211.909 231.350 –0.03414

Westcoast

May 388.458 409.925 –0.02254

LeafletEast coast Feb. 215.917 262.491 –0.06699East coast May 315.465 498.204 –0.06741Central Feb. 221.230 22004.3 –0.30072Central May 297.659 1215.61 –0.09064West

coastFeb. 303.005 1274.25 –0.07264

Westcoast

May 961.168 1345.42 –0.04221

Table 3. Model parameters for the negativeexponential function Y = a – b*e(–kx) for Cycasmicronesica seed diameter (mm) expansionfitted for three locations.

Location a b k

East coast 61.5874 82.0228 –0.01342Central 62.1592 85.1844 –0.01176West coast 61.7251 73.8347 –0.00975

Fig. 3. (A) Length of Cycas micronesica megasporophyll as a function of days after emergence. Noparameter from non-linear models was significant, so data are from all locations. (B) Seed diameter asa function of days after emergence. The effect of location was significant for two of the three non-linearmodel parameters, so data are exclusively from a central Guam habitat.

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leaf and strobili development among seasonsand locations.

The shape and timing of C. micronesicareproductive organ expansion were relativelyindependent of location and season. In con-trast, leaf and leaflet expansion were influencedby these spatial and temporal factors. Thus, ourresults conform to the general principles ofthe Berg hypothesis (Berg, 1959, 1960), whichargues that phenotypic variation in reproductivestructures is decoupled from that of vegeta-tive organs as a result of selection generatedby pollinators.

Horticulturists may exploit this approachin studying organ expansion dynamics forvarious applications. For example, this knowl-edge may inform experimental design forunderstanding the factors that regulate leafexpansion (Dale, 1988) and timing of the pro-cesses related to the transition from hetero-trophic tissue to autotrophic tissue (Turgeon,1989). Cycad organ ontogeny is a relativelyunexplored field, and studies are needed todetermine when cell division versus cell ex-pansion processes occur within seed integu-ment, embryo, and gametophyte tissues forfemale plants or during the highly contrastingfirst and second height expansion phases ofthe microstrobili development for male plants.

A thorough understanding of cycad leafdevelopment is also of critical importance forhorticulturists. For example, the Cycas-specificChilades pandava Horsfield butterfly ovipo-sitions on expanding tissue exclusively. Lar-vae access to a newly emerging leaf leads toconsumption of the entire structure, includingpetiole and rachis. In contrast, if ovipositionsare delayed until the rachis is partially ex-panded, then larval damage is restricted toa portion of the leaflets. Therefore, timing of

prophylactic treatments within just a few daysdictates whether a portion or all of the leaf areconsumed by this lepidopteran pest.

Knowledge of the timing of reproductiveorgan development is additionally crucial forsuccessful sexual propagation of rare cycadspecies that are represented by only a few in-dividuals. All cycads are dioecious, so a growerthat does not have access to cryostorage fa-cilities must enable synchrony of reproduc-tive phenology among individuals to realizesuccess (Marler, 2010). This can best be donewith a detailed understanding of the timing ofdevelopment and the factors that affect plas-ticity of organ development in relation tofemale ovule receptivity and male pollen dis-semination. The recent advent of efficientinternationalcommunicationsamongcycaden-thusiasts has enabled extensive coordination ofpollen provision among growers despite theirseparation by great geographic distances. Abetter understanding of strobili developmentand the factors that affect the timing of thatdevelopment will greatly increase the successrates for growers working collaboratively topropagate rare cycad species.

Our results reveal great disparity in thewindow of time between reproductive organemergence and pollination with male strobilirequiring 33 to 40 d more than female strobilito reach the pollination phase. The most ac-curate method for projecting the appropriatedate for pollination stage would depend onrecording the emergence date of strobili.Megastrobili receptivity can be expected atDay 28, and the initiation of a 5- to 6-d windowof pollen shed can be expected for micro-strobili at Day 62. If the emergence date ismissed, our results illuminate two other meth-ods for accurate estimation. Megasporophyll

extension rates of 8 mm�d–1 occurred for thefirst few days; therefore, length of developingsporophylls could be measured and then Day0 could be retrospectively estimated using thisstable extension rate. The female aromaticstage at Day 28 could then be projected withaccuracy because of the limited variabilityof ultimate megasporophyll length. In con-trast, microstrobili height was highly hetero-geneous, so use of the growth rate within theinitial linear phase to retrospectively estimateDay 0 would be less accurate. However, theappearance of the microstrobili just beforeinitiation of the final doubling of height (Fig.1J) is distinguishable with full size and matureappearance of the surface of each microspo-rophyll. At this stage, the sporophylls appearto adhere in a manner similar to the carpelsthat comprise the syncarp of pineapple [Ananascomosus (L.) Merr.]. Daily inspection of themicrostrobili base at this stage until looseningof sporophylls begins can be used to accu-rately estimate �7 d until initiation of pollendehiscence.

Season and habitat interactively influ-enced leaf development. The primary abioticfactors influenced by season on Guam are rain-fall and photoperiod. The major rainy seasonis July to December, and the major dry seasonis January to June. Thus, soil moisture reservesduring the February flush were less limitingthan during the May flush. Heterogeneity ofphotoperiod or soil moisture reserves may haveaccounted for the significant seasonal differ-ences. The locations vary in direct abiotic stress-ors with the east coast location receivingdesiccating trade winds and chronic aerosoldeposits of oceanic mist. These localized char-acteristics may have accounted for the signif-icant location differences.

We have shown the use of this approachfor comparing season and location diversityin relation to the timing of organ develop-ment of a single species. Temporal compar-isons with this approach could be extended toannual cycles, and spatial comparisons couldbe extended to multiple islands or countriesfor species with wide ranges. Taxonomic com-parisons could be extended to congenericsrather than disjunct locations of a single spe-cies. Finally, we propose that this approachcould be used to understand any changes inorgan development that result from distur-bances. This may include the patterns of short-term recovery from abiotic disturbances suchas tropical cyclones (Marler and Hirsh, 1998)or fire (Keppel, 2001; Negron-Ortiz andGorchov, 2000). Furthermore, it may includethe influence of biotic disturbances such asinvasions of exotic herbivores (Marler andMuniappan, 2006) or various forms of humandisturbance (Perez-Farrera et al., 2006).

As a group, cycads are important as a re-sult of unique traits such as their basalposition in seed plant phylogenetic relations(Norstog and Nicholls, 1997). However, re-search on organ development of cycads hasbeen ignored in relation to other horticulturalspecies that historically have commandedgreater economic value on the national or in-ternational market. The three families, 10

Fig. 4. (A) Height of Cycas micronesica microstrobili as a function of days after emergence. Location andseason did not influence the development of microstrobili height; therefore, data are from all locationsand seasons. Data are standardized for each replication as quotient of ultimate absolute height, whichranged from 35.3 to 56.5 cm. (B) Microstrobili diameter as a function of days. The three non-linearmodel parameters were not influenced by location or season; therefore, data are from all locations andseasons. Data are standardized for each replication as a quotient of ultimate absolute diameter, whichranged from 11.1 to 14.5 cm.

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genera, and 300+ species offer much in pro-viding answers to horticultural questions ofimportance to biologists, conservationists, andecologists. Although the concepts that under-lie our objectives are ecumenical among bi-ologists, we are the first to use this empiricalapproach to describe cycad organ develop-ment. We intend for this report to evoke newideas for improving cycad conservation efforts.Toward that end, we hope that our approachmay serve as an archetype for an informa-tive mechanism to study cycad growth anddevelopment.

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