Hydrobiologia 326/327 : 229-234, 1996 .

229S. C. Lindstrom & D. J. Chapman (eds), Fifteenth International Seaweed Symposium .©1996 Kluwer Academic Publishers. Printed in Belgium.

Organellar DNA restriction fragment length polymorphism (RFLP) andnuclear random amplified polymorphic DNA (RAPD) analyses ofmorphotypes of Gracilaria (Gracilariales, Rhodophyta) from Chile

Mariela Gonzalez l , Rolando Montoya2 , Arturo Candia 1 , Patricia Gomez 2 & Manuel Cisternas'' Departamento de Botanica, Facultad de Ciencias Naturales y Oceanograficas2Departamento de Biologia Molecular, Facultad de Ciencias Biologicas, Universidad de Concepcion, Casilla2407, Concepcion, Chile ; e-mail: mgonzale@halcon.dpi.udec.cl

Key words: Gracilaria, morphotypes, RAPD analysis, RFLP analysis, seaweed

Abstract

The extreme phenotypic variability recognized among the species of Gracilaria has highlighted the need for theapplication of refined methods to help solve taxa identifications . In Chile, there still exists uncertainty about theexact number of Gracilaria species. Our investigations are centered on DNA analyses of morphotypes collectedfrom different geographical locations, namely Lenga and Isla Santa Maria, Region VIII (36°00' S to 38°00' S),and Maullin, Region X (39°30' S to 43°40' S) . These two regions of Chile are considered as areas of confluenceof G. chilensis, G. verrucosa, and a species of Gracilariopsis . In this study four morphotypes, from a naturalbed located in Maullfn, were analyzed for RFLP of plastid DNA and the results compared with data of fourmorphotypes from a bed in Lenga . The DNA banding patterns from each enzyme digest were identical irrespectiveof morphotypes and/or locations. In an attempt to unravel the nature of the morphological differences found amongLenga and Maullin morphotypes, RAPD analyses of nuclear DNA were also performed ; however, no polymorphismhas been found yet. Therefore, the data of this study, as well as concurrent data from preliminary interfertility tests,suggest that all morphotypes belong to a single taxon, Gracilaria chilensis .

Introduction

During the past decade interest in solving the system-atics of gracilarioid marine algae has revived, mostlybecause of the economic importance as agar producers(Fredericq & Hommersand, 1989a, 1989b ; Santelices& Doty, 1989) .

In Chile, most of the research undertaken onGracilaria has dealt with its production ecology incommercial beds, including aspects of populationdynamics, persistence and natural propagation of thethalli, abiotic and biotic factors that affect growth andproductivity, and models of exploitation (Santelices &Fonck, 1979; Pizarro, 1986 ; Santelices, 1989) . Basicaspects of the biology of Gracilaria and specifically itstaxonomy still need to be clarified (Santelices, 1989) .Kim (1970), in his revision of the Chilean speciesof Gracilaria, recognized two species of this genus

that compose the natural beds that are commerciallyexploited : Gracilaria lemaneiformis (Bory) Weber vonBosse [now Gracilariopsis lemaneiformis (Weber vonBosse) Dawson, Acleto et Foldvik], recognized fromCoquimbo (29°56' S) to Chiloe (42°30' S ; Etcheverry,1958; Kim, 1970), and Gracilaria verrucosa (Hud-son) Papenfuss, identified from Dichato (36°33' S) toQuetalmahue (41°52' S ; Romo et al., 1979) .

Subsequent studies by Bird et al. (1986) indicat-ed that most of the Gracilaria harvested in Chile[from Coquimbo, Cerro-Verde (37°44' S), and Maullin(41 °3l' S)] corresponded to another species, Gracilar-ia chilensis Bird, McLachlan et Oliveira, and thatG. verrucosa (a North Atlantic species) probablyis absent from the Chilean flora as well as else-where in the Pacific . According to these authors,G. chilensis shares a number of characteristics withG. lemaneiformis to the degree that morphological-

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ly they are indistinguishable . However, they differin the location of the spermatangia (cortical textorii-type conceptacles in G. chilensis versus superficiallayers in G. lemaneiformis) and in the ocurrence ofbasal absorbing filaments between the gonimoblastsand the cystocarp floor (present in G. chilensis; absentin G. lemaneiformis ; Fredericq & Hommersand, 1990) .These species may have been mistaken in previousstudies in Chile, and some reports of G. lemaneiformismay actually refer to G. chilensis . There is, howev-er, no question that G. lemaneiformis is an importantcomponent of the Chilean flora (Bird et al., 1986) .

More recently, Candia & Reyes (1993) and Candia(pers. comm., 1994) recognized the existence of a num-ber of morphotypes in beds from the VIIIth (ca 36° S to38° S) and Xth Regions (ca 39°30' S to 43°40' S) thatdiffered in length and thickness of thalli, branching pat-tern, color, and in the ocurrence of reproductive struc-tures. Moreover, Santelices & Ugarte (1990) found anunidentified species of Gracilaria from samples col-lected from Maullfn. Besides the phenotypic variabilityusually found among the species of Gracilaria, San-telices & Varela (1993) also have reported the existenceof intra-clonal variation in G. chilensis, a factor thatshould be taken into account in understanding the tax-onomic limits and phylogenetic relationships amongspecies of Gracilaria .

Basic knowledge of the biological identity of pop-ulations, species, and in some cases even genera ofcultivated agarophytes in Chile is necessary for theapplication of specific management programs in areasof exploitation, for beds declared genetic reserves, andfor selecting species with appropiate commercial prop-erties of agar (Abbott, 1979 ; Santelices & Doty, 1989) .

Recently, research on algal systematics has beengreatly facilitated by the use of molecular techniques .These tools provide a means of characterizing indi-viduals, populations and species, and in many cases,of determining unequivocally the systematic affinitiesof organisms . In particular, restriction fragment lengthpolymorphisms (RFLPs) have been used to delineatespecies of Gracilaria and Gracilariopsis (Goff & Cole-man, 1988 ; Bird et al ., 1990). More recently, Bird et al .(1990, 1992) and Goff et al. (1994) have differentiatedspecies of Gracilariales by sequencing, respectively,the 18S rDNA gene and the ITS and 5 .8S rDNA geneof the ribosomal repeat region .

The present study uses molecular approaches tosolve taxonomic problems of Gracilaria in Chile .Restriction fragment length polymorphism patterns ofplastid DNA of four morphotypes collected in a bed

from Maullfn were characterized and compared withthe patterns of four morphotypes collected from a bedat Lenga (36°45' S) (Gonzalez et al., 1995). In anattempt to unravel the nature of the morphologicaldifferences found among Lenga and Maullfn morpho-types, the present study also includes random amplifiedpolymorphic DNA (RAPD) analysis of nuclear DNA,as this technique has been shown to discriminate organ-isms at species and/or population level (Williams et al.1990; Welsh & McClelland, 1990 ; Goodwin & Annis,1991 ; Baird et al., 1992; Wilde et al ., 1992 ; Patwaryet al ., 1993) .

Materials and methods

Four to five hundred grams of gametophytic thalli ofeach morphotype were collected from different areasof a bed located in Maullfn River (41°36' S ; 73'38'W),Xth Region, Chile . According to Candia (pers. comm .,1994) the morphotypes were designated as follows :MI : thalli of intermediate thickness (0.5-1 .5 mm), 10-30 cm in length, profusely branched, growing on rocks ;M2: thalli thin (0 .3-1.0 mm), profusely branched, 30-80 cm in length, growing in sand ; M3 : thalli thick (1-2.5 mm), 40-130 cm in length, growing in sand ; M4 :similar to M2 but olive-green . All other morphotypeswere brown-reddish .

Thallus preparation : DNA extraction and plastidDNA purification techniques were as in Rice & Bird(1990) with some modifications . One hundred gramsfresh weight were used for each morphotype, and thelysis buffer solution used was 4% SDS, 0.2 M NaCl,0.05 M Tris pH 8.0 and 0.1 M EDTA. After the CsCIultracentrifugation in Hoechst dye (H 33258), the dyeand CsCI were removed, and the plastid and nuclearDNA precipitated . After precipitation and subsequentwashes in 70% ethanol, the DNA samples were air-dried and resuspended in the appropiate amount of TEto give a final concentration of 10 ng DNA ml -1 .

Ten ml aliquots of the ultracentrifugation upperband DNA from each morphotype from Maullfn (20 ngml-1 ) were digested with 2 or 10 units of the followingendonucleases : BamH I, Bgl II, Cla I, Hae III, Eco RI,Pst I, Pvu II, Sal1, Xba I and Xho I, following the pro-tocol suggested by the suppliers . The digested DNAwas electrophoresed for 16 h at 20-30 V on gels of0.7 and 1 .0% agarose in TAE buffer. After staining inethidium bromide (0 .5 mg ml -1 ), the gels were exam-ined and photographed on a UV transilluminator . A

Table 1 . Oligonucleotide (l0-mer) primers used inthe RAPD analyses .

Primer

Sequence

P1 5' ACGTATCTGC 3'P2 5' ACAACTGCTC 3'P3 5' TGACTGACGC 3'P4 5' AGCAGCCTGC 3'P5 5' GCATATTCCG 3'P6 5' GGTCTCTCCC 3'P17 5' CCTGGGCCTC 3'P30 5' CCGGCCTTAG 3'P50 5' TTCCCCGCGC 3'P61 5' TTCCCCGACC 3'P100

5' ATCGGGTCCG 3'

Polaroid MP4 camera with a Wratten 23A filter and aPolaroid Type 665 film was used to photograph gels .

For RAPD analysis, nuclear DNA (lower band) offour morphotypes from Lenga (namely LI : estuarine,intermediate thickness, brownish; L2: marine, thin,brown-reddish ; L3 : marine, thick, brown-reddish ; L4 :marine, thick, olive-green) and the four morphotypesfrom Maullin purified through a CsCI-Hoechst gradi-ent were used. Eleven oligodeoxyribonucleotides of10 bases each were used to prime PCR reactions (P1to P6, P17, P30, P50, P61, P100; Table 1) . Theywere obtained from Oligop6ptido (Centro de Sintesis yAnAlisis de Biomol6culas, Universidad de Chile, San-tiago, Chile) .

Typical amplifications were performed in 25 tl vol-umes containing Taq DNA polymerase buffer (PerkinElmer), 200 mM of each deoxynucleotide (dATP,dCTP, dGTP, dTTP), 0 .2 mM primer, 60 ng genomicDNA, 1 .5 mM Mg2+ ion and 1 .0 unit of Taq DNA poly-merase (Perkin Elmer) . The amplifications were per-formed in a Perkin Elmer Thermal Cycler programmedfor 40 cycles of 1 min at 94 °C, 1 min at 42 °C, and2 min at 72 °C . Reaction products were separated byelectrophoresis on a 2% agarose gel . The gels werestained and photographed as for RFLPs .

Results

The total DNA isolated from each morphotype andfractionated on a Cesium chloride-Hoechst gradientgave three bands : high, intermediate, and low densi-ty, visible under UV light (Figure 1) . Aliquots of theintermediate and low density (upper) fractions from

Figure 1 . CsCI-Hoechst gradient of one of the Maullin morphotypesof Gracilaria. (1) organelle DNA, (2) unknown DNA, (3) nuclearDNA, (4) RNA.

CsCI gradients were digested with different restric-tion endonucleases. Distinctive restriction fragmentpatterns between the intermediate and low density frac-tions were obtained (Figure 2), but patterns were identi-cal when the digested intermediate or low density frac-tions were compared among morphotypes (Figures 3 &4). When restriction fragment analysis on the organel-lar DNA (upper fraction) was performed among themorphotypes from Lenga and Maullin identical pat-terns were obtained (Figure 5) .

Four of the 11 RAPD primers (P4, P30, P50, P100)gave clear and reproducible patterns at the annealingtemperature used (42 °C), amplifying one or more por-tions of the genomic DNA. However, none of themshowed any polymorphism among the four Lenga (Fig-ures 6 & 7) or the four Maullin morphotypes or betweenmorphotypes from both localities .

Discussion

The low- and high-density bands of DNA obtainedby ultracentrifugation with CsCI-Hoechst gradient cor-respond to organellar and nuclear DNA, respectively(Goff & Coleman, 1988 ; Bird & Rice, 1990) . Theorigin of the intermediate band is unknown, but dueto its position on the gradient closest to the nuclear

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Figures 2-7 . Figure 2 . Restriction fragment pattern of low (lanes 2, 4 & 6) and intermediate (lanes 3, 5 & 7) density fractions from MaullinGracilaria digested with Hind III (lanes 2 & 3), Bgl II (lanes 4 & 5), Xba I (lanes 6 & 7) . Lane 1 is lambda DNA digested with Hind III.Figure 3 . Restriction fragment pattern of intermediate density fraction of the four morphotypes of Gracilaria from Maullin digested with Sal I .Marker is lambda DNA digested with Sal I . Figure 4 . PstI digestion patterns of organellar DNA (upper fraction) of the four morphotypes ofGracilaria from Maullin . Marker is lambda DNA digested with EcoR I and Hae III. Figure 5 . Bg II, Cla I, PstI and Xba I digestion patternsof organellar DNA (upper fraction) from Lenga and Maullin morphotypes of Gracilaria . Marker as in Figure 4 . Figures 6-7. Bands obtainby RAPD amplification of genomic DNA of the four morphotypes of Gracilaria from Lenga . Reactions were performed with primer P30 andprimer P 100 . Marker is lambda digested with Hue III .

band, it might correspond to ribosomal nuclear DNA(A. W. Coleman, pers . comm., 1994) . To identify thisband, southern hybridization on nitrocellulose mem-branes and radioactive probing with a nuclear rDNArepeat (pHA) are in progress in our laboratory. Thesame approach is being taken with the low densityband to examine the assumption that it respresents pri-marily plastid DNA .

The identical organellar DNA restriction fragmentpatterns found among the Maullin morphotypes andbetween the Lenga and Maullfn morphotypes suggestthat all eight samples belong to a single genetic group .Moreover, the same fractioning pattern was found inGracilaria chilensis by Bird et al. (1990) using thesame endonucleases (Bam HI, Kpn I and Kba I) . Con-current data from preliminary interfertility tests (Can-dia & Reyes, 1993 ; Candia, pers . comm., 1994) amongthe eight morphotypes, as well as anatomical studiesof the reproductive structures (Candia, 1988 ; Laf6n,1992) lead us to conclude that all eight correspond toGracilaria chilensis .

The RAPD analysis performed with genomic DNAof the four morphotypes from Lenga and four morpho-types from Maullin did not allow us to discriminateamong them . No differences in the number or the sizeof nuclear fragments randomly amplified were found .Patwary et al. (1993) used 165 primers and found only37 yielding polymorphisms among isolates of Gelidi-um vagum Okamura. Nine of the 11 primers we usedwere among the successful ones used by Patwary et al.(1993). Obviously, a much higher number of primerswill have to be used to detect polymorphisms withinand between populations of G. chilensis .

Acknowledgements

We thank Dr H . L . Barrales and especiallyDr Annette W. Coleman for reading and criticallyreviewing the manuscript . Financial support was pro-vided by the Fondecyt 1930428 grant to A . Candia andD.I.U.C. 91 .32.26-1 grant to M . Gonzalez .

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