BIOLOGIJA. 2006. Nr. 1. P. 53–59 © Lietuvos mokslų akademija, 2006 Assessment of genetic polymorphism in natural populations of ostrich fern... 53 © Lietuvos mokslų akademijos leidykla, 2006 Assessment of genetic polymorphism in natural populations of ostrich fern (Matteuccia struthiopteris L., Dryopteridaceae) in Lithuania by using RAPD markers A. Stapulionytė1, J. R. Lazutka1, Randomly amplified polymorphic DNA analysis was carried out to examine genetic diversity, population structure and polymorphism level among and J. R. Naujalis1, A. Odland2 within seven natural populations of ostrich fern (Matteuccia struthiopteris L., Dryopteridaceae) in Lithuania. In total, 43 plants were analysed using 10 1 Department of Botany and Genetics, polymorphic decanucleotide primers of random sequence. 73 polymorphic Vilnius University, 21 M. K. Čiurlionio St., bands ranging in size from 470 to 2600 bp were derived using selected pri- LT-03101 Vilnius, Lithuania mers. The average value for Shannon’s phenotypic diversity index for all seven populations was I = 0.0498 (SD = 0.0534, P = 0.02), mean heterozigo- 2 sity was 0.033 (SD = 0.038, P = 0.03), total gene diversity H = 0.3385 Telemark University College, t (SD = 0.0164), gene diversity among populations G = 0.9009 and gene flow Hallvard Eikas plass, N-3800 Bø, Norway st Nm = 0.0550. PCA and UPGMA analysis revealed a high degree of interpo- pulation differentiation. Genetic differences among populations were due to the absence or presence of the same alleles in different populations, but not to the frequency of alleles in each population. Analysis of molecular variance (AMOVA) revealed that 9% of the total molecular variance was due to an individual variation within the populations and 91% to the differences among Φ populations ( PT = 0.909, P = 0.001, df = 6). A weak but statistically signi- ficant correlation (r = 0.354, P = 0.047) between Nei’s genetic distances and the geographic location of populations was established using the Mantel test. The very low intrapopulation differentiation shows that clonal propagation is prevailing in populations of ostrich fern in Lithuania. The parameters of genetic diversity and a correlation of genetic distance with the geographic location revealed the existence of population isolation by distance forasmuch as migration is very low. Key words: clonality, genetic diversity, Matteuccia struthiopteris, RAPD, population genetic structure INTRODUCTION rophylls [2–4]. Ostrich fern often forms extensive mo- no-dominant stands as the light at the ground level Matteuccia struthiopteris (L.) Todaro (Dryopteridace- in the most vigorous and dense stands of Matteuccia ae) is a hemicryptophytic fern, which has circumpo- is reduced to a level insufficient for the growth of lar distribution in the north temperate and sub-bo- competitors [2]. M. struthiopteris populations are long- real zones of the northern hemisphere [1, 2]. Its lived and theoretically they are immortal due to con- area spreads from North America, through Europe tinuous clonal growth [5]. Stands of Matteuccia spre- to East Asia. In Europe it is mostly confined to ad asexually by the colonization of surrounding soil northeastern parts and usually along floodplains, by stolons developed on the rhizomes. Ramets (erect springy sites, and its occurrence may be considered rootstocks with a projecting crown of fronds) con- as an indicator of moist eutrophic soils [2]. Matteuc- nected to the mother plant by rhizome may geneti- cia is the only fern species in Lithuania with the cally represent the same individual (genet) in a po- dimorphic fronds: sterile trophophylls and fertile spo- pulation. Plants of sexual origin have not been re- corded in dense fern stands [4, 6]. The lack of se- E-mail: [email protected] xually reproduced new Matteuccia plants have also 54 A. Stapulionytė, J. R. Lazutka, J. R. Naujalis, A. Odland been reported from other areas [7], and this feature (Naujoji Akmenė, Šilutė and Telšiai districts), one po- may be related to the location of population accor- pulation in the eastern part (Anykščiai distr.), and two ding to the species areal [8]. Plants of Matteuccia populations in the central part of Lithuania (Kaišiado- disperse by air and water dispersal of spores, and rys and Panevėžys districts). The last – Pilis island – water dispersal of crowns and/or stolons from popu- population is different from the rest because it is loca- lations along riverbanks. Establishment of young ted on the island in Lake Plateliai (Þemaitija regional plants from gametophytes has not been studied, for park). The tops of fronds (10 cm) and croziers (coi- in spite of the prodigious production of spores per led young fronds) from the top of the rootstocks of fertile frond, very few spores germinate [5]. 10–12 ferns growing at a distance no less than 3 m Ostrich fern is a quite rare and endangered spe- were collected for analysis from each population. cies in Lithuania. Populations of this species are cle- Plants were picked up from the whole area occupied arly restricted and sparse in the country. This spe- by this species going lengthways the population. Plant cies is widely known for its ornamental value in the material was collected in August–September of 2004 whole world and as an economic fern in America and July–August of 2005. In total, 43 samples collec- where edible fiddleheads are a spring vegetable [2, ted from seven populations were analysed using 5, 9]. There is no threat for ostrich fern to be ex- RAPD (Table 1). terminated in Lithuania because of its nutritive qu- DNA extraction and amplification. Genomic DNA ality as a vegetable, but the distribution of this spe- was extracted from 135 mg of frond or 100 mg of cies is too narrow to be indifferent and this is a crozier tissue using liquid nitrogen and the Genomic critical case of interest. M. struthiopteris is protected DNA Purification Kit (Fermentas, Lithuania) in ac- by law in the majority of Central European coun- cordance with recommendations of manufacturer. tries [1]. There have been carried out different stu- DNA concentration and purity were measured spec- dies on the physiology [10] and propagation [11], trophotometrically (BioPhotometer, Eppendorf, Ger- morphology [4] and anatomy [12], structural varia- many). Ten decanucleotide primers (Roth, Germany) tions [13], demography [4] and composition of ost- of random sequence (GC 70–80%) were chosen from rich fern populations [1, 14]. Although some syste- three primer kits (Kit 270, Kit 380, Kit 470). Ampli- matic analyses and phytogeographic studies have be- fication was performed in a thermocycler (Master- en published [2], the genetic composition of Mat- cycler personal 5332, Eppendorf, Germany) and the teuccia populations remains unclear. Only a few ar- amplified DNA bands were separated electrophore- ticles can be found to deal with the ostrich fern tically under the conditions described earlier [14]. population genetic structure or genetic diversity us- Amplification was performed at least in two inde- ing allozyme systems [9]. The present study is a new pendent experiments for each sample. Data from cle- insight to the attributes of M. strutiopteris populations ar, unambiguous and reproducible bands were used that have been studied genetically very modestly up for analysis. to now. As there is no available specific information Data analysis. Amplified DNA bands were esti- about the genetics of this species, the RAPD me- mated as distinct DNA loci with two alleles. Mono- thod was chosen as the most appropriate. morphic loci had only one allele (present = 1) while polymorphic loci had both (present = 1 and absent MATERIALS AND METHODS = 0) alleles. The number of polymorphic loci, measures of he- Plant material. Seven natural populations of ostrich terozygosity and the Shannon’s index of phenotypic fern (M. struthiopteris) were analysed in Lithuania using diversity (I), genetic distances (GD) among popula- RAPD markers: three populations in western Lithuania tions, parameters of genetic diversity (Ht, GST, h) and Table 1. Sampling locations (districts in brackets), coordinates and sample sizes of individuals analyzed in populations of ostrich fern (Matteuccia struthiopteris L.) in Lithuania No Population n Latitude Longitude NE 1 Anykščiai (Anykščiai distr.)NE 7 55°34' 25°05' 2 Krekenava (Panevėžys distr.)NE 8 55°35' 24°11' 3 Rambynas (Šilutė distr.)W 4 55°02' 22°06' 4 Pilis island (Telðiai distr.)W 5 56°02’32,1'’ 21°50’50,0'’ 5 Plokštynė (Telšiai distr.)W 7 56°02’35,3'’ 21°50’49,5'’ 6 Venta (N. Akmenė distr.)NW 7 56°09' 22°35' 7 Lomena (Kaiðiadorys distr.)C 5 54°57' 24°26' total 43 NE – northeastern, C – central, W – western, NW – northwestern part of Lithuania. Assessment of genetic polymorphism in natural populations of ostrich fern... 55 gene flow (Nm) were computed with PopGene1.31 of bands were more than 50% frequent in all seven [16] assuming all loci to be dominant and in Hardy– populations. Five private alleles each present in only Weinberg equilibrium. Genetic distances among in- one population were detected. Two private alleles were dividual ferns were estimated according to the Nei specific for Anykščiai population, and the other three and Li formula [17] using TREECON for Windows were specific for Krekenava, Plokštynė and Venta po- V 1.3b [18]. UPGMA (Unweighted Pair-Group Met- pulations respectively (Fig. 1). There were 14 zero al- hod for Arithmetic Averages) cluster analysis based leles (data not shown) which were absent only in on these genetic distances generated a dendrogram one population, but they were not considered as pri- representing the relationships among the populations vate given that fewer mutations contribute to the [18]. GenAlEx_6b4 [19] software package was used occurrence of 0 allele than of the allele that is pre- for the performance of the principal coordinates ana- sent. Data in Table 3 show a very low number of lysis (PCA) – to plot the relationship between gene- polymorphic loci for each population – the mean is tic distances matrix elements based on their princi- only seven.