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Protistology 3 (4), 243-250 (2004)
Protistology
Genetic diversity and relationships between wild and cultivated populations of the sea lettuce, Enteromorpha prolifera, in Korea revialed by RAPD markers
Man Kyu Huh1, Hak Young Lee2, Bok Kyu Lee1 and Joo Soo Choi1
1 Department of Molecular Biology, Dongeui University, The Republic of Korea
2 Department of Biology, Chonnom National University, The Republic of Korea
Summary
RAPD analysis was conducted to estimate genetic diversity and population structure of the wild (natural) and cultivated sea lettuce, Enteromorpha prolifera. The objectives of this study were to estimate the levels of genetic diversity in the wild and cultivated populations and to describe how the genetic variation of this species is distributed within and among its populations. In wild sea lettuce, 93.2% of loci at the species level showed polymorphism. The cultivated populations were found to have fewer alleles per locus (1.20 vs. 1.23), fewer effective alleles per locus (1.37 vs. 1.39), lower percentage of polymorphic locus (37.0 vs. 39.2), and lower gene diversity (0.119 vs. 0.136) than wild populations. These genetic diversity parameters indicate that the cultivated populations are genetically depauperate relative to their presumptive progenitor and the domestication process has eroded the level of genetic variation of this species. Nevertheless, its genetic diversity is higher than average values for species with similar life history. The sexual reproduction, perennial nature, high fecundity, and colonization process are proposed as possible factors contributing to high genetic diversity.
Key words: Enteromorpha prolifera, genetic diversity, population structure, sea lettuce
Introduction
Technological advances, especially in the area of biochemical genetics, have also contributed to an increased interest in plant populations (Hamrick, 1982). For example, Hamrick et al. (1992) demonstrated that species which were widespread, long lived, pri-
marily outcrossed by wind pollination maintained higher levels of intrapopulation genetic variation than species with other combinations of these characteristics.
Wild relatives of cultivated plants represent an interesting system from both agricultural and evolutionary viewpoints (Doebley, 1989). Wild relatives usually
© 2004 by Russia, Protistology
store great amounts of genetic variation (Clegg et al., 1984; Schmit and Debouck, 1991), which may be of present or future interest for plant improvement programs (Bussell, 1999; Beebe et al., 2000).
Sea lettuce, Enteromorphaprolifera, is abundant in East Asian marine ecosystems. Cultivation of this species for side dishes has been very popular in the southern coast of Korea. Although there are many morphological and physiological studies (Sohn, 1993; Hwang et al., 1998), genetic diversity and population structure of this species have not yet been investigated. Therefore, detailed studies, in particular at the DNA level, of genetic diversity of wild populations of the sea lettuce, and genetic relationships between wild and cultivated sea lettuces are necessary from the viewpoint of plant evolution.
Although molecular and biochemical approaches are now increasingly being applied to address taxonomic and phylogenetic relationships within animals and plants in Korea (Huh and Huh, 2002), no population genetics studies have been conducted, especially on the population genetic structure of algal species. This knowledge, however, is crucial for developing programs aimed at protection and preservation of genetic resources of plant species. The Korean populations of E. prolifera are typically small and distributed in patches.
The objectives of this study were to estimate the level of genetic diversity in the species, and to describe how its genetic variation is distributed within and among its populations.
In this study five cultivated and six natural (wild) populations of the sea lettuce from Korea were analyzed for RAPD markers. RAPD assay has been useful in determining genetic relationships among closely related species (Quiros et al., 1991; Demeke et al., 1992). RAPD analysis is quick, robust, and requires minimal preliminary work (Molnar et al., 2000). With the use of RADP we expected to successfully assess the genetic relationships between the local populations of cultivated sea lettuce and the wild populations of this species in Korea.
Material and methods
Plant materials
Five cultivated and six wild populations, representing the geographical range of the sea lettuce in Korea, were used in this study (Fig. 1). From April 2001 to July 2002, twenty plants from each population were randomly sampled and their thallus were used for molecular analysis. The distance between the selected individuals was about 5 m to avoid including individuals emanating from the same rhizome.
Fig. 1. The location of the population studied. CE1-CE5 - cultivated sea lettuce populations; WE1-WE6 - wild sea lettuce populations.
A wild population of Undaria pinnatifida from Busan, Korea was used for the outgroup sample.
GENOMIC DNA ISOLATION AND RAPD ANALYSIS
DNA was extracted using the plant DNA Zol Reagent (Life Technologies Inc., Grand Island, New Ybrk, U.S.A.) according to the manufacturer’s protocol. Forty arbitrarily chosen 10-mer primers, the kit C (0PC-01 to 20) and the kit D (0PD-01 to 20) of Operon Technologies (Alameda, Co.) were used. All the reactions were repeated twice and only reproducible bands were scored for analyses. To analyze the DNA ofindividuals, we selected twelve decamer primers that produced RAPD bands both in cultivated and wild populations in a preliminary test (Table 1).
Amplification reactions were performed in 0.6 ml tubes containing 25 ці of the reaction buffer; 10 mM Tris-HCl, pH 8.8, 50 mM MgCl2, 100 цМ each of dATP, dCTP, dGTP, dTTP, 0.2 mM primer, 2.1 units Taq DNA polymerase, and 25 ng of genomic DNA. The amplification products were separated by electrophoresis on 1.5% agarose gels, stained with ethidium bromide, and photographed under UV light using Alpha Image TM (Alpha Innotech Co., U.S.A). A 100 bp ladder DNA marker (Pharmacia) was used in the end of lane for the estimation of fragment size.
Table 1. List of decamer oligonucleotide utilized as primers, their sequences, and associated polymorphic fragments amplified in the sea lettuce test array
No. of Primer Sequence (5’ to 3’) No. of Polymorphic fragments detected Fragment size range (bp)
OPCOI TTCGAGCCAG 4 750 - 1910
OPC02 GTGAGGCGTC 2 800 - 1720
OPC07 GTCCCGACGA 3 1150 - 2180
OPDOI ACCGCGAACG 2 550 - 1960
OPD02 CGACCCAACC 14 780 - 1880
OPD03 GTCGCCGTCA 12 450 - 1760
OPD05 TGAGCGGACA 3 660 - 2250
OPD08 GTGTGCCCCA 8 650 - 2110
OPD10 GGTCTACACC 13 470 - 1120
OPD14 CTTCCCCAAG 7 550- 1200
OPD17 TTTCCCACGG 3 910 - 2570
OPD19 CTGGGACTT 2 1200 - 2830
Data analysis
All monomorphic and polymorphic RAPD bands visible by eye were scored and only unambiguously scored bands were used in the analyses. Each polymorphic RAPD band was given a score of 1 for presence or
0 for absence. Several standard genetic parameters were estimated using the computer program, POPGENE ver. 1.31 (Yeh et al., 1999). The percentage of polymorphic loci (Pp), mean number of alleles per locus (A), effective number of alleles per locus (AE), Nei’s (1973) gene diversity (H), and Shannon’s Information index (I) (Lewontin, 1972).
The degree of polymorphism was quantified using phenotypic diversity (Bowman et al., 1971):
Ho = - p> log p>
where p. is the frequency of a particular phenotype
1 (King and Schaal, 1989).
HO can be calculated and compared for different populations (Paul et al., 1997). Let
HPOP = 1/n HO
be the average diversity over the n different populations and let
HSP = - p log p
be the diversity of species calculated from the phenotypic frequencies p in all the populations considered together (Paul et al., 1997). Then the proportion of diversity within populations, HPOP/HSP, can compared with that between populations (HSP —
HPOP)/ HSP.
The estimation of genetic similarity (GS) between the genotypes was based on the probability that an amplified fragment from one individual will also be present in another (Nei and Li, 1979). GS = 2 x Number of shared fragment between A and B / (Number of
fragment in A + Number of fragment in B). GS was converted to genetic distance (1-GS) (Le Thierry et al., 2000). Homogeneity of variation among populations was tested by Bartlett’s statistics (SAS, 1989).
Genetic differentiation measured by GST among populations was also calculated. Furthermore, gene flow (Nm) between the pairs of populations was calculated from GST values by Nm = 0.5(1/GST - 1) (McDermott and McDonald, 1993).
Cluster analyses
A genetic distance matrix was used to construct a dendrogram with the unweighted pair group method with arithmetic average (UPGMA) method in the neighbor algorithm of the Phylogeny Inference Package (PHYLIP ver. 3.57, Felsenstein, 1993). One thousand bootstrap resamplings over band phenotypes in the original data gave the support values for branches in the tree.
Results
From the 40 decamer primers used for a preliminary RAPD analysis, twelve primers produced good amplification products both in quality and variability (Table 1). Of all bands observed, 93.2% were polymorphic among all populations and the remaining primers either did not amplify or showed unclear amplification across all genotypes.
In a simple measure of intrapopulation variation, i.e. the percentage ofpolymorphic bands, the CE3 population exhibited the lowest variation (35.6%). The WE2 population showed the highest one (46.6%) (Table 2).
The average number of alleles per locus (A) was 1.392 across populations, varying from 1.356 to 1.466. The effective numbers of alleles per locus at the population level (Ae) and at the species level were 1.230 and 1.326, respectively. The mean genetic diversity within populations was 0.136. In comparing the two groups (cultivated and wild populations), all measures (PP, A, Ae, H, and I) differed significantly from zero
(TaPble 2).E
Total genetic diversity value (HT) was 0.213 (±0.024) and the interlocus mean variation of genetic diversity within populations (HS) was 0.136 (±0.014) (data not shown).
The phenotypic frequency of each band was calculated and used in estimating genetic diversity (HO) within populations (Table 3). The mean HO of wild sea lettuce populations (0.905) was significantly higher than
Table 2. Measures of genetic variability for RAPDs generated among 11 populations of sea lettuce
Pop. Polymorphic loci Pp 4 H I
Cultivated populations
CE1 28 38.4 1.384 1.195 0.120 0.185
CE2 27 37.0 1.370 1.183 0.116 0.179
CE3 26 35.6 1.356 1.177 0.111 0.171
CE4 27 37.0 1.370 1.205 0.121 0.183
CE5 27 37.0 1.370 1.215 0.128 0.193
Mean 27 37.0 1.370 1.195 0.119 0.182
Wild populations
WE1 27 37.0 1.370 1.207 0.126 0.190
WE2 34 46.6 1.466 1.341 0.189 0.275
WE3 32 43.8 1.438 1.272 0.157 0.234
WE4 29 39.7 1.397 1.241 0.140 0.208
WE5 29 39.7 1.397 1.249 0.146 0.217
WE6 29 39.7 1.397 1.240 0.142 0.212
Mean 30 41.1 1 .41 1 1.258 0.150 0.223
t-test 2.526* 2.526* 2.946* 3.061* 2.970*
Total mean 28.6 39.2 1.392 1.230 0.136 0.204
Species 68 93.2 1.932 1.326 0.214 0.345
* p < 0.05.
that of cultivation populations (0.789) (paired t test). Although the Korean populations were small, isolated, and patchily distributed for wild sea lettuce, they maintained a high level of genetic diversity (HSP =
1.961).
An assessment of the proportion of diversity present within populations, Hpop/ HSP, indicated that 24.1% of the total genetic diversity was among populations. Thus, the three quarters ofgenetic variation (75.9%) resided within populations (Table 4). The average number of individuals exchanged between populations per generation (Nm) was estimated to be low (0.870).
A similarity matrix based on the proportion of shared fragments (GS) was used to evaluate relatedness among 11 populations (Table 5). The estimate of GS ranged from 0.850 between CE3 and WE3 to 0.993 between CE1 and CE2.
Clustering of sea lettuce populations, using the UPGMA algorithm, was performed based on the matrix of calculated distances (Fig. 2). The phylogenetic tree showed two distinct groups; cultivated and wild sea lettuce populations were well separated from each other. The tree also shows genetic differentiation among local populations for both cultivated and wild sea lettuce. Although WE2 population was located at an unexpected position on the tree, geographically close populations were situated in close positions on the phylogenetic tree.
Fig. 2. A dendrogram showing the genetic relationships among eleven populations of sea lettuce based on RAPD analysis. For explanation of symbols see Fig.1.
Discussion
Genetic diversity and domestication
The sea lettuce is one of the dominant plants in marine habitats along the continental shelves and in estuaries and bays in the colder parts of the temperate oceans (Pearson, 1995). Genetic diversity in E. prolifera is higher than that of most plant species on land. The same trend is observed at the population level.
Genetic diversity of E. prolifera is comparable with that of other marine algal species, although the use of different methods and parameters (e.g., isozyme [codominant marker] and RAPD [dominant marker], the number of loci, the populations sampled, the enzyme systems studied) may preclude meaningful direct comparisons. In E. prolifera, Pp, A, and HE were 93.2, 1.93, and 0.214, respectively. Hwang et al. (1998) analyzed eleven taxa of Porphyra at similar regions in Korea by starch gel electrophoresis. For Porphyra, the corresponding mean values were 21.7, 1.4, and 0.062, respectively. For Undariapinnatifida, P, A, and HE were 44.4, 1.78, and 0.169, respectively (Huh and Huh, 2002). Thus, E. prolifera showed higher levels of poly-
Table 3. Estimates of genetic diversity (HO) within populations of sea lettuce
Primer Populations Total
CE1 CE2 CE3 CE4 CE5 Mean WE1 WE2 WE3 WE4 WE5 WE6 Mean Mean
OPC01 1.342 0.651 0.994 1.241 0.693 0.984 1.035 0.950 0.937 0.637 0.693 1.099 0.892 0.958
OPC02 0.000 0.000 0.000 0.000 0.000 0.000 0.586 0.691 0.000 0.000 0.000 0.000 0.213 0.142
OPC07 0.000 0.000 0.349 0.586 0.500 0.287 0.500 0.000 0.500 0.637 0.637 0.500 0.462 0.429
OPD01 0.320 0.000 0.000 0.000 0.000 0.064 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.036
OPD02 0.693 0.673 0.693 1.127 1.892 1.016 2.055 1.763 1.251 1.365 0.898 1.237 1.428 1.364
OPD03 1.895 1.938 1.71 1 0.898 1.584 1.605 1.537 1.910 1.709 1.866 1.851 1.782 1.776 1.670
OPD05 0.000 0.000 0.335 0.000 0.868 0.241 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.096
OPD08 1.073 2.110 2.019 2.572 1.224 1.800 1.073 2.805 2.883 2.084 2.673 2.642 2.360 2.114
OPD10 1.366 1.305 1.434 0.679 0.673 1.091 1.097 1.306 0.953 1.517 1.036 1.061 1.162 1.076
OPD14 1.430 1 .436 1.470 1.634 1.768 1.548 1.354 1.585 1.580 1.688 1.709 1.541 1.576 1.588
OPD17 1.072 0.586 0.000 1.055 1.061 0.755 0.925 0.500 1.067 1.040 0.970 1.080 0.930 0.974
OPD19 0.000 0.000 0.000 0.000 0.354 0.071 0.000 0.000 0.322 0.000 0.000 0.000 0.054 0.075
Mean 0.766 0.725 0.751 0.816 0.885 0.789 0.847 0.959 0.934 0.903 0.872 0.912 0.905 0.877
morphic and genetic diversity than the taxa of Porphyra and genetic diversity overall and to maintain more variation
U. pinnatifida by Wilcoxon’s signed-rank test (p< 0.05). within their populations than the species with higher
The relatively high level of genetic variation found proportion of asexual reproduction (Hamrick and
in E. prolifera is consistent with several aspects of its Godt, 1989; Huh, 2001). In E. prolifera, the contents
biology. Firstly, the breeding system of E. prolifera has of each cell in the female filaments round up and
an important role in genetic variability. Species with become eggs; the cellular contents ofthe male filaments
predominantly sexual reproduction tend to have higher migrate by amoeboid mobility into the oogonia, where
syngamy takes place. The zygotes remain dormant, often for a year or more, and finally divide by meiosis. Three of the meiospores disintegrate; the other germinates and grows into a vegetative filament (Pearson, 1995). Secondly, long-lived perennial species, like E. prolifera, generally maintain relatively higher levels of variation than annuals (Hamrick and Godt, 1989). As populations of E. prolifera are older, opportunities for accumulation of mutations should be high (Ledig, 1986). Thirdly, plant species with high fecundity usually maintain high genetic diversity (Huh, 2001). Finally, high genetic diversity is associated with the species’ colonizing success (Hamrick and Godt, 1989). Colonizing species are often expected to be markedly depauperate in genetic variation within populations due to founder effects and genetic drift (Hamrick et al., 1992). E. prolifera has maintained a considerable amount of variation during the colonization process. During colonization, individuals with high genetic diversity may survive in the course of natural selection.
The level of genetic variation found in the Korean populations of the sea lettuce was high; the average percentage of polymorphic bands was 37.0% for the
Table 4. Partitioning of the genetic diversity into within and among populations of sea lettuce
Primer Hpop Hsp Hpop/Hsp (Hsp"Hpop)/Hsp
OPC01 0.958 1.309 0.732 0.268
OPC02 0.142 0.33 0.431 0.569
OPC07 0.429 0.479 0.895 0.105
OPD01 0.036 0.637 0.056 0.944
OPD02 1.364 2.399 0.569 0.431
OPD03 1.670 2.319 0.720 0.280
OPD05 0.096 0.209 0.461 0.539
OPD08 2.114 3.518 0.601 0.399
OPD10 1.076 1.938 0.555 0.445
OPD14 1.588 3.301 0.481 0.519
OPD17 0.974 1.002 0.973 0.027
OPD19 0.075 0.209 0.359 0.641
Mean 1.169 1.961 0.759 0.574
Table 5. Similarity matrix (above diagonal) of 9 populations based on RAPD using Nei and Li (1979) and genetic distances (below diagonal) according to Le Thierry et al. (2000)
Pop. CE1 CE2 CE3 CE4 CE5 WE1 WE2 WE3 WE4 WE5 WE6
CE1 - 0.993 0.986 0.927 0.873 0.896 0.874 0.860 0.870 0.896 0.906
CE2 0.007 - 0.987 0. 928 0. 875 0.900 0. 878 0. 863 0. 869 0. 895 0.903
CE3 0.014 0.013 - 0.914 0.862 0.885 0.878 0.850 0.869 0.888 0.890
CE4 0.076 0.075 0.090 - 0.941 0.920 0. 879 0. 899 0.893 0.951 0.950
CE5 0.136 0.134 0.148 0.061 - 0. 883 0. 875 0.860 0.894 0.934 0.930
WE1 0.110 0.105 0.123 0.083 0.125 - 0. 882 0.867 0.867 0.898 0.895
WE2 0.135 0.130 0.130 0.130 0.134 0.125 - 0.865 0.893 0.914 0.904
WE3 0.151 0.147 0.162 0.106 0.151 0.142 0.145 - 0.884 0.895 0.890
WE4 0.139 0.141 0.141 0.114 0.114 0.143 0.114 0.123 - 0.944 0.929
WE5 0.109 0.11 0.119 0.051 0.067 0.108 0.090 0.111 0.058 - 0.984
WE6 0.099 0.103 0.117 0.052 0.073 0.116 0.101 0.116 0.073 0.016 -
cultivated populations and 39.2% for the wild populations (Table 2). RAPD marker diversity ofcultivated sea lettuce, E. prolifera (mean H= 0.119) is significantly different from that of wild sea lettuce (mean H = 0.136, p < 0.05) (Table 2). The comparison ofbanding patterns between cultivated and wild sea lettuce revealed that six bands were not shared between these two categories. Six bands were found to be specific to wild sea lettuce, whereas none, to cultivated sea lettuce. Thus, the domestication processes via artificial selection have eroded the levels of genetic diversity in cultivated sea lettuce. It is in good agreement with the concept that most inland crops show a reduced level of polymorphisms as compared to their presumed progenitors (Doebley, 1989). Other studies also found that wild species usually maintain higher level of polymorphisms compared to cultivated species (Abo-elwafa and Shimada, 1995; Chan and Sun, 1997). But in other species such as barley and common buckwheat, cultivated populations have higher genetic variability (Brown, 1978; Ohnishi, 1998). In addition, the domestication process has not eroded the levels of genetic variation in soybean (Kiang and Gorman, 1983). Ultimately, high variability levels of the wild species are expected because they were not subject to any of the selection pressures of domestication, and the maintenance of higher genetic variability would favor their survival under natural conditions (Chan and Sun, 1997). Populations of long-lived woody plants, composed of cohorts established at different times and occupying relatively large areas, can be genetically differentiated both temporally and spatially (Knowles, 1980; Schnabel and Hamrick, 1990).
Since young, softer “leaves” of the sea lettuce are used as food, wild populations should be relatively older than cultivated ones that are not “allowed” to grow old. As mentioned above, in older populations there may be more opportunities for accumulation of mutations (Ledig, 1986). Therefore, wild populations demonstrate considerably higher variation than cultivated ones.
Population structure
Although we did not analyze further subdivision of local populations, we may infer that RAPD variation that resided mainly within wild sea lettuce populations is maintained in patchily distributed subpopulations or demes, either by random drift of neutral alleles or micro-environmental selection for adaptive alleles (Ruckelshaus, 1998).
However, no great local difference in RAPD variation of cultivated populations was observed. Gene flow between populations was not relatively great. The estimated Nm was 0.870. However, gene flow among cultivated populations was high (Nm = 2.184). Hence, we can expect weak or low gene flow from cultivated populations to wild ones. Cultivation of the sea lettuce is very common in Korea. To cultivate it, farmers move sea lettuces from place to place to forage on good products. Cutting of blades and vegetative filament dispersal by farmers may be one of the mechanisms of gene flow among cultivated populations. This occasional movement of plants may have resulted in high gene flow chance and little spatial genetic differentiation.
On the other hand, judging from the distribution of natural populations, the level of gene flow in wild sea lettuce populations is mainly caused by male filament dispersal via sea current supplemented by short distance flow of pollen grains. We frequently found parts of sea lettuce among flotsam on beaches and rocks, and found that such plants can be germinated. This hypothesis on diffusion of wild sea lettuce by sea currents, however, needs to be tested by future works.
Phylogenetic relationships between cultivated and
WILD SEA LETTUCE
The phylogenetic tree shown in Fig. 2 clearly distinguishes two clades, the cultivated and the wild clade.
Cultivated populations show a close relationship between their phylogenic and geographical positions. The wild coastal populations of the Korean Peninsula are relatively small and maintain more genetic variation than the cultivated ones. The uneven distribution of locality-specific bands could be explained by isolation —by distance and might reflect migration from the WE2 and WE3 populations to southeast and northwest regions of the Korean Peninsula by sea current. It is unclear why the wild sea lettuce has not invaded inland regions.
In a phylogenetic tree based on RAPD variability, the position of the Korean populations in the UPGMA tree and their geographical position match almost comple-tely (Fig. 2). However, it is relevant to stress that RAPD markers used allowed us to discriminate among all populations, even those that could not be distinguished on the basis of allozyme analysis (data not shown). Some authors have considered RAPD-based analysis to be more definitive in separation of clusters at the species level than isozyme-based analysis (Heun et al., 1994; Bartish et al., 2000), mostly because RAPDs yield lower coefficients of variation than isozyme, thus allowing for a higher level ofdiscrimination of the cultivated and the wild populations of the sea lettuce. Thus, RAPD markers are very effective in classifying natural populations of the wild and the cultivated sea lettuce in Korea.
To a large extent, the allelic composition ofcultivated populations represents a subset of wild ones. In wild populations 68 polymorphic loci were detected, 56 of which were also detected in cultivated ones. No unique alleles were found in cultivated populations. These 56 loci are, in general, the most common wild populations alleles at their respective loci, which is what would be expected if cultivated populations alleles were a random subset of wild populations alleles. The Korean cultivated sea lettuce is a part of the Korean wild sea lettuce. Cultivated populations and wild sea lettuce are grouped together. Namely, cultivated sea lettuce is almost certainly derived from wild populations in this study. However, we did not find any examples of close connection between a cultivated and a wild population of the sea lettuce.
The information on the phylogenetic relationships of E. prolifera and closely related species is very valuable for the taxonomy of the genus Enteromorpha, the studies of the origin of cultivated sea lettuce, and future sea lettuce breeding.
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Address for correspondence: Man Kyu Huh. Department of Molecular Biology, Dongeui University, 995 Eomgwangno, Busanjin-gu, Busan, 614-714, The Republic of Korea. E-mail: mkhuh@dongeui.ac.kr
Editorial responsibility: Alexander Yudin
