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Protistology 4 (3), 245-250 (2006)
Protistology
Genetic diversity and phylogenetic relationships in five Porphyra species revealed by RAPD analysis
Man Kyu Huh 1, Bok Kyu Lee 2 and Hak Young Lee 2
1 Department of Molecular Biology, Dong-eui University, Busan, Korea
2 Department of Biology, Chonnam National University, Kwangju, Korea
Summary
The genus Porphyra comprises red algae known as laver. They are distributed throughout Asia and used as food in Korea and other countries. Five species of Porphyra (P. tenera, P. yezoensis, P. seriata, P. suborbiculata and P. dentata) are regarded as ecologically and economically important in Korea. We used random amplified polymorphic DNA (RAPD) to investigate their genetic diversity and phylogenetic relationships. The analysis of ten primers revealed 97 loci, of which 70 were polymorphic (72.2%). P. seriata had the highest genetic diversity (0.147), and P. dentata, the lowest one (0.059). An indirect estimate of the number of migrants per generation (Nm = 0.212) indicated that gene flow was very low among the populations studied. The classification of Porphyra species based on the DNA markers does not fully coincide with the classification based on morphology.
Key words: Porphyra, random amplified polymorphic DNA, phylogenetic relationships
Introduction
Seaweeds are an important source of food in China, Korea, and Japan. According to Xia and Abbott (1987), most families in northeastern China eat a seaweed dish at least once a day and over 100 million pounds of seaweed are consumed annually all over the country. Of the 73 seaweed species listed by Xia and Abbott as food items in China, 47 are red algae, 15 are green algae, and 11 are brown algae. Of the 28 genera of red algae
contributing to the Chinese diet, four genera account for half of the species: Porphyra (8 spp.), Gracillaria (6 spp.), Gelidium (5 spp.) and Hypnea (4 spp.). Although many of the same species are used in Korea, some are different. The Chinese prefer seaweeds as a hot dish, broth or soup, whereas the Koreans would rather cook them beforehand and then eat cold or dry as boiled rice wrapping or in Korean salad. Five species of Porphyra are abundant in Korean marine areas (Hwang and Lee, 2002). They are known as laver and eaten with boiled
© 2006 by Russia, Protistology
rice. The Korean populations of five Porphyra species are typically distributed in patches. The cultivation of these species has been very popular in the south and eastern coast of Korea (Lee and Kang, 1986). Laver is known to be one of the sources of potassium and calcium in organic form as well as carotene (Pearson, 1995). Thus, these species play an important role in Asian ecology and economy.
Although molecular and biochemical approaches are now increasingly being applied to taxonomic and phylogenetic relationships within animals and plants in Korea, no population genetic studies of algal species have been conducted. Taxonomy of Porphyra mostly relies on morphological characteristics and allozyme analysis (Hwang et al., 1998). However, the resolving power of morphological characteristics is restricted, mainly because of the small number of characters available. Efficient methods to clarify the taxonomic status of several species are much needed.
RAPD assay has been useful in determining genetic relationships among closely related species (Demeke et al., 1992; Beebe et al., 2000). It is quick, robust and requires minimal preliminary work (Kresovich et al., 1992; Molnar et al., 2000). We expected that RAPD analyses could be used to assess the amount and structure of genetic diversity within and between natural populations and to discriminate all the tested genotypes more finely than the allozymes (Huh and Ohnishi, 2001). Hence it would be possible to characterize genetic relationships in local natural populations of Porphyra in Korea.
Therefore, the objectives of this study were to determine the amount of genetic diversity in the genus Porphyra and to describe how species-specific markers are distributed among species. Insights into the relative gene diversity within and between wild populations of Porphyra would be useful in plant breeding as well as for development of strategies of ex situ conservation of genetic resources.
Material and Methods
Plant material
Material was obtained from five species of the genus Porphyra: P. tenera, P. yezoensis, P. seriata, P. suborbiculata, and P. dentata in Korea (Table 1). The algae were identified according to Hwang and Lee (2002). Forty plants of each species were randomly gathered from each species during the period from 2004 to 2005. The population of every species was divided into two subpopulations (patches) to examine molecular variation within and between subpopulations. Undaria pinnatifida (Harvey) Suringar was used as the outgroup.
Dna extraction
The genomic DNA of the 240 samples including the outgroup was extracted from fresh blades using the plant DNA Zol Kit (Life Technologies Inc., Grand Island, New York, U.S.A.) according to the manufacturer's protocol.
RAPD ANALYSIS
Twenty arbitrarily chosen 10-mer primers, 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 of individuals, we selected 10 decamer primers that produced RAPD bands in five species in a preliminary test (Table 2).
Amplification reactions were performed in 0.6 ml tubes containing 25 ^l of the reaction buffer; 10 mM Tris-HCl, pH 8.8, 50 mM MgCl2, 100 ^M each of dATP, dCTP, dGTP, dTTP, 0.2 mM primer, 2.1 units Taq DNA polymerase, and 25 ng of genomic DNA. A 100 bp ladder DNA marker (Pharmacia) was used for the estimation of fragment size. 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., USA).
Statistical analyses
All RAPD bands were scored by eye and only unambiguously scored bands were used in the analyses. Since RAPDs are dominant markers, it was assumed that each band corresponded to a single character with two alleles, presence (1) and absence (0) of the band, respectively.
The following genetic parameters were calculated using a POPGENE computer program (ver. 1.31) developed by Yeh et al. (1999): the percentage of polymorphic loci (PP); mean numbers of alleles per locus (A); effective number of alleles per locus (Ae); and gene diversity (H) (Lewontin, 1972). The degree of polymorphism was quantified using Shannon's index (H0) of phenotypic diversity (Bowman et al., 1971).
To analyze the organization of variability within Porphyra, we examined the genetic variation by partitioning the total genetic diversity (HT) into an intraspecies component (HS) and an inter-species one (GST). 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).
To elucidate the extent of genetic divergence between the populations, Nei's genetic identity (GI) and genetic distance (D) were calculated for each pairwise
Table 1. Collection sites of the algae used in the study.
Species Locality of populations
Porphyra tenera P. yezoensis P. seriata P. suborbiculata P. dentata Undaria pinnatifida Myeonggi-dong, Gangseo-gu, Busan Province Heuidong-ri, Jindo-gun, Chonlanam-do Province Samcheon, Sacheon-ci, Gyeongsangnam-do Province Gokyeumdo, Wando-gun, Chonlanam-do Province Jeongdo-ri, Wando-gun, Chonlanam-do Province Jeongdo-ri, Wando-gun, Chonlanam-do Province
combination ofpopulations (Nei, 1973). Homogeneity of variance among species was tested by Bartlett's statistics.
A phylogenetic tree was constructed by the neighbor-joining (NJ) method using the NEIGHBOR program in PHYLIP version 3.57 (Felsenstein, 1993).
Results
From the 20 decamer primers used for a preliminary RAPD analysis, ten primers yielded good amplification products both in quality and variability (Table 2). Overall, 97 fragments were generated among the Porphyra array tested. At the species level, 70 of the 97 loci (72.2%) showed detectable polymorphism in at least one species. Invariant fragments ranged from 1 to 6 per primer.
In a simple measure of intra-species variability by the percentage of polymorphic bands, P. seriata
Table 2. List of decamer oligonucleotide used as primers, their sequences, and associated polymorphic fragments amplified in the Porphyra representatives.
Primer Sequence(5' to 3') Number of bands Number of polymorphic bands Percentage of polymorphic bands
OPD01 ACCGCGAACG 7 4 57.1
OPD02 CGACCCAACC 13 12 92.3
OPD03 GTCGCCGTCA 11 8 72.7
OPD04 TCTGGTGAGG 6 4 66.7
OPD05 TGAGCGGACA 12 6 50.0
OPD06 ACCTGAACGG 10 7 70.0
OPD07 TTGGCACGGG 8 5 62.5
OPD08 GTGTGCCCA 13 10 76.9
OPD09 CTCTGGAGAC 7 5 71.4
OPD10 GGTCTACACC 10 9 90.0
Total 97 70 72.2
exhibited the highest variation (38.1%). P. dentata showed the lowest variation (15.5%) (Table 3). The mean numbers of alleles per locus (A) and effective number of alleles per locus (Ae) revealed the same trends for P. seriata and P. dentata.
The phenotypic frequency of each band was calculated and used to estimate genetic diversity (H) within species. The mean Hwas 0.103 across species, varying from 0.059 to 0.147. Shannon's index of phenotypic diversity (H0) was highest in P. seriata (0.219), and showed significant difference (paired t test). P. dentata had the lowest expected diversity (0.088).
As typical populations of Porphyra are small, isolated, and patchily distributed, they maintain a low level of total genetic diversity (HT = 0.109) (Table 4). The mean HT was 0.109 across species, varying from 0.226 in P. seriata to 0.022 in P. yezoensis. Measures of total genetic variability (paired t test) showed significant difference.
It was shown that most of the genetic variation (64%) resided within species. Inter-species diversity accounted for 36% of the total genetic diversity (Table 4). The average number of individuals exchanged between populations per generation (Nm) was estimated to be very moderate or low (0.926).
Genetic identity (GI) based on the proportion of shared fragments was used to evaluate relatedness among five species. GI ranged from 0.490 to 0.966 (Table 5).
Clustering of species, using the NJ algorithm, was performed based on the matrix of calculated distances (Fig. 1). The phylogenic tree showed three distinct groups: P. dentata and P. yezoensis, P. tenera and P. subor-biculata, and P. seriata. P. seriata is resolved as sister group to P. tenera and P. suborbiculata.
OPD4-01, OPD4-03 and OPD6-
02 loci can be recognized as unique loci of P. seriata. OPE05-03, OPE05-07, and OPE08-07 loci were not found in P. tenera, P. suborbiculata, and P. seriata. Thus, these loci can be used to distinguish the group "P. yezoensis and P. dentata' from other groups. P. yezoensis does not have OPD10-05 locus, so it can be used to distinguish P. dentata from P. yezoensis. P. tenera does not have OPD08-04 locus that is present in P. suborbiculata.
Table 3. Measures of genetic variation for RAPD generated among species. The number of polymorphic loci (Np), percentage of polymorphism (Pp), mean number of alleles per locus (A), effective number of alleles per locus (Ae), gene diversity (H), and Shannon’s information index (Ho).
Species Np Pp A Ae H Ho
P. tenera 22 22.68 1.227 1.165 0.094 0.137
P. yezoensis 32 32.99 1.330 1.214 0.129 0.191
P. seriata 37 38.14 1.381 1.241 0.147 0.219
P. suborbiculata 21 21.65 1.217 1.138 0.084 0.124
P. dentata 15 15.46 1.155 1.097 0.059 0.088
Mean 25.4 26.18 1.262 1.171 0.103 0.151
t-test * * ns * ** **
ns: Not significant; * = p < 0.05; ** = p < 0.01.
Discussion
Genetic diversity and population structure
Estimates of genetic diversity in Porphyra obtained from RAPD analysis can be compared with those obtained from allozyme analysis. One may also try to compare genetic diversity in Porphyra and in other marine algae, though differences between investigations (in the number of loci, populations sampled, and the enzyme systems studied) may preclude meaningful direct comparisons. The percentage of polymorphic loci (Pp) and mean numbers of alleles per locus (A) obtained for Porphyra in the present investigation are 26.2% and 1.3, respectively. Hwang with coauthors analyzed eleven taxa of Porphyra by starch gel electrophoresis. In that study, Pp and A were 21.7 and 1.4, respectively (Hwang et al., 1998). Estimates ofpolymorphism ofboth RAPD and isozyme markers are, therefore, very similar for the genus. It can be concluded that RAPD and isozymes reveal similar patterns of genetic diversity. These markers in Porphyra probably experience similar evolutionary forces.
A striking feature revealed in this study is a high intra-species variation (64%). Taking into account the sexual reproduction and outcrossed mating systems of Porphyra, the mean identity value of 0.664 shown for the five species is higher than it could be expected for
congeneric species (Ham-rick et al., 1992). However, this high value is not especially surprising if we consider that the Porphyra collections were made over a narrow geographic area. Mean genetic identity of the Porphyra species is slightly higher than expected. It is probable that directional movement toward genetic similarity in a relatively homogeneous habitat operates in the populations of Porphyra.
An indirect estimate of gene flow based on GST was 0.926. It is similar to or lower than that in eelgrass, Zostera marina, where it was shown to be 1.1-2.8 (Ruckelshaus, 1998). Z. marina is a perennial angiosperm inhabiting soft-bottom marine habitats, ranging from the intertidal zone to depths of approximately 15 m in temperate latitudes (Den Hartog, 1970). An Nm value greater than 1.0 is considered necessary to prevent divergence resulting from genetic drift (Wright, 1951). Although the level of gene flow in Porphyra is not high enough to counter-balance genetic drift, these values were lower than those obtained for other species (Hamrick et al., 1992).
Phylogenetic relationships within Porphyra
In the generated phylogeny, P. dentata and P. tenera clades are separated from each other, these two species being also distinguishable from each other in blades. The position of P. dentata, P. suborbiculata, and P. seriata on the phylogenetic tree matches very well their classification according to morphological characters. However, our results show P. dentata and P. yezoensis to be closely related, while previous morphological taxonomic studies did not consider these two species as very close relatives. P. dentate is an androdioeious species, whereas P. yezoensis is a monoecious one (Miura, 1988).
Since species of Porphyra show a wide range of morphological and geographical variation, it is difficult
Table 4. Total genetic diversity (HT), intra-species genetic diversity (HS), inter-species genetic diversity (GST) and gene flow (Nm) between the pairs of species.
Locus HT Hs GST Nm
P. dentata 0.066 0.035 0.474 0.556
P. yezoensis 0.022 0.015 0.333 1.000
P. seriata 0.226 0.151 0.333 1.000
P. suborbiculata 0.059 0.042 0.290 1.222
P. tenera 0.172 0.109 0.370 0.852
Mean 0.109 0.070 0.360 0.926
t-test ** ** ns *
ns: Not significant; * = p < 0.05; ** = p < 0.01.
------ P deiilalti
------ P yezoensis
--------- -------------- P tenera
---------- P suborbiculata
--------------- P seriata
---------------------------------- Undaria pinnatif/da
___ 0 01 chances
Fig. 1. A phylogenetic tree of five Porphyra species based on RAPD analysis.
Table 5. Nei’s unbiased genetic identity values of among Porphyra species (above diagonal) and genetic
distances among species (below diagonal).
Locus P. dentata P. yezoensis P. seriata P. suborbiculata P. tenera
P. dentata - 0.737 0.698 0.667 0.556
P. yezoensis 0.305 - 0.652 0.572 0.966
P. seriata 0.572 0.560 - 0.593 0.490
P. suborbiculata 0.360 0.428 0.522 - 0.696
P. tenera 0.384 0.035 0.713 0.362 -
to elucidate phylogenetic relationships using morphological characteristics (Hwang and Lee, 2002). Molecular approaches could be applied for this purpose. At present there is good agreement about the relationships of Porphyra species derived from allozyme analysis (Hwang et al., 1998). Though a small number ofspecies in our study does not allow a detailed discussion of relationships within the Porphyra complex, it shows that further RAPD analysis will certainly provide an enhanced view on the phylogeny of this genus. Additional molecular experiments such as AFLP, SSR, and ITS would be also useful.
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Address for correspondence: Man Kyu Huh. Department of Molecular Biology, Dong-eui University 995 Eomgwangno, Busanjin-gu, Busan 614-714, Korea. E-mail: mkhuh@deu.ac.kr
Editorial responsibility: Alexander Yudin
