The Extent of Clonality and Genetic Diversity in the Rare Caldesia

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Aquatic Botany 84 (2006) 301–307 www.elsevier.com/locate/aquabot

The extent of clonality and genetic diversity in the rare Caldesia grandis (Alismataceae): Comparative results for RAPD and ISSR markers Jin-Ming Chen a, Wahiti Robert Gituru b, Yu-Hang Wang a, Qing-Feng Wang a,* a Laboratory of Plant Systematics and Evolutionary Biology, College of Life Sciences, Wuhan University, Wuhan, 430072 Hubei, PR China b Botany Department, Jomo Kenyatta University of Agriculture and Technology, P.O. Box 62000-00200, Nairobi, Kenya Received 30 April 2005; received in revised form 10 October 2005; accepted 30 November 2005

Abstract Genetic variation and clonal diversity of three natural populations of the rare, highly clonal marsh herb Caldesia grandis Samuelsson were investigated using random ampliﬁed polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers. Both of the markers worked effectively in clone identiﬁcation of C. grandis. RAPD markers detected more diversity than ISSR markers in the three populations examined. Of the 60 RAPD primers screened, seven produced highly reproducible bands. Using these primers, a total of 61 DNA fragments were generated with 52 (85.25%) being polymorphic indicating considerable genetic variation at the species level. Analysis of molecular variance (AMOVA) showed that a large proportion of genetic variation (81.5%) resided within populations, while only a small proportion (18.5%) resided among populations. With the use of 52 polymorphic RAPD markers, we were able to identify 127 genets among 342 samples from three populations. The proportion of distinguishable genets (PD: mean 0.37), Simpson’s diversity index (D: mean 0.91), and evenness (E: mean 0.78) exhibited high levels of clonal diversity compared to other clonal plants. These results imply that sexual reproduction has played an important role at some time during the history of these populations. Nevertheless, the high level of diversity could have been also partially generated from somatic mutations, although this is unlikely to account for the high diversity generally found among C. grandis genets. # 2006 Elsevier B.V. All rights reserved.

Keywords: Caldesia grandis; Clonal structure; Genetic diversity; ISSR; RAPD; Rare plant

1. Introduction 1998). A genet is composed of all tissue originating from one zygote, whereas a ramet is a potentially independent individual In the angiosperms, vegetative propagation is extremely derived from a single genet (Richards, 1986; Eriksson, 1993). widespread and common (Albert et al., 2003). Most perennial For a clonal plant population, the genetically effective ﬂowering plants combine sexual reproduction with some form population size cannot be determined from counting the of asexual reproduction through vegetative propagation, for number of ramets present; what appears to be a ‘‘large’’ example by rhizomes, bulbils, layering, tillering, or rooting of population may be in fact be ‘‘small’’ in terms of genotypes surface runners (Cook, 1983; Richards, 1986; Eckert et al., (Esselman et al., 1999). Populations of clonal plants consisting 1999). Clonal growth is almost ubiquitous in aquatic or wetland of few genets tend to be subject to similar genetic processes that plants (Sculthorpe, 1967; Cook, 1990). affect any small population, such as genetic drift and inbreeding Clonal plants present special problems for the analysis of (Barrett and Kohn, 1991). In order to obtain information about genetic variation in populations because individuals can be population dynamics and evolution in clonal plants, the recognized at two different organizational levels: genets and effective population size cannot be determined just by counting ramets (Kays and Harp, 1974; Harper, 1977; Escaravage et al., ramets; genets as well as ramets must be studied (Eriksson, 1993). Any study of the conservation biology of clonal plants should be concerned with the number of genetic individuals * Corresponding author. Tel.: +86 27 68752869; fax: +86 27 68752869. (genets) found within the ramets of a population (Sipes and E-mail address: [email protected] (Q.-F. Wang). Wolf, 1997; Esselman et al., 1999).

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Caldesia grandis Samuelsson is a perennial, erect marsh by scattered patches of Viburnum macrocephalum. C. grandis herb that belongs to the aquatic family Alismataceae. This in BH population is found in four patches in BH marsh species is confined to mountainous bogs and marshes in (2580703000N; 09883304700E) in Yunnan Province. A total of 342 Southeast Asia and has been found in China and the eastern individuals were sampled for RAPD with 134 from LPH Himalayas (Cook, 1996). C. grandis in China has been reported population, 82 from GH, and 126 from BH. A subset from Hubei, Hunan, Guangdong and Yunnan Provinces in comprising 96 of the 342 individuals were examined for ISSR Mainland China as well as from the island of Taiwan (Gituru markers: 36 from LPH, 12 from GH and 48 from BH. Leaves of et al., 2002). The species is rare, occurring as small populations plants from LPH, GH and BH were collected from different in China. In our recent field investigation, only three natural plots and these plots represent almost the full spatial extent of populations including one population in Yunnan Province and the species within the populations (there are very few two populations in Hunan Province were found in Mainland individuals occurred between the sampled plots). We sampled China. C. grandis is a self-compatible species, which can every individual for each plot in the three populations except for reproduce both sexually by selfed and out-crossed seeds and plots 4 in GH population; in plot 4 of GH population sampled vegetatively through bulbils that commonly occur in the plants grew at least 1.5 m apart (the number of the individuals inflorescences (Gituru et al., 2002). C. grandis produces many in this plot is relative large). flowers and seeds, but seedling recruitment is rarely observed (Gituru et al., 2002). 2.2. DNA extraction Identification of different clones in populations of clonal plants have been greatly facilitated by the use of molecular Total genomic DNA was isolated from 0.5 g of silica-dried markers, such as allozymes (Widen et al., 1994) and leaf tissue following the procedure described by Fu et al. polymerase chain reaction (PCR)-based markers like random (2003). amplified polymorphic DNA (RAPD) (Esselman et al., 1999; Persson and Gustavsson, 2001; Hangelbroek et al., 2002; Albert 2.3. RAPD amplification et al., 2003), inter-simple sequence repeat (ISSR) (Esselman et al., 1999; Li and Ge, 2001) and amplified fragment length Reactions were carried out in a volume of 25 ml containing polymorphism (AFLP) (Albert et al., 2003; Escaravage et al., 0.25 mmol/l each of dNTP, 2.5 mlof10 Taq buffer 1998; Suyama et al., 2000). Although allozymes analysis has [10 mmol/l Tris–Hcl (PH 8.3), 1.5 mM MgCL2 and 50 mM long been used to identify clones and to study population KCL], 1 mmol/l primer, 1 U Taq Polymerase (Tian Yuan genetics of clonal plants, it usually underestimates genetic Biotech) and 40 ng of DNA template. Amplification of polymorphism and has a limited ability to distinguish genetic genomic DNA was made on a PTC-100TM thermocycler (MJ individuals (Esselman et al., 1999; Wang et al., 1999). The Research, Inc.), and commenced with 4 min at 94 8C, followed PCR-based DNA markers evolve rapidly enough to be variable by 45 cycles of 1 min at 94 8C, 1 min annealing at 34 8C, and within a population, thus they are suited for detecting genotypic 2 min extension at 72 8C, and a final extension cycle of 7 min at diversity (Esselman et al., 1999). In the present study we 72 8C. Amplification products were resolved electrophoreti- employ RAPD and ISSR markers (1) to genetically identify C. cally on 1.5% agarose gels run at 100 V in 0.5 TBE (Tris– grandis clones as well as to estimate the diversity of these boric acid–EDTA), visualized by staining with ethidium clones; (2) to estimate the genetic diversity in C. grandis bromide, and photographed under ultraviolet light. Sixty populations; (3) to partition the genetic diversity among and RAPD primers from Genbase Co. Ltd. (Shanghai, China) were within populations. screened on six randomly selected individuals. The six samples were amplified twice with the same primer. Seven primers that 2. Materials and methods produced clear and 100% reproducible fragments were selected for further analysis (Table 2). 2.1. Study sites and sampling 2.4. ISSR amplification During July and August 2004, three wild populations of C. grandis occurring in three marshes Lang Pan Hu, Guai Hu, and PCR reactions were conducted in volumes of 25 ml Bei Hai (referred to as LPH, GH and BH populations) in Hunan containing 0.25 mM each of dNTP, 2.5 mlof10 Taq buffer and Yunnan Provinces in China were sampled. In Hunan [10 mmol/l Tris–Hcl (PH 8.3), 1.5 mM MgCL2 and 50 mM Province, C. grandis is found in two populations (LPH and GH) KCL], 1 mM primer, 1U Taq polymerase (Tian Yuan Biotech) occurring in two marshes close to the center of Mangshan and 60 ng of DNA template. A PTC-100TM thermocycler (MJ Nature Reserve in Hunan Province. The LPH marsh Research) was used with the thermocycle program set at: 94 8C (2485200000N; 11284301900E) is densely covered with a thick for 2 min, followed by 35 cycles of 30 s at 94 8C, 1 min at mat comprised mainly of the peat moss Sphagnum cuspidatum 55 8C, 1.5 min at 72 8C, and ending with 7 min at 72 8C. and the fern Cyclosorus acuminatus. A sluggish stream flows Amplification products were electrophoretically resolved on across the marsh and the marsh is subdivided by patches of the 1.5% agarose gels run at 100 V in 0.5 TBE (Tris–boric acid– bamboo Sinarundinaria nitida. The GH marsh (2580301200N; EDTA) buffer. Gels were stained with ethidium bromide. Sixty- 11380001000E) is about 8 km from LPH marsh and is subdivided five ISSR primers (SBS Genetech. Co. Ltd.) were screened 中国科技论文在线 http://www.paper.edu.cn

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twice on six samples of C. grandis for unambiguous, clear and Table 1 100% reproducible band patterns. Five primers were selected Clonal diversity in three populations of Caldesia grandis (N = sample size; G = number of genotypes; PD = proportion of distinguishable genets; for further analysis (Table 2). D = Simpson index; E = Fager index)

2.5. Data analysis NG PD DE LPH 134 46 0.34 0.80 0.56 RAPD and ISSR bands were scored as present (1) or absent GH 82 46 0.56 0.95 0.79 (0) for each sample. For both RAPD and ISSR data, the Jaccard BH 126 38 0.30 0.97 0.98 Mean – 43.3 0.37 0.91 0.78 coefficient was employed to calculate pairwise band simila- Species 342 127 0.37 0.96 0.91 rities for samples using the program NTSYSpc 2.02 (Rohlf, 1998). A group of samples showing identical band patterns were considered as belonging to the same genet. population. Three genotypes (clone 20, clone 44, clone 71) were Four measures of genotypic diversity were calculated present in both LPH and GH populations. The LPH population (Fager, 1972; Ellstrand and Roose, 1987): (1) G, the number contained one large clone (clone 3), which consist of 59 ramets of genotypes detected; (2) PD, the proportion of distinguishable and is distributed in four plots (Plots 1–4) (Fig. 1). All BH genets genets, PD = G/N; (3) Simpson’s index of diversity corrected were unique to this marsh. From the five ISSR primers, 22 for finite sample size (Pielou, 1969): D =1 [Ni (Ni 1)/ polymorphic markers were generated (Table 2). A total of 25 (N 1)], where Ni is the number of samples of the ith different genotypes were observed among the 96 samples genotypes; (4) The genotypic evenness (Fager, 1972), analyzed. When RAPD and ISSR markers were combined, no E =(D Dmin)/(Dmax Dmin), where Dmin =(G 1)(2N additional multilocus genotype was detected. The results showed G)/N (N 1) and Dmax =(G 1)N/G(N 1). that the ISSR markers were not more informative than RAPD Genetic diversity was measured as the percentage of markers. For this reason, the subsequent analyses were only polymorphic loci (PPL) at the plot, the population and the carried out with the RAPD data. species level and as the Shannon’s diversity index (I) Based on RAPD data analyses, the proportion of distin- (Lewontin, 1972) using POPGENE program 1.31 (Yeh et al., guishable genotypes was 0.34 for the LPH population, 0.56 for 1997). the GH population and 0.30 for the BH population (mean Analysis of molecular variance (AMOVA) was performed PD = 0.37). The values of D and E were 0.80 and 0.56 for the using squared Euclidean distances (Excoffier et al., 1992) LPH population, 0.95 and 0.79 for the GH population, and 0.97 among all plants and among all genets to partition the variation and 0.98 for the BH population (mean D = 0.91, E = 0.78) into hierarchical components. Variance was apportioned to the (Table 1). following components: among populations, among plots and UPGMA cluster analyses revealed most genets did not group within populations/plots. The AMOVA was performed at the by plots (Fig. 2) and there was no distinct genetic differentiation ramet-level, thus including all samples, as well as at the genet- between populations. level where each genet was represented only once. At the genet- level one copy of a genet (ramet) represented the genet. This 3.2. Genetic diversity and structure copy was taken from the plot where it was most abundant, or when more than one plot had equally high numbers of copies it A total of 61 bands ranging in size from 100 to 1800 bp were was taken at random from one of these plots. The Genetic obtained. Of all loci observed, 85.25% (PPL) were polymorphic analyses were performed with WINAMOVA program 1.55 in the 342 individuals investigated, the Shannon’ index (I) was (Excoffier, 1993). Input files for this program were generated using AMOVA-PREP (Miller, 1998). Significance tests were Table 2 performed using 1000 permutations. Number of bands scored from RAPD and ISSR primers A clustering analysis of all genets was done using the Primers Number of Number of unweighted pair group method with an arithmetic average scorable bands polymorphic loci (UPGMA) using NTSYSpc 2.02 (Rohlf, 1998). RAPD 50-CCTGGGTGGA-30 88 3. Results 50-CCTGGGTTTC-30 97 50-GCCCGGTTTA-30 11 10 0 0 3.1. Clonal diversity 5 -CTCCCTGAGC-3 74 50-TTCCCGGAGC-30 10 9 50-TTCCGGGTGC-30 88 We regard ramets with the same RAPD or ISSR profile as 50-TTCCGCGGGC-30 86 having the same genotype and belonging to the same clone. ISSR 0 0 From the seven RAPD primers, 52 polymorphic markers were 5 -(AC)8T-3 12 4 0 0 generated (Table 2). A total of 127 different genotypes or clones 5 -(AC)8C-3 12 5 0 0 were identified among the 342 samples analyzed (Table 1). Of 5 -(AC)8G-3 12 5 0 0 these, 38 genotypes were specific to the BH population, 43 were 5 -(AT)8(CG)C-3 10 2 50-(AG) (CG)T-30 14 6 specific to the LPH population and 43 were specific to the GH 8 中国科技论文在线 http://www.paper.edu.cn

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Fig. 1. Spatial distribution of the 127 clones of Caldesia grandis at the sampling sites. Samples belonging to the same clone are represented by the same number. The ramets representing the different clones are placed randomly.

0.243. Genetic diversity varied among populations with PPL showed less variation (0.116) (Table 3). The PPL values among values ranging from 40.98% in BH to 60.66% in LPH (Table 3). plots ranged from 4.92% (Plot 1 in LPH) to 49.18% (Plot 4 in The Shannon’ index (I) indicated that the GH population GH) and the Shannon’ index ranged from 0.031 (Plot 1 in LPH) had the greatest variation (0.202), while the LPH population to 0.216 (Plot 3 in GH). 中国科技论文在线 http://www.paper.edu.cn

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Table 3 RAPD analysis of genetic diversity in natural populations of C. grandis (numbers in parentheses are standard deviations; PPL: percentage of polymorphic loci; H: Nei’s gene diversity; I: Shannon’s information index) Population Number of PPL HI polymorphic loci LPH population 37 60.66 0.1096 (0.1173) 0.116 (0.1866) Plot 1 3 4.92 0.0219 (0.0969) 0.0313 (0.1388) Plot 2 10 16.39 0.0401 (0.0914) 0.0672 (0.1531) Plot 3 6 9.84 0.0389 (0.1198) 0.0575 (0.1761) Plot 4 29 47.54 0.1178 (0.1481) 0.1915 (0.2269) Plot 5 27 44.26 0.1210 (0.1515) 0.1942 (0.2337) GH population 32 52.46 0.1244 (0.1556) 0.2018 (0.2319) Plot 1 12 19.67 0.0589 (0.1248) 0.0930 (0.1936) Plot 2 17 27.87 0.0870 (0.1497) 0.1356 (0.2271) Plot 3 26 42.62 0.1437 (0.1922) 0.2164 (0.2767) Plot 4 30 49.18 0.1210 (0.1607) 0.1944 (0.2367) BH population 25 40.98 0.1178 (0.1657) 0.1843 (0.2465) Plot 1 10 16.39 0.0674 (0.1578) 0.0980 (0.2268) Plot 2 5 8.20 0.0367 (0.1253) 0.0523 (0.1777) Plot 3 15 24.59 0.0965 (0.1788) 0.1417 (0.2571) Plot 4 19 31.15 0.0783 (0.1442) 0.1250 (0.2145) Species level 52 85.25 0.1368 (0.1184) 0.2416 (0.1736)

According to the AMOVA, most of the variance was found 4. Discussion between individuals within plots 72.5%, a small but signiﬁcant amount among plots (13.1%) and among populations (14.4%). Both the RAPD and ISSR molecular markers have been used Within-population variability accounted for 81.5% of the in population genetic studies and in detecting clonal diversity molecular variation when carried out at the ramet level and (Li and Ge, 2001; Esselman et al., 1999; Parsons et al., 1997). 75.1% at the genet level (Table 4). Researchers who have compared RAPD and ISSR methods have found that ISSR markers exhibit higher levels of polymorphism or reproducibility compared with RAPD markers (Fang and Roose, 1997; Esselman et al., 1999; Parsons et al., 1997; Qian et al., 2001). The results of the present study showed that the ISSR markers were not more

Table 4 Analyses of molecular variance (AMOVA) of RAPD data from three C. grandis populations, considered at the ramet level and genet level (d.f. = degree of freedom; The P values for the corresponding F-tests were all signiﬁcant (P < 0.001)) Source of variation d.f. Variance % of total component variance Ramet level Among all populations 2 0.64 14.4 Among plots within 10 0.59 13.1 populations Within plots 130 3.25 72.5 Among all populations 2 0.83 18.5 Within all populations 140 3.65 81.5 Among all plots 12 1.07 24.7 Within all plots 130 3.25 75.3

Genet level Among all populations 2 0.65 13.7 Among plots within 10 0.65 13.7 populations Within plots 114 3.43 72.6 Among all populations 12 1.14 24.9 Fig. 2. Dendrogram of 38 clones of C. grandis in BH population based on Within all populations 114 3.43 75.1 RAPD markers generated using the UPGMA clustering method. 中国科技论文在线 http://www.paper.edu.cn

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informative than RAPD markers; however, both of the markers colonization event exhibit a rapid loss of genets, resulting in a worked effectively in detecting clones in C. grandis popula- few large clones. Our survey of three populations of C. grandis tions. The ISSR markers provided an opportunity for checking showed that very few large clones occurred in the populations the power of the RAPD markers to identify clones. and that most of the clones (52%, a total of 66 clones) consist of The genotypic diversity values obtained for C. grandis are only a single ramet (Fig. 1). Therefore it seems probable that in comparable to those recorded for other clonal plants. The these populations repeated seedling recruitment (RSR, Eriks- proportion of genotypes detected (mean PD = 0.37), the son, 1993) occurs and that it may even be frequent in these genotypic diversity (mean D = 0.91) and the evenness value populations. Nevertheless, the high level of diversity may also (mean E =0.78)inC. grandis were, however, all higher than be partially maintained by the initial seedling recruitment (ISR, the clonal plant average (PD = 0.17, D =0.62andE =0.68) Eriksson, 1993). After an initial establishment only a few (Ellstrand and Roose, 1987). However, it is noteworthy that seedlings in a short period is sufficient to maintain or increase most of the studies from which the clonal plant average were clonal diversity (Soane and Watkinson, 1979; Watkinson and derived were based on a small number of allozyme loci, which Powell, 1993). may account partially for their lower D,PDandE values Another possible explanation for the clonal diversity found (Hangelbroek et al., 2002; Persson and Gustavsson, 2001). The in C. grandis populations is somatic mutations. Clonal plants estimates of genotypic diversity werewithin the range of those can be very long-lived thus somatic mutations may eventually values obtained in studies on clonal plants using RAPD lead to some variation (Persson and Gustavsson, 2001). Some markers (mean PD = 0.44 and D = 0.74, see Hangelbroek et al., researchers have considered RAPD markers to represent 2002). segments of DNAwith non-coding regions and to be selectively It was assumed that genetic diversity was lower for clonal neutral (Bachmann, 1997; Landergott et al., 2001). Similar plants than for non-clonal plants (Harper, 1977). However, a opinions have been reported with regard to ISSR markers growing body of data indicated that populations of clonal plants (Esselman et al., 1999). Some studies have shown that RAPD could maintain considerable amounts of genetic diversity markers are found throughout the genome and may be (Ellstrand and Roose, 1987; Hamrick and Godt, 1989; Eckert associated with functionally important loci (Penner, 1996). and Barrett, 1993; Widen et al., 1994). Our survey of three However, there is little information to indicate that ISSR populations of C. grandis revealed a high level of genetic markers are functionally important, consequently, the ISSR variation at the species level with 85.25% of loci being bands may be expected to evolve at a fast evolutionary rate and polymorphic. AMOVA analysis showed that most of the genetic that owing to the resultant hypervariability they may provide variation (81.5%) was resided within populations, and only a higher estimates of genetic diversity in a population compared small proportion of the variation (18.5%) was resulted from to RAPD markers (Esselman et al., 1999). Results of the present differentiation among populations. The pattern of genetic study, however, indicated that, although both markers were variation found among C. grandis populations appear to be effective, greater RAPD diversity was observed compared to similar those studies on long-lived, late-successional species ISSR diversity. Therefore, the molecular marker variation that have a mixed or outcrossing breeding system and observed is more likely a reflection of genetic diversity dependent on animals or wind for their pollination (Hamrick generated by sexual reproduction (recombination) rather than and Godt, 1996; Bartish et al., 1999; Nybom and Bartish, 2000). somatic mutations. The high diversity found among C. grandis Field observation indicates that C. grandis can produce genets can, therefore, not be attributed to somatic mutations many flowers from early July to late September with a peak in alone. August and the effective pollinators were bees (Insecta; The insights gained from exploring the genetic variation of Hymenoptera). Both pollen viability and the seed set in open- populations of rare species are important in developing a sound pollinated controls were high (65.44% and 71.78%, respec- program for protecting them. For a species with only 20% tively, Gituru et al., 2002). However, no seed germination was variation residing among populations, the samples taken from observed. The failure of the seeds of C. grandis to germinate two populations are adequate to capture 95% of the total genetic was attributed probably to the existence of highly specific diversity of the species (Hamrick et al., 1991). AMOVA germination requirements and also the presence of fungal analysis in the present study showed that almost as much growth that frequently occurred on the mature fruits (Gituru variation exists within plots as that in the whole population, also et al., 2002). Although seedlings are very rarely observed in as much variation exists among plots within populations as populations of C. grandis, the genetic variation revealed in this among populations, thus each population or plot contains a lot study pattern among C. grandis populations is similar to results of genetic diversity. Damage to the habitats is a main reason for of studies on other non-clonal, outcrossing plants as well as their rarity of many plant species; it is therefore, a good other clonal plant species with low levels of seedling conservation strategy to protect more of these habitats. recruitment (Jonsson et al., 1996; Persson and Gustavsson, Fortunately, all the extant C. grandis populations in China 2001). This indicates that sexual reproduction has played an are located in gazzeted Natural Reserves, and which has so far important role at some time during the history of the protected the populations from damage. However, there is an populations. urgent need to pay more attention to the diminishing the According to Watkinson and Powell (1993) populations in population sizes that can potentially result in genetic drift and which there is no further seedling recruitment after an initial inbreeding. 中国科技论文在线 http://www.paper.edu.cn

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