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Aquatic Botany 87 (2007) 1–6 www.elsevier.com/locate/aquabot

A comparison of the extent of genetic variation in the endangered natans and its widespread congener S. trifolia Jin-Ming Chen a, Wahiti Robert Gituru b, Qing-Feng Wang a,* a Laboratory of Systematics and Evolutionary Biology, College of Life Sciences, Wuhan University, Wuhan 430072, Hubei, PR b Botany Department, Jomo Kenyatta University of Agriculture and Technology, P.O. Box 62000-00200, Nairobi, Kenya Received 13 May 2006; received in revised form 28 November 2006; accepted 1 December 2006

Abstract Genetic variation and clonal diversity of 14 populations of the endangered clonal herb Sagittaria natans and its widespread congener S. trifolia were investigated using inter-simple sequence repeat (ISSR) markers. Using nine effective ISSR primers, a total of 92 DNA fragments were generated with 54 (percentage of polymorphic loci, PPL: 58.7%) being polymorphic. A higher level of genetic diversity among populations was found in S. natans (PPL: 48.9%) than S. trifolia (PPL: 32.6%). Analysis of molecular variance (AMOVA) showed that in each species a similar proportion of the total genetic variation resided within and among populations, and that between species there was a moderate genetic differentiation (Gst: 0.601). With the use of 54 polymorphic ISSR markers, we identified 116 genets among 138 samples from five S. natans populations, and 93 genets among 215 samples from nine S. trifolia populations. The proportion of distinguishable genets (PD: mean 0.82) and Simpson’s diversity index (D: mean 0.95) for S. natans exhibited higher degree of clonal diversity compared to S. trifolia (PD: mean 0.42; D: mean 0.89). Sexual reproduction might have been played an important role in maintaining and increasing the clonal diversity in both species. Recent and on-going decimation of S. natans populations in the region appear not to have had a major impact on genetic diversity in this rare plant. # 2007 Published by Elsevier B.V.

Keywords: Clonal structure; Genetic diversity; ISSR; Population differentiation; Sagittaria

1. Introduction one potential limitation of these studies is that the included taxa are treated as independent samples, ignoring their phylogenetic As more species dwindle toward extinction, the rare and relatedness (Gitzendanner and Soltis, 2000). As a result, narrowly distributed species have in recent years received more growing interest in population genetic and conservation biology attention especially with regard to their viability (Gitzendanner has directed attention to comparisons of widespread and and Soltis, 2000). The survival of a species depends not only on restricted congeneric species (Karron et al., 1988; Soltis and habitat and geographic factors but is ultimately linked to the Soltis, 1991; Lewis and Crawford, 1995; Ge et al., 1999; He genetic variation available to a species (Dodd and Helenurm, et al., 2000; Dodd and Helenurm, 2002; Park, 2004). Closely 2002), thus knowledge of the genetic aspects of rarity within related species are likely to share many life-history features due rare and endangered species is critical to their conservation and to recent common ancestry, making it easer to identify the management (Schaal et al., 1991; Hamrick and Godt, 1996). causes of differences in genetic diversity (Dodd and Helenurm, Several previous surveys of genetic variation in have 2002). Recent comparative studies of genetic variation between shown that rare, endemic, or narrowly distributed plants tend to rare and widespread species have demonstrated that several rare maintain low degree of genetic variability due to the impact of species are as polymorphic as their widespread congeners genetic drift, the founder effect, and directional selection with (Gitzendanner and Soltis, 2000; Dodd and Helenurm, 2002). high levels of inbreeding (Franklin, 1980; Karron, 1987; Thus, it is difficult to make generalizations that species with Hamrick and Godt, 1989; Ellstrand and Elam, 1993). However, limited geographic range always have low genetic diversity. Sagittaria trifolia is a perennial, erect marsh herb that belongs to the aquatic family . Sagittaria trifolia * Corresponding author. Tel.: +86 27 68752869; fax: +86 27 68752560. is one of the most widespread species in the genus Sagittaria E-mail address: [email protected] (Q.-F. Wang). ranging from north Beikal in Russia to Southeast and

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Southeast Europe (Chen, 1989). Sagittaria natans is a float- Table 1 leaved plant species and is confined to bogs, rivers, and streams Locations of the populations of S. natans and S. trifolia sampled in this study in Northeast Europe and Northeast Asia (Chen, 1989). Liaoning Populations Location Latitude (N)/Longitude (E) Province of China is the southern-most location of S. natans.In S. natans China S. natans has been reported from Liaoning, Jilin, and EKH Erkahe, Inner Mongolia 49830009.700/117845010.000 Helongjiang provinces and also in Inner Mongolia (Chen, SQ Shiquan, Helongjiang 4983201400/120840028.900 1989). However, in our recent field investigation, only five DH Dunhua, Jilin 43820003.900/128813022.800 0 00 0 00 natural populations including one population in Jilin Province, WSL Wusuli, Helongjiang 46848 32.0 /134800 54.0 LHP Lianhuapao, Helongjiang 46844040.800/134801023.000 one population in Inner Mongolia and three populations in Helongjiang Province were found in China. Sagittaria natans is S. trifolia LDS Liudingshan, Jilin 43819026.900/128813050.600 now considered to be rare and threatened or endangered in MS Mishan, Helongjiang 45830040.000/131851020.000 China and is listed among the second category of the key HL Hulin, Jilin 45847035.000/133835045.300 protected wild plants (Yu, 1999). Both S. natans and S. trifolia MJG Majiagang, Helongjiang 45827004.000/132816053.400 are self-compatible species, which can reproduce both sexually XKH Xingkaihu, Helongjiang 45827004.000/13281604000 0 00 0 00 by selfed and out-crossed seeds and vegetatively through corms BC Bacha, Helongjiang 47825 47.0 /133818 32.0 TJ Tongjiang, Helongjiang 48820056.400/134824059.400 (Chen, 1989). Phylogenetic studies of Sagittaria species using RH Raohe, Helongjiang 46844038.800/134801022.000 morphological and molecular data found that S. trifolia and S. DJC Dongjingcheng, Helongjiang 44806054.000/129811024.000 natans are sister species (Chen, 1989; Chen and Wang, unpublished data). In recent years, inter-simple sequence repeats (ISSRs) have powdered in liquid nitrogen, mixed with 2 mL extraction buffer been successfully employed to reveal genetic diversity and [1.4 M NaCl, 100 mM Tris–HCl (pH 8.0), 20 mM EDTA, 2% identify the different clones in populations of clonal plants (V/V) CTAB, and 2% 2-mercaptoethanol] at 65 8C, and (Esselman et al., 1999; Li and Ge, 2001; Chen et al., 2006). The incubated at 65 8C for 30 min with gentle shaking every 5 min. principal goal of this study was to (1) investigate and compare the Proteins were extracted twice with 2 mL of chloroform: amount and distribution of genetic diversity in the endangered isoamylalcohol (24:1), then centrifuged at 10,000 g for species S. natans with its widespread congener S. trifolia using 2 min. RNase (10 mgmL1) was added to the supernatant and ISSR markers, (2) to genetically identify S. trifolia and S. natans incubated for 2 h at 37 8C. The mixture was centrifuged at clones as well as to estimate the genetic diversity of these clones. 10,000 g for 2 min. The sediment was washed twice in 70% This information was intended to contribute to a better ethanol, air-dried, and resuspended in 100 mL 0.1 TE, and understanding of the causes of rarity of S. natans. then stored at 20 8C.

2. Materials and methods 2.3. ISSR PCR amplification

2.1. Plant materials Reactions were carried out in a volume of 25 mL containing 0.25 mM of each dNTP, 2.5 mLof10 Taq buffer [10 mM The plant specimens of S. natans used in this investigation Tris–HCl (pH 8.3), 1.5 mM MgCl2 and 50 mM KCl], 1 mM were obtained from five populations throughout the natural primer, 1 U Taq Polymerase (Tian Yuan Biotech) and 40 ng of range of the species in China. In each of the five populations, a DNA template. Amplification of genomic DNA was made on a sample of 19 or more plants was taken. A total of 138 PTC-100TM thermocycler (MJ Research, Inc.), and commenced individuals of S. natans from the five populations were included with 4 min at 94 8C, followed by 35 cycles of 1 min at 94 8C, in the study. For S. trifolia, a total of 215 individuals from nine 1 min annealing at 55 8C and 2 min extension at 72 8C, and a populations (18 or more individuals were sampled in each final extension cycle of 7 min at 72 8C. Amplification products population) in Northeast China that were in sympatric to S. were resolved electrophoretically on 1.5% (W/V) agarose gels natans populations were sampled. Details on collection site are run at 100 V in 0.5 Tris–boric acid–EDTA (TBE), visualized given in Table 1. About 5 g of fresh leaves was harvested from by staining with ethidium bromide, and photographed under each plant and immediately dried in a ziplock plastic bag ultraviolet light. Sixty-five ISSR primers (SBS Genetech. Co. containing about 70 g of silica gel. To estimate genetic Ltd., Shanghai, China) were screened on eight randomly variability within populations we only used plants sampled at selected individuals (four samples of each species). The eight least at 3-m distances from each other. The samples were stored samples were amplified twice with the same primer. Nine at room temperature until DNA was isolated in the laboratory at primers that produced clear and 100% reproducible fragments Wuhan University. were selected for further analysis (Table 2).

2.2. Total DNA extraction 2.4. Data analysis

Total genomic DNA was isolated from 0.5 g of silica-dried ISSR bands were scored as present (1) or absent (0) for each leaf tissue using a modification of the CTAB extraction sample. The Jaccard coefficient was employed to calculate procedure of Doyle and Doyle (1987). Leaf material was pairwise band similarities for samples using the program 中国科技论文在线 http://www.paper.edu.cn

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Table 2 Table 3 Name of primers and sequences of nine effective ISSR primers used in the Clonal diversity in populations of S. natans and S. trifolia (N = sample size; present study G = number of genotypes; PD = proportion of distinguishable genets; D = Simpson index; E = Fager index) Primers Sequences (50–30) Population NGPD DE SBS808 (AG)8C SBS809 (AG)8G S. natans SBS810 (AC)8T EKH 24 22 0.92 0.99 0 SBS812 (AC)8G SQ 24 6 0.25 0.75 0.76 SBS817 (AT)8G DH 19 19 1.0 1.0 0 SBS826 (GA)8T WSL 48 47 0.98 1.0 0 SBS827 (GA)8C LHP 23 22 0.96 1.0 0 SBS834 (AT)8(CG)C Mean – – 0.82 0.95 0.15 SBS840 (TA)8(AT)G Species level 138 116 0.84 0.99 0.67 S. trifolia LDS 26 9 0.35 0.87 0.88 NTSYSpc 2.02 (Rohlf, 1998). A group of samples showing MS 24 14 0.58 0.94 0.82 HL 26 11 0.42 0.91 0.88 identical band patterns were considered as belonging to the MJG 26 15 0.58 0.93 0.76 same genet. Four measures of genotypic diversity were XKH 22 7 0.26 0.89 0.98 calculated (Fager, 1972; Ellstrand and Roose, 1987): (1) G, BC 25 16 0.64 0.95 0.77 the number of genotypes detected; (2) PD, the proportion of TJ 22 7 0.32 0.84 0.86 distinguishable genets, PD = G/N; (3) Simpson’s index of RH 26 6 0.23 0.80 0.87 DJC 18 8 0.44 0.89 0.86 diversityP corrected for finite sample size (Pielou, 1969): Mean – – 0.42 0.89 0.85 D ¼ 1 ½NiðNi 1Þ=NðNi 1Þ , where Ni is the number of Species level 215 93 0.43 0.99 1.0 samples of the ith genotypes; (4) The genotypic evenness (Fager, 1972), E =(D Dmin)/(Dmax Dmin), where Dmin = (G 1)(2N G)/N(N 1) and Dmax =(G 1)N/G(N 1). S. trifolia populations. Based on ISSR data analyses, the Genetic diversity was measured as the percentage of proportion of distinguishable genotypes ranged from 0.25 to 1.0 polymorphic loci (PPL) at the population and the species (mean PD = 0.82) for S. natans populations and from 0.23 to level and as Nei’s gene diversity (Nei, 1978) and the Shannon’s 0.64 for S. trifolia populations (mean PD = 0.42). For the S. diversity index (I)(Lewontin, 1972) using POPGENE program natans populations the values of D ranged from 0.75 to1.0 and 1.31 (Yeh et al., 1997). the values of E ranged from 0 to 0.76 (mean D = 0.95, Analysis of Molecular Variance (AMOVA) was performed E = 0.15). For the S. trifolia populations the values of D ranged using squared Euclidean distances (Excoffier et al., 1992) from 0.80 to 0.99 and the values of E ranged from 0.76 to 0.98 among all samples to partition the variation into hierarchical (mean D = 0.89, E = 0.85) (Table 3). components (between or within species, among or within populations for each species). The genetic analyses were 3.2. Genetic diversity and structure performed with WINAMOVA program 1.55 (Excoffier, 1993). Input files for this program were generated using AMOVA- A total of 92 bands ranging in size from 100 to 1800 bp were PREP (Miller, 1998). Significance tests were performed using obtained. Of all loci observed, 48.9% (PPL) were polymorphic 1000 permutations. in the 138 individuals of S. natans and 32.6% were polymorphic To further investigate phenetic relationships among popula- in the 215 individuals of S. trifolia. Genetic diversity varied tions or among individuals, the binary ISSR matrix was used to among populations with PPL values ranging from 4.4% to compute pairwise band similarity coefficients of Nei (1978). 41.3% in S. natans populations and from 7.6% to 18.5% in S. Clustering analysis of all populations and all individuals were trifolia populations (Table 4). The Shannon’ index (I) indicated performed using the unweighted pair group method with an that the WSL population had the greatest variation (0.197), arithmetic average (UPGMA) using NTSYSpc 2.02 (Rohlf, while the SQ population showed least variation (0.039) for S. 1998). natans (Table 4). In S. trifolia populations the I values among populations ranged from 0.028 in the RH population to 0.101 in 3. Results the MS population. The Nei’s gene diversity index (H) displayed similar trends to those of the PPL values both at 3.1. Clonal diversity population and species level in the two species (Table 4). AMOVA revealed that in S. natans a greater proportion of We regard ramets with the same ISSR profile as having the the genetic variance resided between individuals within same genotype and belonging to the same clone. From the nine populations (51.4%), while a smaller proportion (48.6%) ISSR primers, 54 polymorphic markers were generated. A total resided among populations. For S. trifolia, within-population of 209 different genotypes or clones were identified among the variability accounted for 50.6% of the molecular variation 353 samples analyzed (Table 3). Of these, 116 genotypes were while among-population variation accounted for 49.4% specific to S. natans populations and 93 were specific to (Table 5). An examination of the proportion of diversity 中国科技论文在线 http://www.paper.edu.cn

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Table 4 Genetic diversity in populations of S. natans and S. trifolia (numbers in parentheses are standard deviations; PPL = percentage of polymorphic loci; I = Shannon’s information index; H = Nei’s gene diversity) Population No. of PPL HI polymorphic (%) loci S. natans EKH 14 15.2 0.058 (0.143) 0.086 (0.209) SQ 4 4.4 0.016 (0.082) 0.039 (0.112) DH 19 20.7 0.059 (0.134) 0.092 (0.199) WSL 38 41.3 0.129 (0.181) 0.197 (0.262) LHP 32 34.8 0.108 (0.173) 0.164 (0.255) Species level 45 48.9 0.150 (0.191) 0.228 (0.274) S. trifolia LDS 16 17.4 0.056 (0.134) 0.085 (0.198) MS 17 18.5 0.068 (0.156) 0.101 (0.224) Fig. 1. UPGMA dendrogram of Nei’s (1978) genetic distance between popula- HL 11 11.9 0.032 (0.099) 0.051 (0.150) tions and species of Sagittaria. MJG 14 15.2 0.044 (0.116) 0.070 (0.175) XKH 9 9.8 0.033 (0.105) 0.051 (0.157) BC 15 16.3 0.049 (0.117) 0.076 (0.180) the proportion of genotypes detected in S. natans (mean TJ 9 9.8 0.036 (0.118) 0.053 (0.170) PD = 0.82) and the genotypic diversity (mean D = 0.95) were RH 7 7.6 0.017 (0.071) 0.028 (0.109) DJC 7 7.6 0.030 (0.107) 0.044 (0.156) higher than the average values for S. trifolia (mean PD = 0.42, Species level 30 32.6 0.082 (0.151) 0.129 (0.223) mean D = 0.89). Hangelbroek et al. (2002) estimated the expected genotypic diversity parameters (PD and D)in herbaceous clonal plants to be 0.08–0.94 (mean PD: 0.44) between species indicated that a somewhat greater amount of and 0.00–1.00 (mean D: 0.74), respectively. In the present study variation (60.1%) occurred between species whereas 39.9% the genotypic diversity (mean PD and D values) detected in S. occurred within species. trifolia were closely similar to the estimated values for A cluster analysis (UPGMA) indicated that except for herbaceous clonal plant species. For S. natans the levels of individuals from DH population that formed a separate cluster, genotypic diversity (mean PD and D values) detected in this the individuals from the other four S. natans populations (EKH, study were higher than the estimated values for herbaceous WSL, LHP, DH) clustered together. Individuals from the nine clonal plant species. populations of S. trifolia clustered together. While those from Field observations indicated that both S. natans and S. the two Sagittaria species clustered distinctly separate from trifolia produced many flowers from early July to late each other (data not presented). The UPGMA dendrogram of September with a peak in August and that the effective Nei’s (1978) genetic distance between populations separated pollinators were bees (Insecta; Hymenoptera). Both pollen the two species at a genetic distance of 0.23 (Fig. 1). viability and seed set in open-pollinated controls were high. Seed germination was observed in the field and also in the 4. Discussion laboratory (Chen and Wang, unpublished data). Our survey of a total of fourteen populations of both S. natans and S. trifolia The genotypic diversity values obtained in this study for S. showed that most of the clones consist of only a single ramet; natans are comparable to those obtained for the nine moreover only very few large clones were detected in these populations of its widespread congener S. trifolia. However, populations. In the absence of further seedling recruitment a rapid loss of genets resulting in a few large clones would be Table 5 expected to follow an initial colonization event (Watkinson and Analysis of molecular variance for individuals of S. natans and S. trifolia using Powell, 1993). Therefore, it seems probable that repeated ISSR markers (significance tests after 1000 permulations) seedling recruitment (RSR, Eriksson, 1993) occurs in these Source of variation Variance % of total P-value populations and that it may even be frequent. Observations of component variance successful seed germination both in the field and in the S. natans laboratory (Chen and Wang, unpublished data) further attests to Among populations 3.78 48.6 <0.001 the presence of seedling recruitment as a feature in the Within populations 4.00 51.4 <0.001 propagation strategies in these two species. Quantitative S. trifolia estimates of outcrossing rates in natural populations of S. Among populations 1.98 49.4 <0.001 trifolia indicated that the mating system of this species was Within populations 2.03 50.6 <0.001 dominated by outcrossing mechanisms (Wang and Chen, 2000). S. natans + S. trifolia A similar mating system pattern was found in S. natans (Chen Between species 8.20 60.1 <0.001 and Wang, unpublished data). Sexual reproduction may Within species 5.45 39.9 <0.001 therefore have played an important role in establishment of 中国科技论文在线 http://www.paper.edu.cn

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S. natans and S. trifolia populations, and the high levels of population, which indicated the existence of gene flow among clonal diversity observed might have been maintained or the populations. AMOVA also showed that roughly similar increased mainly by sexual recombination. That higher degree amounts of genetic variation resided within and among of clonal diversity were found in S. natans than in S. trifolia populations. Considering that with the exception of individuals may indicate that the former species possesses lower ability to growing in the WSL and RH populations (which occur in ponds colonize via asexual reproduction than its more widespread beside Wusuli River) all the extant S. natans populations in the congener S. trifolia. Similarly the lower degree of genetic study area are separated from each other by large geographical variability in S. trifolia may point towards a more limited role of distances, it is unlikely that high degree of gene flow presently sexual reproduction in the reproductive strategy of this species occur between these isolated populations. The considerably than in S. natans. These assertions are presently still a matter of high gene flow indicated in the UPGMA dendrogram might be conjecture and although the in-depth investigations into the indicative of an earlier period of more pronounced gene flow breeding systems of both species that would be required to when the species had a more continuous distribution. validate or disapprove these assertions was not within the scope The present situation where S. natans displays higher degree of the present study, our findings would form a sound basis for of genetic variation than its more widespread congener species further research in this area. S. trifolia is most likely attributed to multiple causes rather than Unlike many other endangered plant that exhibited lower to a single event. The present-day S. natans populations have, degree of genetic diversity than its widespread congener (see rather unexpectedly, maintained high degree of genetic Gitzendanner and Soltis, 2000), the endangered S. natans diversity in spite of the decline in the number of individuals maintains higher degree of genetic diversity than its widespread in recent years associated with increasing intensity of human congener S. trifolia at both population and species level. Our activity in the area, and especially the effects of establishment findings were in accordance with several cases of rare and of industries. There is, however, real danger that if the trend endangered species containing high levels of genetic diversity toward a decline in numbers is left unchecked, genetic even when they have extremely narrow geographical distribu- consequences associated with small isolated populations tion (Lewis and Crawford, 1995; Ge et al., 1999). Several including genetic drift and inbreeding will diminish genetic factors should be included in attempts to account for the diversity in S. natans. This would reduce the species adaptive observed higher degree of genetic diversity in the endangered ability thereby increasing its susceptibility to changes in the species S. natans. As stated above, sexual reproduction may biotic and abiotic environments. Appropriate conservation have played an important role in establishment of both S. natans strategies are required for the long-time survival of this species. and S. trifolia populations. However, S. natans may possess a Since damage to the habitats is the prevalent causative factor for higher ability to sexual recombination than S. trifolia. The more the decline in populations and number of individuals, limited sexual recombination in S. trifolia may contribute to the conservation strategies should aim at protecting more habitats. lower degree of genetic diversity than the endangered species S. Conservation efforts including artificially promoting gene flow natans. within and between populations as well as establishing an ex Another possible explanation for the observed higher degree situ conservation program are also recommended for this of genetic variation in S. natans than in S. trifolia might be that species. the historical distribution of S. natans probably differs dramatically from its modern-day distribution range. That this Acknowledgements might hold true even within relatively recent history is demonstrated by the fact that by 1989 S. natans was widely The authors thank Yang Chun-Feng, Zhang Xiao-Lin, Zheng distributed in Northeast Europe and Northeast Asia (Chen, Yi-Hong and Du Zhi-Yuan for their help in fieldwork and Xia 1989), which is not the case presently. In Northeast China, S. Jing and Huang Fang for their assistance in the laboratory. This natans was found widely distributed in bogs, rivers and streams study was supported by grants from the National Natural during the past decade (Chen, 1989). Most S. natans Science Foundation of China (No.: 30370098 and No.: populations in this region have presently been extirpated 30570111) and the Program for New Century Excellent Talents largely due to increased human activity associated with the rise in University (From Ministry of Education, People’s Republic in human population and industrialization in recent years. It of China) granted to WQF (NCET-05-0619) however appears that the remaining populations of S. natans have not suffered heavy losses of genetic diversity. Erosion of References genetic diversity in the species may, however, have not been readily discernible due to initial high levels of diversity in the Chen, J.K., 1989. Systematic and Evolutionary Biology Studies on Chinese species arising from frequent recombination, given the frequent Sagittaria. Wuhan University Press, Wuhan. sexual reproduction in the life history of this taxon. The clonal Chen, J.M., Gituru, W.R., Wang, Y.H., Wang, Q.F., 2006. The extent of habit of this species may also have contributed to retention of clonality and genetic diversity in the rare Caldesia grandis (Alismata- genetic diversity in the remnant populations of S. natans by ceae): comparative results for RAPD and ISSR markers. Aquat. Bot. 84, 301–307. ‘‘fixing’’ some of the genetic variation. Dodd, S.C., Helenurm, K., 2002. 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