Conservation genetics of the endangered terrestrial orchid Liparis japonica in Northeast China based on AFLP markers

Levels of genetic diversity and population genetic structure of the rare, endangered terrestrial orchid Liparis japonica were examined for eight natural populations (n = 185) in Northeast China using six AFLP primer pairs, where this species has experienced severe habitat loss and fragmentation. Based on 406 DNA bands, a high level of genetic diversity was found at the species level with the PPB of 85.47 %, while the genetic diversity at the population level was low (PPB = 47.48 %). A significantly high degree of population differentiation was found with 42.69 % variation existed among populations as measured by AMOVA, indicating potential restricted gene flow. The genetic distances between populations were independent of the corresponding geographic distances, and the genetic relationship of individuals had no significant correlation with their spatial distribution. The restricted gene flow might be impacted by reduced population size, habitat destruction and fragmentation. The results in this study suggested that habitat protection and keeping a stable environment are critical for the conservation of L. japonica species.     

A comparison of the population genetic structure of parasitic Viscum album from two landscapes differing in degree of fragmentation

Parasite populations do not necessarily conform to expected patterns of genetic diversity and structure. Parasitic plants may be more vulnerable to the negative consequences of landscape fragmentation because of their specialized life history strategies and dependence on host plants, which are themselves susceptible to genetic erosion and reduced fitness following habitat change. We used AFLP genetic markers to investigate the effects of habitat fragmentation on genetic diversity and structure within and among populations of hemiparasitic Viscum album. Comparing populations from two landscapes differing in the amount of forest fragmentation allowed us to directly quantify habitat fragmentation effects. Populations from both landscapes exhibited significant isolation-by-distance and sex ratios biased towards females. The less severely fragmented landscape had larger and less isolated populations, resulting in lower levels of population genetic structure (F _ST = 0.05 vs. 0.09) and inbreeding (F _IS = 0.13 vs. 0.27). Genetic differentiation between host-tree subpopulations was also higher in the more fragmented landscape. We found no significant differences in within-population gene diversity, percentage of polymorphic loci, or molecular variance between the two regions, nor did we find relationships between genetic diversity measures and germination success. Our results indicate that increasing habitat fragmentation negatively affects population genetic structure and levels of inbreeding in V. album, with the degree of isolation among populations exerting a stronger influence than forest patch size.     

Landscape genetic structure of <Emphasis Type="Italic">Olea europaea</Emphasis> subsp. <Emphasis Type="Italic">cuspidata</Emphasis> in Ethiopian highland forest fragments

Understanding patterns of genetic diversity at the landscape scale will enhance conservation and management of natural populations. Here we analyzed the genetic diversity, population connectivity, and spatial genetic structure among subpopulations and age groups of Olea europaea subsp. cuspidata, a cornerstone species of the Afromontane highlands. The study was conducted at the landscape level within a radius of approximately 4 km, as well as on a fine scale (intensive study plot) of less than 300 m radius. In total 542 samples from four natural subpopulations in northwestern Ethiopia were analyzed using ten nuclear microsatellite markers. Inbreeding was higher in smaller populations. No genetic difference was detected among cohorts of different tree sizes in the intensive studied plot. Average population differentiation was low but significant (F _ST ?=?0.016). Landscape genetic analysis inferred two groups: the most distant subpopulation WE located less than 4 kms from the other three subpopulations formed a separate group. Sixty-four percent of the total migrants were shared among the three latter subpopulations, which are spatially clustered. Immigrants were non-randomly distributed inside of the intensive study plot. Significant spatial genetic structure (SGS) was found both at the landscape scale and in the intensive study plot, and adults showed stronger SGS than young trees. An indirect estimate of 220 m as mean gene dispersal distance was obtained. We conclude that even under fragmentation migration is not disrupted in wild olive trees and that large protected populations at church forests are very important to conserve genetic resources. However, the higher level of inbreeding and evidence for population bottlenecks in the small populations, as well as the persisting heavy pressure on most remaining populations, warrants quick action to maintain genetic diversity of wild olive in the Ethiopian highlands.     

Comparative population genetics of two dominant plant species of high Andean wetlands reveals complex evolutionary histories and conservation perspectives in Chile’s Norte Chico

High Andean wetlands are naturally fragmented ecosystems that are impacted by anthropogenic activities. Although they constitute important reservoirs of biodiversity and ecosystem service providers, many aspects of their ecology are still unknown. In this study, we investigated the population genetic structure of two dominant and highly interactive plant species of high altitude wetlands, Patosia clandestina (Juncaceae) and Carex gayana (Cyperaceae), in 21 high Andean wetlands of Chile’s Norte Chico. Using rbcL gene sequences and AFLP markers, we found that both species displayed low levels of within-wetland genetic diversity, high inter-population genetic differentiation, and spatially-dependent genetic variation arising from isolation-by-distance. The distance at which populations become genetically independent was of the same order of magnitude in both species (125–175 km). Despite these similarities, idiosyncratic spatial patterns were detected. C. gayana in the three most northeastern wetlands demonstrated marked differences relative to the rest of the populations, with the latter group following a latitudinal stepping-stone pattern. In P. clandestina, a genetic barrier was found to divide the northern and southern populations into two balanced groups, and spatial genetic variation was consistent with a hierarchical island model. The data indicate that each of the two species likely responded to different geological and ecological events, resulting in the definition of unique evolutionarily significant units in both. These results suggest that the implementation of global conservation programs at regional scales would likely result in the loss of important components of biodiversity in these ecosystems, and underscore the need for caution in designing effective conservation strategies.     

Genetic structure and seed-mediated dispersal rates of an endangered shrub in a fragmented landscape: a case study for Juniperus communis in northwestern Europe

Background

Population extinction risk in a fragmented landscape is related to the differential ability of the species to spread its genes across the landscape. The impact of landscape fragmentation on plant population dynamics will therefore vary across different spatial scales. We quantified successful seed-mediated dispersal of the dioecious shrub Juniperus communis in a fragmented landscape across northwestern Europe by using amplified fragment length polymorphism (AFLP) markers. Furthermore we investigated the genetic diversity and structure on two spatial scales: across northwestern Europe and across Flanders (northern Belgium). We also studied whether seed viability and populations size were correlated with genetic diversity.

Results

Unexpectedly, estimated seed-mediated dispersal rates were quite high and ranged between 3% and 14%. No population differentiation and no spatial genetic structure were detected on the local, Flemish scale. A significant low to moderate genetic differentiation between populations was detected at the regional, northwest European scale (PhiPT = 0.10). In general, geographically nearby populations were also genetically related. High levels of within-population genetic diversity were detected but no correlation was found between any genetic diversity parameter and population size or seed viability.

Conclusions

In northwestern Europe, landscape fragmentation has lead to a weak isolation-by-distance pattern but not to genetic impoverishment of common juniper. Substantial rates of successful migration by seed-mediated gene flow indicate a high dispersal ability which could enable Juniperus communis to naturally colonize suitable habitats. However, it is not clear whether the observed levels of migration will suffice to counterbalance the effects of genetic drift in small populations on the long run.     

Fine-scale social and spatial genetic structure in Sitka black-tailed deer

The spatial extent of Sitka black-tailed deer (Odocoileus hemionus sitkensis) populations below the regional scale is relatively unknown, as is dispersal between populations. Here, we use noninvasive samples to genotype 221 Sitka black-tailed deer in three watersheds on Prince of Wales Island, Alaska, separated by a maximum of 44 km, using traditional and spatial genetic approaches. We find that despite geographic proximity, multiple lines of evidence suggest fine-scale genetic structure among the three study sites. The 2 most geographically distant watersheds differed significantly in genetic composition, suggesting an isolation-by-distance pattern. Within study sites, deer exhibited spatial genetic structure within a radius of 1,000 m. Based on a reduced sample of known-sex individuals, females exhibited positive spatial genetic structure within a radius of 500 m but males showed no structure. Moreover, females were more likely to be related to their 5 nearest female neighbors, regardless of distance, than were males. Evidence indicates dispersal by both sexes although it may be more common, or dispersal distances are greater, in males. Nonetheless, analysis of assignment indices and comparison of sex-specific correlograms found no evidence of sex-biased dispersal between watersheds. Patterns of spatial relatedness and connectivity suggest limited dispersal among Sitka black-tailed deer, creating genetic structure on a fine spatial scale, perhaps as small as the watershed.     

Patterns of geographic distribution have a considerable influence on population genetic structure in one common and two rare species of Rhododendron (Ericaceae)

Genetic diversity is essential for species to sustain their populations and evolutionary potential. In order to develop effective conservation strategies for rare species, it is necessary to understand differences in patterns of genetic diversity between common and rare species. Data about population genetic structure is important to design effective conservation strategies for rare species. In this study, we compared the genetic diversity and population genetic structure of a common species, Rhododendron weyrichii, to those of two rare species, Rhododendron sanctum and Rhododendron amagianum, with different geographic distributions. We analyzed five microsatellite loci in 16 populations of R. weyrichii, 9 populations of R. sanctum, and 6 populations of R. amagianum. As expected, the level of genetic diversity indicated by allelic richness and gene diversity was lower for the rare species R. sanctum than for the common species R. weyrichii. However, there was no statistically significant difference in genetic diversity between R. weyrichii and the other rare species, R. amagianum. Analyses of the isolation-by-distance pattern, neighbor-joining trees, and Bayesian clustering indicated that R. sanctum had a strong population genetic structure whereas R. amagianum exhibited very weak genetic structure among populations and that there was moderate population genetic structure for R. weyrichii. Therefore, the degree and pattern of population genetic structure in each species was unrelated to its rarity and instead merely reflected its geographic distribution.     

Contrasting spatial genetic structure in Annona crassiflora populations from fragmented and pristine savannas

In continuous populations, fine-scale genetic structure tends to be stronger in species with restricted pollen and seed dispersal. However, habitat fragmentation and disturbances can affect genetic diversity and spatial genetic structure due to disruption in ecological processes, such as plant reproduction and seed dispersal. In this study, we compared the genetic diversity and fine-scale spatial genetic structure (SGS) in two populations of Annona crassiflora (Annonaceae) in a pristine savanna Reserve (ESECAE) and in a fragmented disturbed savanna area (PABE), both in Cerrado biome in Central Brazil. The analyses were based on the polymorphism at 10 microsatellite loci. Our working hypothesis was that SGS is stronger and genetic diversity is lower in population at fragmented area (PABE) than at pristine area (ESECAE). Both populations presented high levels of polymorphism and genetic diversity and showed no sign of bottleneck for both Wilcoxon sign-rank test for heterozygosity excess (p > 0.05) and coalescent analyses (growth parameter g not different from zero), but population at fragmented area showed higher fixation index and stronger SGS. Besides, populations are significantly differentiated (F _ST = 0.239, R _ST = 0.483, p < 0.001 for both). Coalescent analyses showed high historical effective population sizes for both populations, high gene flow between ESECAE and PABE and recent time to most recent common ancestor (~37 k year BP). Our results suggest that despite the high genetic diversity, fragmentation and disturbance may have been affecting populations of this species increasing mating between closely related individuals leading to high fixation index and strong SGS.     

AFLP and ISSR analysis reveals high genetic variation and inter-population differentiation in fragmented populations of the endangered Litsea szemaois (Lauraceae) from Southwest China

Litsea szemaois (Lauraceae) is an endemic and endangered species from the tropical rain forests of Xishuangbanna, southern Yunnan, SW China, but habitat fragmentation, especially exacerbated by rubber planting, has caused a decline in population size of the species. AFLP and ISSR were used to investigate the genetic diversity and population structure of eight populations from across its known distribution. Three AFLP and ten ISSR primer combinations produced a total of 203 and 77 unambiguous and repeatable bands respectively, of which 164 (80.8%) and 67 (87.0%) were polymorphic for the two markers. These two markers showed that Litsea szemaois exhibits comparatively high genetic diversity at species level (heterozygosity (hs) = 0.2109) relative to some other Lauraceae. Most of the genetic variation was partitioned within populations, but genetic differentiation between populations was significant and relatively high (Φ _st = 0.2420, θ^B = 0.1986) compared with other tropical plants. The genetic characteristics of L. szemaois may be related to its outbreeding system, insect pollination and fragmented distribution. Because L. szemaois is dioecious and slow to mature, ex situ conservation across its genetic diversity is unlikely to succeed, although seedlings grow well under cultivation. Thus, in situ conservation is very important for this endangered species, especially as only 133 adult individuals are known in the wild. In particular, the Nabanhe and Mandian populations should be given a high conservation priority due to their higher genetic diversity, larger population size and better field condition, but wider sampling is required across all populations to determine additional areas with significant genetic conservation value.     

Population genetic structure of wild daffodils (Narcissus pseudonarcissus L.) at different spatial scales

We studied the population genetic and clonal structure of the endangered long-lived perennial plant Narcissus pseudonarcissus using random amplified polymorphic markers. Estimates for mean gene diversity within 15 populations of N. pseudonarcissus of three neighbouring geographical regions were high in comparison to other long-lived perennials (H _eN = 0.33). The genetic diversity of the two smallest populations (<200 plants) was significantly reduced, indicating loss of genetic variability due to drift. The analysis of the population genetic structure revealed a significant genetic differentiation both between regions (Φ_ST = 0.06) and between populations within regions (Φ_ST = 0.20). However, there was incomplete correspondence between geographical regions and the population genetic structure. In order to preserve the overall genetic variation in wild populations of N. pseudonarcissus, management measures should thus aim to protect many populations in each region. The spatial genetic structure within populations of N. pseudonarcissus was in agreement with an isolation by distance model indicating limited gene flow due to pollinator behaviour and restricted seed dispersal. The very restricted spatial extent of clonal growth (<5 cm) and the high level of clonal diversity indicate that clonal growth in N. pseudonarcissus is not an important mode of propagation and that management measures should favour sexual reproduction in order to avoid further reductions in the size and number of populations.     

Population genetic diversity and geographical differentiation of MHC class II DAB genes in the vulnerable Chinese egret (<Emphasis Type="Italic">Egretta eulophotes</Emphasis>)

Major histocompatibility complex (MHC) genes are excellent markers for the study of adaptive genetic variation occurring over different geographical scales. The Chinese egret (Egretta eulophotes) is a vulnerable ardeid species with an estimated global population of 2600–3400 individuals. In this study, we sampled 172 individuals of this egret (approximately 6 % of the global population) from five natural populations that span the entire distribution range of this species in China. We examined their population genetic diversity and geographical differentiation at three MHC class II DAB genes by identifying eight exon 2 alleles at Egeu-DAB1, eight at Egeu-DAB2 and four at Egeu-DAB3. Allelic distributions at each of these three Egeu-DAB loci varied substantially within the five populations, while levels of genetic diversity varied slightly among the populations. Analysis of molecular variance showed low but significant genetic differentiation among five populations at all three Egeu-DAB loci (haplotype-based ?_ST: 0.029, 0.020 and 0.042; and distance-based ?_ST: 0.036, 0.027 and 0.043, respectively; all P < 0.01). The Mantel test suggested that this significant population genetic differentiation was likely due to an isolation-by-distance pattern of MHC evolution. However, the phylogenetic analyses and the Bayesian clustering analysis based on the three Egeu-DAB loci indicated that there was little geographical structuring of the genetic differentiation among five populations. These results provide fundamental population information for the conservation genetics of the vulnerable Chinese egret.     

Chloroplast DNA-based genetic diversity and phylogeography of Pyrus betulaefolia (Rosaceae) in Northern China

Pyrus betulaefolia Bunge, considered as an intermediate between oriental and occidental pear groups, is one of the most important wild pear species. The number of its populations is decreasing because of habitat destruction, fragmentation, and continuous exploitation, so protection and conservation measures are urgently needed. Assessment of its genetic diversity and phylogeography are imperative for its efficient conservation. Two chloroplast DNA intergenic fragments were used to detect genetic diversity and phylogeography of 320 individuals from 18 wild P. betulaefolia populations. Haplotype variation, genetic differentiation, and historical events of the populations were estimated. The results showed that P. betulaefolia populations sampled in northern China contained a high level of genetic diversity (H _T?=?0.826). A significant isolation-by-distance value (r?=?0.587, P?<?0.001, 1,000 permutations) among all 18 populations indicated a correlation between genetic divergence and geographic distance. Four population groups were identified in a neighbor-joining tree based on the genetic distance. Analyses of molecular variation showed that the genetic variation mainly existed among population groups, representing 64.61 % of the total variation. Phylogeographic analyses indicated that the populations of P. betulaefolia experienced a scenario of rapid range expansion, which probably occurred between 608,000 and 204,580 years ago. Meanwhile, both the restricted gene flow with isolation by distance and allopatric fragmentation were crucial processes responsible for shaping the genetic patterns of P. betulaefolia. The occurrence of specific haplotypes might be ascribed to an ancestral introgression or joint retention of an ancestral polymorphism with other Pyrus species at the northern edge of the distribution of P. betulaefolia. Three populations displaying a high level of haplotype diversity and unique haplotypes were assumed to be relict populations of Quaternary glaciation and should have conservation priority. Three additional large populations should also be preferentially protected by building natural preservation zones.     

Fine-scale spatial genetic structure and clonal distribution of the cold-water coral Lophelia pertusa

Determining the spatial genetic structure within and among cold-water coral populations is crucial to understanding population dynamics, assessing the resilience of cold-water coral communities and estimating genetic effects of habitat fragmentation for conservation. The spatial distribution of genetic diversity in natural populations depends on the species’ mode of reproduction, and coral species often have a mixed strategy of sexual and asexual reproduction. We describe the clonal architecture of a cold-water coral reef and the fine-scale population genetic structure (<35 km) of five reef localities in the NE Skagerrak. This study represents the first of this type of analysis from deep waters. We used thirteen microsatellite loci to estimate gene flow and genotypic diversity and to describe the fine-scale spatial distribution of clonal individuals of Lophelia pertusa. Within-population genetic diversity was high in four of the five reef localities. These four reefs constitute a genetic cluster with asymmetric gene flow that indicates metapopulation dynamics. One locality, the Säcken reef, was genetically isolated and depauperate. Asexual reproduction was found to be a highly important mode of reproduction for L. pertusa: 35 genetic individuals were found on the largest reef, with the largest clone covering an area of nearly 300 m².     

Genetic diversity and population structure of the serpentine endemic Ni hyperaccumulator Alyssum lesbiacum

Alyssum lesbiacum is a well-established Ni hyperaccumulator, endemic to serpentine soils of Lesbos Island (Greece). A total of 95 individuals were collected from 17 plots encompassing the only four large populations of this species. ISSR (Inter-simple sequence repeat) markers were used to assess genetic diversity and population structure of A. lesbiacum to provide initial guidance for the development of successful management and conservation measures. A total of ten primers produced 82 bands, 96.34 % being polymorphic. The largest part of total diversity was found within populations, rather than among them. AMOVA analysis partitioned the largest part of diversity within plots (66 %), while 15 % contained among plots within populations and 19 % among populations. Principal coordinates analysis along with dendrogram based on genetic distances among populations showed a high degree of genetic differentiation of the isolated population Loutra. At a smaller scale, distance was not the most significant factor influencing the patterns of genetic diversity, but topography, ecosystem types and connectivity through streams. According to our results, conservation efforts should be organized at the level of watersheds and ecosystem types, considering them as management units. For ex situ conservation and restoration, seed samplings should be representative of the different habitats and watersheds as well as the patches of large and small populations while keeping the east population of Loutra separate from the other three central populations, to avoid any loss of genetic diversity and to preserve the character of the local adapted populations.     

Low genetic diversity of a high mountain burnet moth species in the Pyrenees

The burnet moth Zygaena anthyllidis, endemic to the high elevations of the Pyrenees, is vulnerable to land-use. In order to identify conservation priorities based on an assessment of genetic diversity within populations and gene flow among populations, we examined Z. anthyllidis’ genetic variability and differentiation based on allozyme electrophoresis from seven populations scattered across its entire range. In comparison to other mountain Lepidoptera, the populations studied exhibit a low level of genetic diversity. Remarkable between-population differentiation (F _ST = 0.053), the presence of private alleles, and the lack of significant isolation-by-distance pattern characterises the genetic make-up of the species. We interpreted the pattern of genetic differentiation as a consequence of low dispersal power in combination with insufficient landscape connectivity. Ongoing land-use change might reinforce genetic differentiation due to habitat fragmentation and additionally affect negatively allozyme variability at shifting range margins, i.e. the capacity to adapt to changing environments. We therefore suggest creating a network of suitable habitats at the landscape scale to facilitate genetic exchange and to conserve the species’ overall genetic variability.     

Intrapopulational genetic diversity of Araucaria angustifolia (Bertol.) Kuntze is different when assessed on the basis of chloroplast or nuclear markers

A natural population of Araucaria angustifolia (Bertol.) Kuntze was evaluated to quantify genetic diversity, using nucleotide sequences of chloroplast maturase K genes (matK) and six specific nuclear polymorphic microsatellite loci (nuSSR). Estimates of nucleotide diversity assessed from matK sequences were low (π = 0.001), but they indicated the potential of this gene for evaluating divergence among widespread populations. The matK sequence (1,565 bp) was used for analysis of phylogenetic relationships, enabling clustering of the South American species (A. angustifolia and A. araucana). For analysis of intraspecific genetic diversity, nuSSR were more informative than matK, resulting in a polymorphic loci percentage of 69, a number average of alleles per locus of 11.6, expected genetic diversity 0.741, and significant spatial genetic structure up to 25 m. Our data can help in the selection of genetic sources for conservation of this species, which is at risk of extinction.     

Geographic variation in relict populations: genetics and phenotype of bush-cricket Pholidoptera frivaldskyi (Orthoptera) in Carpathians

A decreasing population size is often causing species extinction, however, relict species persisting in small-sized populations counter this. We analysed spatial genetic variation and past changes in population size at the maternally-inherited mitochondrial DNA level to clarify the origin of all recently known isolated populations of Pholidoptera frivaldskyi occurring in the range of Carpathian Mountains. Along with that we analysed also morphological variation as some phenotypic traits can retain useful information on population genetic structure. We found a relatively low genetic diversity within isolated populations as 778 bp COI gene sequences revealed only 13 unique haplotypes (n = 173 individuals from 10 populations). The spatial analysis of molecular variance identified three geographically homogenous genetic clusters (one in Slovakia and two in Romania) with a high level of differentiation among them, suggesting restricted gene flow, whilst Bayesian skyline simulation reconstructed a negative demographic change through evolutionary time. Inferred genetic pattern clearly coincides with differences in males’ colour phenotype as the extent of pigmentation on the lateral pronotum varied significantly among genetic lineages. We suggest that geographical variation in the species populations has relict-like character and their isolated occurrence is not a result of recent introduction events. Identification of ‘evolutionary units’ may help in the conservation and management of this rare insect species.     

Genetic Variation in Natural Populations of Stipa tenacissima from Algeria

Intermicrosatellite PCR [inter-simple sequence repeat (ISSR)-PCR] markers and cytogenetics criteria were used to assess the level of genetic diversity and genetic structure in 17 populations of Stipa tenacissima (Gramineae) from Algeria. All populations sampled in the steppe area were diploids (2n = 2x = 24), and those sampled in the dry area were hexaploids (2n = 6x = 72). The dendrogram based on ISSR-PCR showed homogeneity within populations and large variability among populations. All individuals of the same population were gathered and formed groups clearly separated in all populations. These groups were separated into two clusters related to biotope, one from the steppe area and the other from the dry area. AMOVA indicated low genetic diversity among populations (30% of variation) and high within populations (70%). This variation pattern would constitute an adaptive strategy to grow in various ecological conditions.     

Genetic structure of the critically endangered plant Tricyrtis ishiiana (Convallariaceae) in relict populations of Japan

Tricyrtis ishiiana is a relic endemic plant taxon of the Convallariaceae that inhabits two nearby gorges in Kanagawa Prefecture, Japan. The distribution range and number of populations are thought to have been reduced to the present refugial populations during the Quaternary climatic oscillations. Because of its showy flowers, this plant has faced illegal removal from its natural habitats for horticultural use and has been designated a critically endangered species (class IA). In this study, we analyzed the genetic structure of the relict populations of T. ishiiana in order to contribute to the conservation strategies of the prefectural government. Our analyses of nine nuclear microsatellite loci detected high genetic diversity (H _E = 0.704 and H _O = 0.541) for the two populations. The two populations were slightly differentiated (R _ST = 0.032), accompanied by faint substructure across the populations (K = 3). In addition, each population exhibited spatial genetic structuring. The relatively low inbreeding coefficient for both populations together (F _IS = 0.233) and each population separately (F _IS = 0.217?C0.246) may be attributable to crossing among descendants within a population along with occasional gene flow between the populations. These results suggested that the extant populations have not experienced a severe bottleneck. The two extant populations were genetically differentiated at a very low level, accompanied by occasional pollen flow via pollinators and/or seed dispersal by gravity in the mountainous environment. Occasional gene exchange between the populations has allowed T. ishiiana to harbor high genetic diversity despite being a relic plant confined to two small refugial populations.