Genetic Diversity of the Endemic and Medicinally Important Plant Rheum officinale as Revealed by Inter-Simpe Sequence Repeat (ISSR) Markers

Rheum officinale Baill., an important but endangered medicinal herb, is endemic to China. Inter-simple sequence repeat (ISSR) markers were employed to investigate the genetic diversity and differentiation of 12 populations of R. officinale. Thirteen selected primers yielded 189 bright and discernible bands, with an average of 14.54 per primer. The genetic diversity was low at the population level, but pretty high at the species level (H = 0.1008, I = 0.1505, PPB = 28.95% vs. H = 0.3341, I = 0.5000, PPB = 95.24%, respectively) by POPGENE analysis. Analysis of molecular variance (AMOVA) showed that the genetic variation was found mainly among populations (74.38%), in line with the limited gene flow (N(m) = 0.2766) among populations. Mantel test revealed a significant correlation between genetic and geographic distances (r = 0.5381, P = 0.002), indicating the role of geographic isolation in shaping the present population genetic structure. Both Bayesian analysis and UPGMA cluster analysis demonstrated the similar results. Our results imply that the conservation efforts should aim to preserve all the extant populations of this endangered species, and cultivation is proposed in this study.     

Genetic Diversity and Differentiation of Dendrocalamus membranaceus (Poaceae: Bambusoideae), a Declining Bamboo Species in Yunnan, China, as Based on Inter-Simple Sequence Repeat (ISSR) Analysis

Dendrocalamus membranaceus Munro is a woody bamboo with a high economic and ecological value that often occurs as natural stands, such as in the large-scale forested areas of China's Yunnan Province. Due to its overexploitation, the habitat of D. membranaceus in Yunnan has been dramatically reduced, and the quality of the stands has declined. As a preliminary analysis in considering the effective protection for these germplasm resources, we assessed the genetic diversity of 12 natural populations in Yunnan, using inter-simple sequence repeat (ISSR) markers. From 10 ISSR primers, we generated 155 bands, of which 153 were polymorphic (98.71%). Compared with other species in the genus, this species demonstrated a greater genetic diversity (S = 0.349) and lower genetic differentiation (G(ST) = 0.252). Our analysis of molecular variance revealed that the genetic differentiation among the populations is significant. A large proportion of the genetic variation (78.95%) resides among the individuals within populations, whereas only 21.05% are found among populations. Mantel tests indicated no significant correlation between genetic and geographic distances among the populations. Given the low sexual reproducibility and characteristics of monocarpic plants, we recommend implementing in situ conservation measures for all of the D. membranaceus populations in Yunnan and collecting sufficient samples for ex situ conservation. Furthermore, the conservation area should be extended to its main natural habitats, the Lancang-Mekong River Valley.     

High Genetic Diversity and Low Differentiation of Michelia coriacea (Magnoliaceae), a Critically Endangered Endemic in Southeast Yunnan, China

Michelia coriacea, a critically endangered tree, has a restricted and fragmented distribution in Southeast Yunnan Province, China. The genetic diversity, genetic structure and gene flow in the three extant populations of this species were detected by 10 inter-simple sequence repeat (ISSR) markers and 11 simple sequence repeat (SSR) markers. Examination of genetic diversity revealed that the species maintained a relatively high level of genetic diversity at the species level (percentage of polymorphic bands) PPB = 96.36% from ISSRs; PPL (percentage of polymorphic loci) = 95.56% from SSRs, despite several fragmental populations. Low levels of genetic differentiation among the populations of M. coriacea were detected by Nei's G(st) = 0.187 for ISSR and Wright's F(st) = 0.090 for SSR markers, which is further confirmed by Bayesian model-based STRUCTURE and PCoA analysis that could not reveal a clear separation between populations, although YKP was differentiated to other two populations by ISSR markers. Meanwhile, AMOVA analysis also indicated that 22.84% and 13.90% of genetic variation existed among populations for ISSRs and SSRs, respectively. The high level of genetic diversity, low genetic differentiation, and the population, structure imply that the fragmented habitat and the isolated population of M. coriacea may be due to recent over-exploitation. Conservation and management of M. coriacea should concentrate on maintaining the high level of genetic variability through both in and ex-situ conservation actions.     

Genetic Diversity and Population Differentiation of Calanthe tsoongiana,a Rare and Endemic Orchid in China

Calanthe tsoongiana is a rare terrestrial orchid endemic to China, and this species has experienced severe habitat loss and fragmentation. Inter-simple sequence repeat (ISSR) markers were employed to assess the genetic diversity and differentiation of six populations of C. tsoongiana. Based on 124 discernible fragments yielded by eleven selected primers, high genetic diversity was revealed at the species level; however, genetic diversity at the population level was relatively low. High-level genetic differentiation among populations was detected based on analysis of molecular variance (AMOVA), indicating potential limited gene flow. No significant relationship was observed between genetic and geographic distances among the sampled populations. These results suggested that restricted gene flow might be due to habitat fragmentation and reduced population size as a result of human activities. Based on the findings, several conservation strategies were proposed for the preservation of this threatened species.     

Conservation Genetics of an Endangered Lady’s Slipper Orchid: Cypripedium japonicum in China

Knowledge about the population genetic variation of the endangered orchid, Cypripedium japonicum, is conducive to the development of conservation strategies. Here, we examined the levels and partitioning of inter-simple sequence repeat (ISSR) diversity (109 loci) in five populations of this orchid to gain insight into its genetic variation and population structure in Eastern and Central China. It harbored considerably lower levels of genetic diversity both at the population (percentage of polymorphic loci (PPL) = 11.19%, Nei’s gene diversity (H) = 0.0416 and Shannon’s information index (I) = 0.0613) and species level (PPL = 38.53%, H = 0.1273 and I = 0.1928) and a significantly higher degree of differentiation among populations (the proportion of the total variance among populations (Φ_pt) = 0.698) than those typical of ISSR-based studies in other orchid species. Furthermore, the Nei’s genetic distances between populations were independent of the corresponding geographical distances. Two main clusters are shown in an arithmetic average (UPGMA) dendrogram, which is in agreement with the results of principal coordinate analysis (PCoA) analysis and the STRUCTURE program. In addition, individuals within a population were more similar to each other than to those in other populations. Based on the genetic data and our field survey, the development of conservation management for this threatened orchid should include habitat protection, artificial gene flow and ex situ measures.     

Genetic Diversity and Population Structure of Siberian apricot (Prunus sibirica L.) in China

The genetic diversity and population genetic structure of 252 accessions from 21 Prunus sibirica L. populations were investigated using 10 ISSR, SSR, and SRAP markers. The results suggest that the entire population has a relatively high level of genetic diversity, with populations HR and MY showing very high diversity. A low level of inter-population genetic differentiation and a high level of intra-population genetic differentiation was found, which is supported by a moderate level of gene flow, and largely attributable to the cross-pollination and self-incompatibility reproductive system. A STRUCTURE (model-based program) analysis revealed that the 21 populations can be divided into two main groups, mainly based on geographic differences and genetic exchanges. The entire wild Siberia apricot population in China could be divided into two subgroups, including 107 accessions in subgroup (SG) 1 and 147 accessions in SG 2. A Mantel test revealed a significant positive correlation between genetic and geographic distance matrices, and there was a very significant positive correlation among three marker datasets. Overall, we recommend a combination of conservation measures, with ex situ and in situ conservation that includes the construction of a core germplasm repository and the implement of in situ conservation for populations HR, MY, and ZY.     

Development of 22 Polymorphic Microsatellite Loci for the Critically Endangered Morato��s Digger Toad, Proceratophrys moratoi

The Morato’s digger toad (Proceratophrys moratoi) inhabits Brazilian moist savannas and is critically endangered due to its very limited geographic distribution, reduced number of isolated populations, and evidence of population decline and local extinctions. With the objective of providing tools for the genetic study of the species, 22 polymorphic microsatellite loci were isolated and screened using DNA extracted from samples of oral mucosa cells obtained from 113 individuals representing five remnant P. moratoi populations in the Brazilian state of São Paulo. These markers presented 2–18 alleles per locus, polymorphism information content (PIC) of 0.02–0.87, observed heterozygosity of 0.02–0.96 and expected heterozygosity of 0.02–0.87. Three of the loci deviated significantly from Hardy–Weinberg equilibrium in one of the populations, possibly due to the presence of null alleles. Significant linkage disequilibrium was also detected between three pairs of loci. The molecular markers developed in this study were able to discriminate each of the individuals sampled (identity analysis). This means that they will be extremely useful for future genetic studies applied to the conservation of P. moratoi, providing a baseline for estimating the levels of genetic diversity, pedigrees, inbreeding, and population structure, which will be essential for the development of effective genetic management programs.     

Genetic Diversity and Differentiation of the Orange-Spotted Grouper (Epinephelus coioides) Between and Within Cultured Stocks and Wild Populations Inferred from Microsatellite DNA Analysis

In the present study, we employed microsatellite DNA markers to analyze the genetic diversity and differentiation between and within cultured stocks and wild populations of the orange-spotted grouper originating from the South China Sea and Southeast Asia. Compared to wild populations, genetic changes including reduced genetic diversity and significant differentiation have taken place in cultured grouper stocks, as shown by allele richness and heterozygosity studies, pairwise F(st), structure, molecular variance analysis, as well as multidimensional scaling analysis. Although two geographically adjacent orange-spotted grouper populations in China showed negligible genetic divergence, significant population differentiation was observed in wild grouper populations distributed in a wide geographical area from China, through Malaysia to Indonesia. However, the Mantel test rejected the isolation-by-distance model of genetic structure, which indicated the genetic differentiation among the populations could result from the co-effects of various factors, such as historical dispersal, local environment, ocean currents, river flows and island blocks. Our results demonstrated that microsatellite markers could be suitable not only for genetic monitoring cultured stocks but also for revealing the population structuring of wild orange-spotted grouper populations. Meanwhile, our study provided important information for breeding programs, management of cultured stocks and conservation of wild populations of the orange-spotted grouper.     

Isolation and Characterisation of 11 Polymorphic Microsatellite Markers in Papaver rhoeas L. (Corn Poppy), a Major Annual Plant Species from Cultivated Areas

Papaver rhoeas, an annual plant species in the Papaveraceae family, is part of the biodiversity of agricultural ecosystems and also a noxious agronomic weed. We developed microsatellite markers to study the genetic diversity of P. rhoeas, using an enriched microsatellite library coupled with 454 next-generation sequencing. A total of 13,825 sequences were obtained that yielded 1795 microsatellite loci. After discarding loci with less than six repeats of the microsatellite motif, automated primer design was successful for 598 loci. We tested 74 of these loci for amplification with a total of 97 primer pairs. Thirty loci passed our tests and were subsequently tested for polymorphism using 384 P. rhoeas plants originating from 12 populations from France. Of the 30 loci, 11 showed reliable polymorphism not affected by the presence of null alleles. The number of alleles and the expected heterozygosity ranged from 3 to 7.4 and from 0.27 to 0.73, respectively. A low but significant genetic differentiation among populations was observed (F_ST = 0.04; p < 0.001). The 11 validated polymorphic microsatellite markers developed in this work will be useful in studies of genetic diversity and population structure of P. rhoeas, assisting in designing management strategies for the control or the conservation of this species.     

Development of Microsatellite Markers Using Pyrosequencing in Galium trifidum (Rubiaceae), a Rare Species in Central Europe

We identify a large number of microsatellites from Galium trfidum, a plant species considered rare and endangered in Central and Western Europe. Using a combination of a total enriched genomic library and small-scale 454 pyrosequencing, we determined 9755 contigs with a length of 100 to 6192 bp. Within this dataset, we identified 153 SSR motifs in 144 contigs. Here, we tested 14 microsatellite loci in 2 populations of G. trifidum. The number of alleles and expected heterozygosity were 1-8 (mean 3.2) and 0.00-0.876 (0.549 on average), respectively. The markers described in this study will be useful for evaluating genetic diversity within and between populations, and gene flow between G. trifidum populations. These markers could also be applied to investigate the biological aspects of G. trifidum, such as the population dynamics and clonal structure, and to develop effective conservation programs for the Central European populations of this species.     

Genetic Diversity of the Critically Endangered Thuja sutchuenensis Revealed by ISSR Markers and the Implications for Conservation

Thuja sutchuenensis Franch. is a critically endangered plant endemic to the North-East Chongqing, China. Genetic variation was studied to assess the distribution of genetic diversity within and among seven populations from the single remnant locations, using inter-simple sequence repeat (ISSR) markers. A total of 15 primers generated 310 well defined bands, with an average of 20.7 bands per primer. The seven populations revealed a relatively high level of genetic diversity in the species. The percentage of polymorphic bands, Nei’s gene diversity and Shannon’s information index at the population and species level were 76.1%, 0.155, 0.252 and 100%, 0.165, 0.295, respectively. A low level of genetic differentiation among populations (G_ST = 0.102), in line with the results of Analyses of Molecular Variance (AMOVA), and a high level of gene flow (N_m = 4.407) were observed. Both the Unweighted Pair Group Method with Arithmatic Mean (UPGMA) cluster analysis and Principal Coordinates Analysis (PCoA) supported the grouping of all seven populations into two groups. In addition, Mantel test revealed no significant correlation between genetic and geographical distances (r = 0.329, p = 0.100). The low genetic differentiation among populations implies that the conservation efforts should aim to preserve all the extant populations of this endangered species.     

Loss of Genetic Diversity in the Cultured Stocks of the Large Yellow Croaker, Larimichthys crocea, Revealed by Microsatellites

The large yellow croaker (Larimichthys crocea) is the most important mariculture fish species in China and the wild stocks of this croaker have collapsed in the past decades due to high fishing pressure and habitat degradation. Due to a lack of wild croaker samples, however, studies concerning the genetic changes of the cultured croaker stocks compared to their wild counterparts were never conducted. Here, we collected three wild populations in the northern and central East China Sea during fisheries survey and investigated the differences in terms of genetic diversity and differentiation between and within cultured stocks and wild populations. Our results demonstrated that the cultured croaker had significantly reduced genetic diversity in contrast to the wild populations, and also presented statistically significant differentiation from the wild, indicating that enhancement of the current wild stock should be conducted with caution. These changes may be caused by founder effects, artificial selection and random genetic drift. With a relatively high level of genetic diversity, the wild populations showed important value for improving the ongoing breeding program of this croaker. Further, we detected no differentiation among the wild populations, suggesting that the wild croaker in the northern and central East China Sea should be considered as one unit for management and conservation.     

Application of Novel Polymorphic Microsatellite Loci Identified in the Korean Pacific Abalone (Haliotis diversicolor supertexta (Haliotidae)) in the Genetic Characterization of Wild and Released Populations

The small abalone, Haliotis diversicolor supertexta, of the family Haliotidae, is one of the most important species of marine shellfish in eastern Asia. Over the past few decades, this species has drastically declined in Korea. Thus, hatchery-bred seeds have been released into natural coastal areas to compensate for the reduced fishery resources. However, information on the genetic background of the small abalone is scarce. In this study, 20 polymorphic microsatellite DNA markers were identified using next-generation sequencing techniques and used to compare allelic variation between wild and released abalone populations in Korea. Using high-throughput genomic sequencing, a total of 1516 (2.26%; average length of 385 bp) reads containing simple sequence repeats were obtained from 86,011 raw reads. Among the 99 loci screened, 28 amplified successfully, and 20 were polymorphic. When comparing allelic variation between wild and released abalone populations, a total of 243 different alleles were observed, with 18.7 alleles per locus. High genetic diversity (mean heterozygosity = 0.81; mean allelic number = 15.5) was observed in both populations. A statistical analysis of the fixation index (F_ST) and analysis of molecular variance (AMOVA) indicated limited genetic differences between the two populations (F_ST = 0.002, p > 0.05). Although no significant reductions in the genetic diversity were found in the released population compared with the wild population (p > 0.05), the genetic diversity parameters revealed that the seeds released for stock abundance had a different genetic composition. These differences are likely a result of hatchery selection and inbreeding. Additionally, all the primer pair sets were effectively amplified in another congeneric species, H. diversicolor diversicolor, indicating that these primers are useful for both abalone species. These microsatellite loci may be valuable for future aquaculture and population genetic studies aimed at developing conservation and management plans for these two abalone species.     

Genetic Variation and Population Differentiation in a Medical Herb Houttuynia cordata in China Revealed by Inter-Simple Sequence Repeats (ISSRs)

Houttuynia cordata is an important traditional Chinese herb with unresolved genetics and taxonomy, which lead to potential problems in the conservation and utilization of the resource. Inter-simple sequence repeat (ISSR) markers were used to assess the level and distribution of genetic diversity in 226 individuals from 15 populations of H. cordata in China. ISSR analysis revealed low genetic variations within populations but high genetic differentiations among populations. This genetic structure probably mainly reflects the historical association among populations. Genetic cluster analysis showed that the basal clade is composed of populations from Southwest China, and the other populations have continuous and eastward distributions. The structure of genetic diversity in H. cordata demonstrated that this species might have survived in Southwest China during the glacial age, and subsequently experienced an eastern postglacial expansion. Based on the results of genetic analysis, it was proposed that as many as possible targeted populations for conservation be included.     

Genetic Diversity and Population Structure: Implications for Conservation of Wild Soybean (Glycine soja Sieb. et Zucc) Based on Nuclear and Chloroplast Microsatellite Variation

Wild soybean (Glycine soja Sieb. et Zucc) is the most important germplasm resource for soybean breeding, and is currently subject to habitat loss, fragmentation and population decline. In order to develop successful conservation strategies, a total of 604 wild soybean accessions from 43 locations sampled across its range in China, Japan and Korea were analyzed using 20 nuclear (nSSRs) and five chloroplast microsatellite markers (cpSSRs) to reveal its genetic diversity and population structure. Relatively high nSSR diversity was found in wild soybean compared with other self-pollinated species, and the region of middle and lower reaches of Yangtze River (MDRY) was revealed to have the highest genetic diversity. However, cpSSRs suggested that Korea is a center of diversity. High genetic differentiation and low gene flow among populations were detected, which is consistent with the predominant self-pollination of wild soybean. Two main clusters were revealed by MCMC structure reconstruction and phylogenetic dendrogram, one formed by a group of populations from northwestern China (NWC) and north China (NC), and the other including northeastern China (NEC), Japan, Korea, MDRY, south China (SC) and southwestern China (SWC). Contrib analyses showed that southwestern China makes the greatest contribution to the total diversity and allelic richness, and is worthy of being given conservation priority.     

Novel Polymorphic Microsatellite Loci for the Korean Black Scraper (Thamnaconus modestus), and Their Application to the Genetic Characterization of Wild and Farmed Populations

In this study, we developed 20 polymorphic microsatellite markers for the Korean black scraper, Thamnaconus modestus (Günther, 1877), Monacanthidae, and used them to compare allelic variation between wild and hatchery populations in Korea. All loci were readily amplified and demonstrated allelic variability, with the number of alleles ranging from 5-35 in the wild population and 5-22 in the farmed population. The average observed and expected heterozygosities were estimated, respectively, as 0.74 and 0.80 in the hatchery samples and 0.78 and 0.81 in the wild ones. These results indicate lower genetic variability in the hatchery population than in the wild population and minor, but significant, genetic differentiation between the two populations (F(ST) = 0.005, P < 0.01). Additionally, cross-amplification was tested in another monacanthid species, Stephanolepis cirrhifer; many loci were found that yielded useful information. The high degree of polymorphism exhibited by the 20 microsatellites will be useful in future aquaculture and population genetic studies for developing conservation and management plans.     

Development and Characterization of New Single Nucleotide Polymorphism Markers from Expressed Sequence Tags in Common Carp (Cyprinus carpio)

The common carp (Cyprinus carpio) is an important aquaculture fish worldwide but only limited single nucleotide polymorphism (SNP) markers are characterized from expressed sequence tags (ESTs) in this species. In this study, 1487 putative SNPs were bioinformatically mined from 14,066 online ESTs mainly from the European common carp, with the occurrence rate of about one SNP every 173 bp. One hundred and twenty-one of these SNPs were selected for validation using PCR fragment sequencing, and 48 out of 81 primers could amplify the expected fragments in the Chinese common carp genome. Only 26 (21.5%) putative SNPs were validated, however, 508 new SNPs and 68 indels were identified. The ratios of transitions to transversions were 1.77 for exon SNPs and 1.05 for intron SNPs. All the 23 SNPs selected for population tests were polymorphic, with the observed heterozygosity (Ho) ranging from 0.053 to 0.526 (mean 0.262), polymorphism information content (PIC) from 0.095 to 0.357 (mean 0.246), and 21 SNPs were in Hardy-Weinberg equilibrium. These results suggest that different common carp populations with geographic isolation have significant genetic variation at the SNP level, and these new EST-SNP markers are readily available for genetics and breeding studies in common carp.     

Development of Seven Microsatellite Markers Using Next Generation Sequencing for the Conservation on the Korean Population of Dorcus hopei (E. Saunders, 1854) (Coleoptera,Lucanidae)

We developed microsatellite markers for genetic structural analyses of Dorcus hopei, a stag beetle species, using next generation sequencing and polymerase chain reaction (PCR)-based genotyping for regional populations. A total of 407,070,351 base pairs of genomic DNA containing >4000 microsatellite loci except AT repeats were sequenced. From 76 loci selected for primer design, 27 were polymorphic. Of these 27 markers, 10 were tested on three regional populations: two Chinese (Shichuan and Guangxi) and one Korean (Wanju). Three markers were excluded due to inconsistent amplification, genotyping errors, and Hardy-Weinberg equilibrium (HWE). By multi-locus genotyping, the allele number, observed heterozygosity and polymorphism information content of seven microsatellite loci were ranged 2‒10, 0.1333‒1.0000, and 0.1228‒0.8509, respectively. In an analysis on the genetic differentiation among regional populations including one Japanese population and one cross-breeding population, the individual colored bar-plots showed that both Chinese populations were closer to each other than to the Far East Asian populations. In Far East Asian populations, Wanju and Nirasaki populations could not be distinguished from each other because the frequency of genetic contents was very similar in some individuals of two populations. Moreover, the cross-breeding population contained all patterns of genetic contents shown in Chinese, Korean, and Japanese populations, compared with the genetic content frequency of each regional population. As a result, we examined whether the cross-breeding population might be a hybrid population, and might contain a possibility of interbreeding with Chinese populations in parental generations. Therefore, these markers will be useful for analyses of genetic diversity in populations, genetic relationships between regional populations, genetic structure analyses, and origin tests.     

Genetic Diversity and Population Structure of the Critically Endangered Yangtze Finless Porpoise (Neophocaena asiaeorientalis asiaeorientalis) as Revealed by Mitochondrial and Microsatellite DNA

Ecological surveys have indicated that the population of the critically endangered Yangtze finless porpoise (YFP, Neophocaena asiaeorientalis asiaeorientalis) is becoming increasingly small and fragmented, and will be at high risk of extinction in the near future. Genetic conservation of this population will be an important component of the long-term conservation effort. We used a 597 base pair mitochondrial DNA (mtDNA) control region and 11 microsatellite loci to analyze the genetic diversity and population structure of the YFP. The analysis of both mtDNA and microsatellite loci suggested that the genetic diversity of the YFP will possibly decrease in the future if the population keeps declining at a rapid rate, even though these two types of markers revealed different levels of genetic diversity. In addition, mtDNA revealed strong genetic differentiation between one local population, Xingchang–Shishou (XCSS), and the other five downstream local populations; furthermore, microsatellite DNA unveiled fine but significant genetic differentiation between three of the local populations (not only XCSS but also Poyang Lake (PY) and Tongling (TL)) and the other local populations. With an increasing number of distribution gaps appearing in the Yangtze main steam, the genetic differentiation of local populations will likely intensify in the future. The YFP is becoming a genetically fragmented population. Therefore, we recommend attention should be paid to the genetic conservation of the YFP.     

Population Genetics of the Endemic Hawaiian Species Chrysodracon hawaiiensis and Chrysodracon auwahiensis (Asparagaceae): Insights from RAPD and ISSR Variation

The genus Chrysodracon has six endemic species in the Hawaii Islands. Chrysodracon hawaiiensis is endemic to Hawaii Island and was described as a distinct species in 1980. It was listed as an endangered species on the International Union for the Conservation of Nature and Natural Resources (IUCN) Red List in 1997. This woody plant species was, at one time, common in exposed dry forests, but it became very rare due to grazing pressure and human development. The tree species Chrysodracon auwahiensis (C. auwahiensis), endemic to Maui and Molokai, still has large adult populations in dry lands of the islands, but unfortunately no regeneration from seed has been reported in those areas for many years. The two endemic species were examined using the molecular technique of random amplified polymorphic DNA (RAPD) and inter simple sequence repeats (ISSR) to determine the genetic structure of the populations and the amount of variation. Both species possess similar genetic structure. Larger and smaller populations of both species contain similar levels of genetic diversity as determined by the number of polymorphic loci, estimated heterozygosity, and Shannon’s index of genetic diversity. Although population diversity of Chrysodracon hawaiiensis (C. hawaiiensis) is thought to have remained near pre-disturbance levels, population size continues to decline as recruitment is either absent or does not keep pace with senescence of mature plants. Conservation recommendations for both species are suggested.