Sky island diversification meets the multispecies coalescent – divergence in the spruce‐fir moss spider (Microhexura montivaga,Araneae, Mygalomorphae) on the highest peaks of southern Appalachia

Microhexura montivaga is a miniature tarantula‐like spider endemic to the highest peaks of the southern Appalachian mountains and is known only from six allopatric, highly disjunct montane populations. Because of severe declines in spruce‐fir forest in the late 20th century, M. montivaga was formally listed as a US federally endangered species in 1995. Using DNA sequence data from one mitochondrial and seven nuclear genes, patterns of multigenic genetic divergence were assessed for six montane populations. Independent mitochondrial and nuclear discovery analyses reveal obvious genetic fragmentation both within and among montane populations, with five to seven primary genetic lineages recovered. Multispecies coalescent validation analyses [guide tree and unguided Bayesian Phylogenetics and Phylogeography (BPP), Bayes factor delimitation (BFD)] using nuclear‐only data congruently recover six or seven distinct lineages; BFD analyses using combined nuclear plus mitochondrial data favour seven or eight lineages. In stark contrast to this clear genetic fragmentation, a survey of secondary sexual features for available males indicates morphological conservatism across montane populations. While it is certainly possible that morphologically cryptic speciation has occurred in this taxon, this system may alternatively represent a case where extreme population genetic structuring (but not speciation) leads to an oversplitting of lineage diversity by multispecies coalescent methods. Our results have clear conservation implications for this federally endangered taxon and illustrate a methodological issue expected to become more common as genomic‐scale data sets are gathered for taxa found in naturally fragmented habitats.     

Phylogeographic pattern of range expansion provides evidence for cryptic species lineages in Silene nutans in Western Europe

As a result of recent or past evolutionary processes, a single species might consist of distinct Evolutionary Significant Units (ESUs), even corresponding to cryptic species. Determining the underlying mechanisms of range shifts and the processes at work in the build-up of divergent ESUs requires elucidating the factors that contribute to population genetic divergence across a species'' range. We investigated the large-scale patterns of genetic structure in the perennial herbaceous plant species Silene nutans (Caryophyllaceae) in Western Europe. We sampled and genotyped 111 populations using 13 nuclear microsatellite loci and 6 plastid single-nucleotide polymorphisms. Broad-scale spatial population genetic structure was examined using Bayesian clustering, spatial multivariate analyses and measures of hierarchical genetic differentiation. The genotypic structure of S. nutans was typical of a predominantly allogamous mating system. We also identified plastid lineages with no intra-population polymorphism, mirroring two genetically differentiated nuclear lineages. No evidence of admixture was found. Spatial trends in genetic diversity further suggested independent leading-edge expansion associated with founding events and subsequent genetic erosion. Overall, our findings suggested speciation processes in S. nutans and highlighted striking patterns of distinct stepwise recolonisation of Western Europe shaped by Quaternary climate oscillations. Two main potential ESUs can be defined in Western Europe, corresponding to Eastern and Western nuclear-plastid lineages. In situ preservation of populations and genetic rescue implying ex situ conservation techniques should take the lineage identity into account. This is particularly true in Great Britain, northern France and Belgium, where S. nutans is rare and where distinct lineages co-occur in close contact.     

Mitochondrial DNA analysis reveals hidden genetic diversity in captive populations of the threatened American crocodile (Crocodylus acutus) in Colombia

Identification of units within species worthy of separate management consideration is an important area within conservation. Mitochondrial DNA (mtDNA) surveys can potentially contribute to this by identifying phylogenetic and population structure below the species level. The American crocodile (Crocodylus acutus) is broadly distributed throughout the Neotropics. Its numbers have been reduced severely with the species threatened throughout much of its distribution. In Colombia, the release of individuals from commercial captive populations has emerged as a possible conservation strategy that could contribute to species recovery. However, no studies have addressed levels of genetic differentiation or diversity within C. acutus in Colombia, thus complicating conservation and management decisions. Here, sequence variation was studied in mtDNA cytochrome b and cytochrome oxidase I gene sequences in three Colombian captive populations of C. acutus. Two distinct lineages were identified: C. acutus‐I, corresponding to haplotypes from Colombia and closely related Central American haplotypes; and C. acutus‐II, corresponding to all remaining haplotypes from Colombia. Comparison with findings from other studies indicates the presence of a single “northern” lineage (corresponding to C. acutus‐I) distributed from North America (southern Florida), through Central America and into northern South America. The absence of C. acutus‐II haplotypes from North and Central America indicates that the C. acutus‐II lineage probably represents a separate South American lineage. There appears to be sufficient divergence between lineages to suggest that they could represent two distinct evolutionary units. We suggest that this differentiation needs to be recognized for conservation purposes because it clearly contributes to the overall genetic diversity of the species. All Colombian captive populations included in this study contained a mixture of representatives of both lineages. As such, we recommend against the use of captive‐bred individuals for conservation strategies until further genetic information is available.     

Allopatric divergence,demographic history,and conservation implications of an endangered conifer <Emphasis Type="Italic">Cupressus chengiana</Emphasis> in the eastern Qinghai-Tibet Plateau

Isolation and demographic history are key factors that affect lineage divergence of tree species in topographic complex areas, such as the Qinghai-Tibet Plateau (QTP), yet few studies have evaluated these factors in a coalescent-based modeling framework. In the present study, we surveyed ten nuclear DNA sequence loci (nDNA) and six nuclear microsatellite loci (nSSR) for an endangered conifer, Cupressus chengiana, throughout its natural range in the eastern QTP. BARRIER analyses revealed a strong genetic barrier between Gansu and Sichuan populations of C. chengiana, and isolation with migration models detected limited gene flow between them, supporting the division of this species into two evolutionary significant units (ESUs). Two independent coalescent-based approaches suggest a Quaternary divergence between ESUs, their consensus age range ((0.09–) 0.59–1.53 (–2.71) Mya) largely overlaps with the time period when the largest glaciation occurred in the QTP. Both demographic inferences, IMa2 and DIYABC, suggest that both ESUs may have experienced a bottleneck or population contraction event during the late Quaternary. A documented massive recent anthropogenic habitat loss and fragmentation may have led to further decrease of the natural distribution of this conifer. We propose that the conservation and management of both natural stands and plantations of C. chengiana should be reconsidered in the light of our findings.     

Consequences of multiple mating‐system shifts for population and range‐wide genetic structure in a coastal dune plant

Evolutionary transitions from outcrossing to selfing can strongly affect the genetic diversity and structure of species at multiple spatial scales. We investigated the genetic consequences of mating‐system shifts in the North American, Pacific coast dune endemic plant Camissoniopsis cheiranthifolia (Onagraceae) by assaying variation at 13 nuclear (n) and six chloroplast (cp) microsatellite (SSR) loci for 38 populations across the species range. As predicted from the expected reduction in effective population size (N_e) caused by selfing, small‐flowered, predominantly selfing (SF) populations had much lower nSSR diversity (but not cpSSR) than large‐flowered, predominantly outcrossing (LF) populations. The reduction in nSSR diversity was greater than expected from the effects of selfing on N_e alone, but could not be accounted for by indirect effects of selfing on population density. Although selfing should reduce gene flow, SF populations were not more genetically differentiated than LF populations. We detected five clusters of nSSR genotypes and three groups of cpSSR haplotypes across the species range consisting of parapatric groups of populations that usually (but not always) differed in mating system, suggesting that selfing may often initiate ecogeographic isolation. However, lineage‐wide genetic variation was not lower for selfing clusters, failing to support the hypothesis that selection for reproductive assurance spurred the evolution of selfing in this species. Within three populations where LF and SF plants coexist, we detected genetic differentiation among diverged floral phenotypes suggesting that reproductive isolation (probably postzygotic) may help maintain the striking mating‐system differentiation observed across the range of this species.     

SNPs selected by information content outperform randomly selected microsatellite loci for delineating genetic identification and introgression in the endangered dark European honeybee (Apis mellifera mellifera)

The honeybee (Apis mellifera) has been threatened by multiple factors including pests and pathogens, pesticides and loss of locally adapted gene complexes due to replacement and introgression. In western Europe, the genetic integrity of the native A. m. mellifera (M‐lineage) is endangered due to trading and intensive queen breeding with commercial subspecies of eastern European ancestry (C‐lineage). Effective conservation actions require reliable molecular tools to identify pure‐bred A. m. mellifera colonies. Microsatellites have been preferred for identification of A. m. mellifera stocks across conservation centres. However, owing to high throughput, easy transferability between laboratories and low genotyping error, SNPs promise to become popular. Here, we compared the resolving power of a widely utilized microsatellite set to detect structure and introgression with that of different sets that combine a variable number of SNPs selected for their information content and genomic proximity to the microsatellite loci. Contrary to every SNP data set, microsatellites did not discriminate between the two lineages in the PCA space. Mean introgression proportions were identical across the two marker types, although at the individual level, microsatellites' performance was relatively poor at the upper range of Q‐values, a result reflected by their lower precision. Our results suggest that SNPs are more accurate and powerful than microsatellites for identification of A. m. mellifera colonies, especially when they are selected by information content.     

Nuclear microsatellite and mitochondrial DNA analyses reveal the regional genetic structure and phylogeographical history of a sanguivorous land leech,Haemadipsa japonica,in Japan

Recent molecular studies have indicated that phylogeographical history of Japanese biota is likely shaped by geohistory along with biological events, such as distribution shifts, isolation, and divergence of populations. However, the genetic structure and phylogeographical history of terrestrial Annelida species, including leech species, are poorly understood. Therefore, we aimed to understand the genetic structure and phylogeographical history across the natural range of Haemadipsa japonica, a sanguivorous land leech species endemic to Japan, by using nine polymorphic nuclear microsatellites (nSSR) and cytochrome oxidase subunit one (COI) sequences of mitochondrial DNA (mtDNA). Analyses using nSSR revealed that H. japonica exhibited a stronger regional genetic differentiation among populations (G'_ST = 0.77) than other animal species, probably because of the low mobility of land leech. Analyses using mtDNA indicated that H. japonica exhibited two distinct lineages (A and B), which were estimated to have diverged in the middle Pleistocene and probably because of range fragmentation resulting from climatic change and glacial and interglacial cycles. Lineage A was widely distributed across Japan, and lineage B was found in southwestern Japan. Analyses using nSSR revealed that lineage A was roughly divided into two population groups (i.e., northeastern and southwestern Japan); these analyses also revealed a gradual decrease in genetic diversity with increasing latitude in lineage A and a strong genetic drift in populations of northeastern Japan. Combined with the largely unresolved shallow polytomies from the mtDNA phylogeny, these results implied that lineage A may have undergone a rapid northward migration, probably during the Holocene. Then, the regional genetic structure with local unique gene pools may have been formed within each lineage because of the low mobility of this leech species.     

Population genetic structure of the endangered crayfish Austropotamobius pallipes in France based on microsatellite variation: biogeographical inferences and conservation implications

1. One important goal in conservation biology is to characterise evolutionary lineages within endangered species before management decisions are taken. Here, we assess population differentiation in the freshwater crayfish Austropotamobius pallipes, an endangered species endemic to western Europe and provide valuable information for the conservation of French populations. 2. Analysis of five microsatellite loci in 44 populations revealed very different within population levels of genetic diversity (0.000 < H₀ < 0.564). Two groups, corresponding to northern and southern French populations, showed a high degree of genetic differentiation in both allele frequencies and allele sizes. Comparison of these results with previous studies of A. pallipes strongly suggests that the divergence between northern and southern populations could have occurred during the last glaciation period of the Pleistocene from one Atlantic and one Mediterranean refuge. 3. Evidence for genetic admixture between these two lineages was revealed by correspondence analyses in southern populations, probably as the result of artificial translocations. 4. French populations appeared significantly differentiated among the different river drainages and were highly structured within rivers. The impact of population size, population bottlenecks and founder events on the population genetic differentiation are discussed. 5. Based on these results, we propose the designation of two evolutionarily significant units for A. pallipes in France. Our data also support the maintenance of separate demographic management strategies for crayfish inhabiting different river systems. However, genetic analyses will have to be combined with demographic and ecological data for sustainable conservation programmes.     

线粒体DNA（mtDNA）多态性在动物保护生物学中的应用

本文从两个方面论述了mtDNA在动物保护生物学中的应用：一是对物种进行遗传多样性的检 测与管理,二是进行与种群统计学数据相关的遗传分析。前者与保护的长期效益（如进化） 密切相关,而后者则主要用于指导短期管理措施的制定。同时,本文重点论述了mtDNA在进 化显著单位(ESUs)和管理单位(MUs)的认定方面的作用。认定ESUs的目的是隔离管理遗传多 样性,它是一系列系统进化史独特的种群,这种独特性同时表现在mtDNA和核DNA上;MUs是 种群统计意义上的生殖隔离单位,具有独特的等位基因频率,与系统发生结构和遗传分歧水 平无关。ESUs与MUs都是保护生物学中保护与管理的重要基本单位。     

Subspecies of the Central American Squirrel Monkey (Saimiri oerstedii) as Units for Conservation

The accurate diagnosis of conservation units now typically includes recognition of genetic diversity and unique evolutionary lineages and is necessary to inform the conservation management of endangered species. We evaluated whether the two currently recognized subspecies of the endangered Central American squirrel monkey (Saimiri oerstedii) in Costa Rica are evolutionarily significant units (ESUs) that should be managed separately in conservation efforts. We used previously published sequences of 50 individuals of Saimiri oerstedii for 880 bp of the mtDNA d-loop and genotypes of 244 individuals for 16 microsatellites and conducted novel analyses to characterize genetic differentiation between subspecies of Saimiri oerstedii. We measured sequence differentiation and inferred an intraspecific molecular phylogeny and a haplotype network, and found consistent results supporting statistically significant divergence and reciprocal monophyly between subspecies. A population aggregation analysis also supported Saimiri oerstedii citrinellus and S. o. oerstedii as diagnosably distinct units. These results confirm previous genetic studies with smaller sample sizes and are consistent with other factors including differences in pelage and morphology and divergence at nuclear markers. Conservation managers should manage these subspecies separately to prevent the loss of genetic diversity via artificially induced outbreeding. High levels of genetic diversity may buffer populations against outside extinction pressures, to which Saimiri oerstedii are vulnerable because of their dwindling habitat and small population size.     

Admixture of genetic lineages of different glacial origin: a case study of <Emphasis Type="Italic">Abies alba</Emphasis> Mill. in the Carpathians

Genetic exchange between divergent lineages of silver fir (Abies alba Mill.) was studied in the Ukrainian Carpathians where two expanding populations originating from different glacial refugia meet. The study included 33 silver fir populations from Ukraine, Romania and Slovakia which were investigated using the maternally inherited mitochondrial nad5-4 marker and biparentally inherited nuclear microsatellites. The boundary between mitochondrial lineages is very sharp; only six populations containing a mixture of different haplotypes were found. Bayesian analysis of population structure based on seven nSSR loci revealed the existence of two clusters which coincided fairly well with mitochondrial lineages. Both haplotype frequencies and proportions of clusters identified by the Bayesian analysis exhibited a clinal transition over the contact zone, with cline widths of 17.6 km for mitochondrial haplotype frequencies (reflecting gene flow by seeds) and 119.6 km for Bayesian clusters based on nSSR (reflecting gene flow by pollen). Allelic richness and gene diversity differ significantly between mitochondrial lineages, the Balkan group being more variable, but an increase in gene diversity towards the boundary between lineages was observed only within the Balkan lineage. The observed patterns are suggested to reflect the postglacial colonization and historical gene flow. They demonstrated that in silver fir as a wind-dispersed species, the colonization front is quite continuous, and the survival of migrant seeds in already established populations is low. The relative contribution of pollen-mediated gene flow to genetic exchange between divergent lineages associated with glacial refugia is much higher than in the case of seeds, but pollen dispersal distance is lower than suggested by earlier studies.     

Comparing RADseq and microsatellites for estimating genetic diversity and relatedness — Implications for brown trout conservation

The conservation and management of endangered species requires information on their genetic diversity, relatedness and population structure. The main genetic markers applied for these questions are microsatellites and single nucleotide polymorphisms (SNPs), the latter of which remain the more resource demanding approach in most cases. Here, we compare the performance of two approaches, SNPs obtained by restriction‐site‐associated DNA sequencing (RADseq) and 16 DNA microsatellite loci, for estimating genetic diversity, relatedness and genetic differentiation of three, small, geographically close wild brown trout (Salmo trutta) populations and a regionally used hatchery strain. The genetic differentiation, quantified as F_ST, was similar when measured using 16 microsatellites and 4,876 SNPs. Based on both marker types, each brown trout population represented a distinct gene pool with a low level of interbreeding. Analysis of SNPs identified half‐ and full‐siblings with a higher probability than the analysis based on microsatellites, and SNPs outperformed microsatellites in estimating individual‐level multilocus heterozygosity. Overall, the results indicated that moderately polymorphic microsatellites and SNPs from RADseq agreed on estimates of population genetic structure in moderately diverged, small populations, but RADseq outperformed microsatellites for applications that required individual‐level genotype information, such as quantifying relatedness and individual‐level heterozygosity. The results can be applied to other small populations with low or moderate levels of genetic diversity.     

Phylogeography of <Emphasis Type="Italic">Leiopelma hochstetteri</Emphasis> reveals strong genetic structure and suggests new conservation priorities

Leiopelma hochstetteri belongs to a singular anuran lineage endemic to New Zealand that diverged from other frogs about 200 million years ago. The species now is reduced to a series of isolated populations in the northern half of the North Island and Great Barrier Island. We have used mitochondrial and nuclear sequence data to examine the genetic affinities of extant populations of L. hochstetteri. Phylogenetic reconstructions reveal that populations are highly structured. Each of the geographically isolated populations harbours independent mtDNA lineages as well as different degrees of nuDNA differentiation. Moreover, molecular dating reveals that this structure originated in the early Pleistocene. This pattern of genetic structure likely results from unfavourable climatic conditions during the Pleistocene combined with the low dispersal ability of the species. Isolated populations in forested refugia existed even in the southern part of the distribution of the species during glacial cycles. Previously published variation in chromosome numbers and isozyme data are consistent with the new evidence. We identified 13 evolutionary significant units (ESUs) that should serve as the focus for future management and conservation of this species.     

Two genetically distinct units of Sinomanglietia glauca (Magnoliaceae) detected by chloroplast PCR‐SSCP

Sinomanglietia glauca is a critically endangered species described from Jiangxi Province in the 1990s. Recently two populations were discovered from Yongshun County of west Hunan Province, about 450 km away from those in Jiangxi. Because of the new findings and the poor reproducibility inherent to RAPD and ISSR markers of previous studies, the population structure of this rare species was reanalyzed with chloroplast PCR‐SSCP (single‐stranded conformation polymorphism), including all of four recorded populations. The results showed that two distinct haplotypes characterized Jiangxi and Hunan populations separately, with no genetic variation occurring within regions. We postulated that this surprising pattern might result from habitat fragmentation and demographic bottlenecks during and/or after the Quaternary glaciation. On the basis of the pronounced genetic structure, two evolutionarily significant units (ESUs) were recommended for effective conservation of S. glauca.     

Discovery Of Genetically Distinct Sympatric Lineages in the Freshwater Mussel Cyprogenia Aberti (Bivalvia: Unionidae)

The western fanshell, Cyprogenia aberti, occurs in the CentralHighlands region of North America and is the only congener ofthe federally endangered fanshell, C. stegaria. Due to a reductionin range size and local extirpations, extant populations ofC. aberti have become increasingly isolated. Consequently, C.aberti has been the focus of numerous conservation efforts,yet no work has examined the degree of genetic variation amongthe species' disjunct populations. Phylogenetic analyses ofnucleotide sequences from two mitochondrial gene portions (CO1and ND1) revealed results important to the conservation managementof C. aberti. First, phylogenetic analyses do not support amonophyletic C. aberti. Second, C. aberti is comprised of asmany as five independent lineages, one of which includes thefederally endangered C. stegaria. Third, analysis recoveredtwo genetically distinct sympatric lineages of C. aberti inthe Ouachita River Drainage. These phylogenetic results suggestthat the five distinct evolutionary lineages of C. aberti shouldbe managed as separate conservation units. This study illustrateswhy it is critical to evaluate genetic variability in endangeredand threatened ‘species’ prior to implementing arecovery program. These data also reveal the value of assessingbiological diversity of nonimperiled taxa before populationsare extirpated and valuable genetic data necessary for reconstructionof evolutionary relationships is lost. (Received 24 October 2005; accepted 8 June 2006)     

Genetic diversity of the Chinese goat in the littoral zone of the Yangtze River as assessed by microsatellite and mtDNA

The objective of this study was to assess the genetic diversity and population structure of goats in the Yangtze River region using microsatellite and mtDNA to better understand the current status of those goat genetic diversity and the effects of natural landscape in fashion of domestic animal genetic diversity. The genetic variability of 16 goat populations in the littoral zone of the Yangtze River was estimated using 21 autosomal microsatellites, which revealed high diversity and genetic population clustering with a dispersed geographical distribution. A phylogenetic analysis of the mitochondrial D‐loop region (482 bp) was conducted in 494 goats from the Yangtze River region. In total, 117 SNPs were reconstructed, and 173 haplotypes were identified, 94.5% of which belonged to lineages A and B. Lineages C, D, and G had lower frequencies (5.2%), and lineage F haplotypes were undetected. Several high‐frequency haplotypes were shared by different ecogeographically distributed populations, and the close phylogenetic relationships among certain low‐frequency haplotypes indicated the historical exchange of genetic material among these populations. In particular, the lineage G haplotype suggests that some west Asian goat genetic material may have been transferred to China via Muslim migration.     

Phylogeography and genetic effects of habitat fragmentation on endangered Taxus yunnanensis in southwest China as revealed by microsatellite data

It is not known how the profoundly complex topography and habitat heterogeneity generated by the uplift of the Qinghai‐Tibetan Plateau (QTP) during the late Tertiary affected population genetic structure of endangered Taxus yunnanensis. In addition, the effects of habitat fragmentation due to anthropogenic disturbance on genetic diversity and population differentiation of this species have not been studied. T. yunnanensis is an ancient tree/shrub mainly distributed in southwest China. Recently, the species has suffered a sharp decline due to excessive logging for its famous anticancer metabolite taxol, resulting in smaller and more isolated populations. To understand the phylogeography and genetic consequences of habitat fragmentation of this endangered species, using 11 polymorphic microsatellites, we genotyped 288 individuals from 14 populations from a range‐wide sampling in China. Our results suggest that two different population groups that were once isolated have persisted in situ during glacial periods in both areas, and have not merged since. Habitat fragmentation has led to significant genetic bottlenecks, high inbreeding and population divergence in this species. The two different population groups of T. yunnanensis could be attributed to restricted gene flow caused through isolation by geographical barriers and by habitat heterogeneity during uplift of the QTP, or the existence of two separate glacial refugia during the Pleistocene. In situ and ex situ conservation of the two Evolutionarily Significant Units (ESUs), artificial gene flow between populations and a comprehensive understanding of the pollination system in this endangered species are suggested from this study.     

Selection and localised genetic structure in the threatened Manauense Harlequin Frog (Bufonidae: Atelopus manauensis)

Knowledge of genome-wide variation and the processes influencing gene flow are critical to managing threatened species. Here, we characterise genetic diversity and the environmental features associated with connectivity for a narrowly distributed and threatened Amazonian frog, Atelopus manauensis. We sampled 94 individuals throughout the upper, middle and lower courses from each of the five major rivers covering the species’ known geographic range and genotyped each individual at 3859 single nucleotide polymorphisms (SNPs). Genetic variation was significantly subdivided into six groups, each mostly containing individuals sampled within the same major river system. The genetic distances among these groups increased with geographic distance, and open forests and the extremities of the altitude gradient were associated with less genetic connectivity. Using F_ST outlier approaches and environmental association analyses, we identified SNPs indicative of localised adaptation, with 28 SNPs significantly associated with forest biomass and altitude. Evidence of divergent selection among the six genetic clusters suggests the presence of six Evolutionary Significant Units (ESUs). Overall, the ESUs were characterized by low contemporary effective population sizes (N_E?<?100) suggesting that genetic variation will be lost by random genetic drift. We demonstrate surprisingly high levels of divergence across the limited distribution of A. manauensis and suggest that each of the six adaptively divergent lineages be considered in conservation planning.

     

Microsatellite evidence for high clonality and limited genetic diversity in Ziziphus celata (Rhamnaceae), an endangered, self-incompatible shrub endemic to the Lake Wales Ridge, Florida, USA

Genetic data are often crucial for designing management strategies for rare and endangered species. Ziziphus celata is an endangered sandhill shrub endemic to the Lake Wales Ridge of central Florida. This self-incompatible clonal species is known from only 14 wild populations, most of which are small (under 100 plants). Focusing on the five populations discovered in 2007, we evaluate the level of genetic diversity and identify clonal lineages within the wild populations of the species with a set of microsatellite loci. To account for somatic mutations and genotyping errors, we identified clonal lineages using a threshold cutoff for pair-wise genetic distances among samples. The microsatellites had up to 18 alleles/locus, and, consistent with outcrossing, samples were highly heterozygous (average population level H _o  = 0.69). Most populations of Z. celata consist of a single clone, and the most diverse population has only 10 clones. Overall Z. celata comprises 41 multi-locus genotypes, and 30 clonal lineages. With nearly 1,000 recorded plants (595 genotyped) and only 30 clonal lineages, Ziziphus celata is highly clonal: clonal richness, R = 0.049. The pair-wise distance method facilitates identification of clonal lineages, avoiding overestimation of clonal diversity. In most cases, the samples that grouped into a lineage were one to four plants differing from a surrounding genotype by a single microsatellite repeat insertion/deletion mutation, consistent with these having arisen via somatic mutations. Our data will enable managers to incorporate extant diversity from wild populations into ex situ collections. Additionally, our research demonstrates the utility of microsatellites for conservation of imperiled species, identifying genotypes of high priority for preservation.     

Protecting evolutionary significant units for the remnant populations of <Emphasis Type="Italic">Berchemiella wilsonii</Emphasis> var. <Emphasis Type="Italic">pubipetiolata</Emphasis> (Rhamnaceae)

Berchemiella wilsonii var. pubipetiolata (Rhamnaceae) is an endangered tree in eastern China. Habitat destruction has resulted in fragmentation of remnant populations and extinction of local populations. AFLP and cpDNA markers were used to determine the population structure of remnant populations of B. wilsonii var. pubipetiolata. Moderate nuclear genomic diversity was found within each of the four remnant populations (H _S = 0.141–0.172), while the cpDNA haplotype diversity in each population ranged from 0.356 to 0.681. Six haplotypes were identified by a combined cpRFLP and cpSSR analysis in a total of 89 individuals. AMOVA revealed significantly AFLP genetic differentiation within and between regions (Φ_SC = 0.196, Φ_CT = 0.396, respectively), and a high cpDNA haplotype differentiation between regions (Φ_CT = 0.849). The results suggest low gene flow between populations of B. wilsonii var. pubipetiolata. Strong genetic divergence between two regional populations as revealed by both AFLP and cpDNA markers provided convincing evidence that two distinct evolutionary lineages existed, and should be recognized as ‘evolutionary significant units’ (ESUs) for conservation concerns.