Development of microsatellite markers for <Emphasis Type="Italic">Metrosideros boninensis</Emphasis> (Myrtaceae), an endangered endemic plant species from the Bonin Islands,Japan

Metrosideros boninensis is an endangered endemic plant species from the Bonin Islands. We isolated and characterized nine microsatellite loci in this species. The expected heterozygosities of these nine markers ranged from 0.127 to 0.768. These markers described here will be useful for investigating the genetic diversity, genetic structure and gene flow, and planning for conservation of M. boninensis.     

Characterization of 18 SNP markers for sperm whale (Physeter macrocephalus)

We report the characterization of 18 new single nucleotide polymorphism (SNP) markers for an endangered species, the sperm whale (Physeter macrocephalus), developed using a targeted gene approach. SNP markers were derived from autosomal regions of the genome using primers originally characterized for genome mapping in other mammals. These SNP markers are the first to be designed for genotyping sperm whale populations and will provide a necessary addition to the genetic tools employed for understanding population structure on a global scale and for developing a conservation management strategy for this endangered species.     

Development and characterization of 24 microsatellite markers in Primula tosaensis,an endangered primrose,using MiSeq

Primula tosaensis (Primulaceae) is an endangered primrose endemic to Japan. In this study, 24 novel microsatellite markers were developed using Illumina MiSeq sequencing to facilitate conservation of this endangered species. The genetic diversity and polymorphisms of these novel markers were measured in 32 individuals from a wild P. tosaensis population. The number of alleles and expected heterozygosities ranged from 2 to 5 (mean = 2.8) and from 0.119 to 0.724 (mean = 0.395), respectively. All loci were in Hardy–Weinberg equilibrium. The markers developed in this study will provide a powerful and practical tool for investigating the population structure and genetic diversity of P. tosaensis.     

Biodiversity assessment using markers for ecologically important traits

Most studies of genetic variation within species to date are based on random markers. However, how well this correlates with quantitative variation is contentious. Yet, functional, or'ecotypic' variation in quantitative traits determines the ecological niche of a species, its future evolutionary potential, and, for livestock, crops and their wild relatives, their usefulness as a genetic resource for breeding. But nowadays we can also assess genetic diversity using markers directly targeted at specific genes or gene families. Such gene-targeted, multilocus profiles of markers can contribute to ex-situ management of genetic resources, ecological studies of diversity, and conservation of endangered species.     

Development of microsatellite markers for <Emphasis Type="Italic">Echinops setifer</Emphasis> (Asteraceae), an endangered grassland plant species in Japan

Echinops setifer is an endangered grassland plant species in Japan. We isolated and characterized nine microsatellite loci in this species. The number of alleles ranged from 3 to 7 and expected heterozygosities from 0.108 to 0.761. These markers described here will be useful for investigating the genetic diversity, genetic structure and gene flow, and planning for conservation of E. setifer.     

Conservation genetics of the yellow-bellied toad (Bombina variegata): population structure,genetic diversity and landscape effects in an endangered amphibian

Revealing patterns of genetic diversity and barriers for gene flow are key points for successful conservation in endangered species. Methods based on molecular markers are also often used to delineate conservation units such as evolutionary significant units and management units. Here we combine phylo-geographic analyses (based on mtDNA) with population and landscape genetic analyses (based on microsatellites) for the endangered yellow-bellied toad Bombina variegata over a wide distribution range in Germany. Our analyses show that two genetic clusters are present in the study area, a northern and a southern/central one, but that these clusters are not deeply divergent. The genetic data suggest high fragmentation among toad occurrences and consequently low genetic diversity. Genetic diversity and genetic connectivity showed a negative relationship with road densities and urban areas surrounding toad occurrences, indicating that these landscape features act as barriers to gene flow. To preserve a maximum of genetic diversity, we recommend considering both genetic clusters as management units, and to increase gene flow among toad occurrences with the aim of restoring and protecting functional meta-populations within each of the clusters. Several isolated populations with especially low genetic diversity and signs of inbreeding need particular short-term conservation attention to avoid extinction. We also recommend to allow natural gene flow between both clusters but not to use individuals from one cluster for translocation or reintroduction into the other. Our results underscore the utility of molecular tools for species conservation, highlight outcomes of habitat fragmentation onto the genetic structure of an endangered amphibian and reveal particularly threatened populations in need for urgent conservation efforts.

     

Small edge populations at risk: genetic diversity of the green lizard (<Emphasis Type="Italic">Lacerta viridis viridis</Emphasis>) in Germany and implications for conservation management

Edge and central populations can show great differences regarding their genetic variation and thereby also in their probability of extinction. This fact might be of great importance for the conservation strategies of endangered species. In this study we examine the level of microsatellite variability within three threatened edge populations of the green lizard subspecies Lacerta viridis viridis (Laur.) in Brandenburg (Germany) and compare the observed variation to other edge and central populations within the northern species range. We demonstrate that the northernmost edge populations contain less genetic variation in comparison to the central population. However, there were no observable significant differences to the other edge population included in this study. Surprisingly, we observed a high genetic differentiation in a small geographical range between the three endangered populations in Brandenburg, which can be explained by processes like fragmentation, isolation, genetic drift and small individual numbers within these populations. We also detected unique genetic variants (alleles), which only occurred in these populations, despite a low overall genetic variation. This study demonstrates the potential of fast evolving markers assessing the genetic status of endangered populations with a high resolution. It also illustrates the need for a comparative analysis of different regions within the species range, achieving a more exact interpretation of the genetic variation in endangered populations. This will aid future management decisions in the conservation of genetic diversity in threatened species.     

Restoration genetics of the vernal pool endemic Lasthenia conjugens (Asteraceae)

Restoration of habitat for endangered species often involves translocation of seeds or individuals from source populations to an area targeted for revegetation. Long-term persistence of a species is dependent on the maintenance of sufficient genetic variation within and among populations. Thus, knowledge and maintenance of genetic variability within rare or endangered species is essential for developing effective conservation and restoration strategies. Genetic monitoring of both natural and restored populations can provide an assessment of restoration protocol success in establishing populations that maintain levels of genetic diversity similar to those in natural populations. California’s vernal pools are home to many endangered plants, thus conservation and restoration are large components of their management. Lasthenia conjugens (Asteraceae) is a federally endangered self-incompatible vernal pool annual with gravity- dispersed seeds. Using the molecular technique of intersimple sequence repeats (ISSRs), this study assessed levels and patterns of genetic variability present within natural and restored populations of L. conjugens. At Travis Air Force Base near Fairfield, California, a vernal pool restoration project is underway. Genetic success of the ecologically based seeding protocol was examined through genetic monitoring of natural and restored populations over a three-year period. Genetic diversity remained constant across the three sampled generations. Diversity was also widely distributed across all populations. We conclude that the protocol used to establish restored populations was successful in capturing similar levels and patterns of genetic diversity to those seen within natural pools. This study also demonstrates how genetic markers can be used to inform conservation and restoration decisions.     

Isolation and characterization of eight polymorphic microsatellite loci for the critically endangered Arenaria nevadensis (Caryophyllaceae)

We report on the isolation and characterization of eight microsatellite markers from enriched libraries for Arenaria nevadensis, one of the most critically endangered plant species in the Iberian Peninsula. These are the first microsatellite loci reported for Arenaria species. The number of alleles ranged from two to eight, and the expected heterozygosity from 0.067 to 0.873. These markers will be useful for characterizing the genetic diversity in A. nevadensis and understanding its population structure, and will provide important genetic data for the conservation and recovery of this species.     

Model‐based conservation planning of the genetic diversity of Phellodendron amurense Rupr due to climate change

Climate change affects both habitat suitability and the genetic diversity of wild plants. Therefore, predicting and establishing the most effective and coherent conservation areas is essential for the conservation of genetic diversity in response to climate change. This is because genetic variance is a product not only of habitat suitability in conservation areas but also of efficient protection and management. Phellodendron amurense Rupr. is a tree species (family Rutaceae) that is endangered due to excessive and illegal harvesting for use in Chinese medicine. Here, we test a general computational method for the prediction of priority conservation areas (PCAs) by measuring the genetic diversity of P. amurense across the entirety of northeast China using a single strand repeat analysis of twenty microsatellite markers. Using computational modeling, we evaluated the geographical distribution of the species, both now and in different future climate change scenarios. Different populations were analyzed according to genetic diversity, and PCAs were identified using a spatial conservation prioritization framework. These conservation areas were optimized to account for the geographical distribution of P. amurense both now and in the future, to effectively promote gene flow, and to have a long period of validity. In situ and ex situ conservation, strategies for vulnerable populations were proposed. Three populations with low genetic diversity are predicted to be negatively affected by climate change, making conservation of genetic diversity challenging due to decreasing habitat suitability. Habitat suitability was important for the assessment of genetic variability in existing nature reserves, which were found to be much smaller than the proposed PCAs. Finally, a simple set of conservation measures was established through modeling. This combined molecular and computational ecology approach provides a framework for planning the protection of species endangered by climate change.     

Conservation genetics of two island endemic Ribes spp. (Grossulariaceae) of Sardinia: survival or extinction?

Measuring levels of population genetic diversity is an important step for assessing the conservation status of rare or endangered plant species and implementing appropriate conservation strategies. Populations of Ribes multiflorum subsp. sandalioticum and R. sardoum, two endangered endemic species from Sardinia, representing the whole genus on the island, were investigated using ISSR and SSR markers to determine levels and structure of genetic variability in their natural populations. Results indicated medium to low genetic diversity at the population level: Nei's gene diversity for ISSR markers ranged from 0.0840 to 0.1316; the expected heterozygosity (H_E) for SSR ranged from 0.4281 to 0.7012. In addition, only one remnant population of R. sardoum showed a high level of inbreeding, in accordance with its very small size. Regarding the structure of the six R. sandalioticum populations, both principal coordinates analysis (PCoA) and STRUCTURE analysis of ISSR and SSR data highlighted low population structure, although two populations appeared to be clearly distinct from the others. The genetic pattern of the two taxa associated with their different ecological positions indicated resilience of R. sandalioticum populations in fresh and humid habitats and uncertain future resistance for the residual R. sardoum population in xeric calcareous stands. Hence, this study highlights the importance of an integrated conservation approach (genetic plus in situ and ex situ conservation studies/measures) for activating management programmes in these endemic and threatened taxa that can be considered as crop wild relatives of cultivated Ribes species.     

Isolation and characterization of polymorphic microsatellite markers for the endangered Korean freshwater fish Hemibarbus mylodon

Korean barbel, Hemibarbus mylodon, is an endangered freshwater species endemic to Korea. In order to undertake a conservation program for this species in Korea, it is essential to evaluate its genetic diversity and population structure. For this purpose, we developed 10 polymorphic microsatellite markers and examined their allelic variation using a total of 60 wild individuals collected from three different localities. The number of alleles per locus ranged from three to 26. The expected and observed heterozygosities per locus ranged from 0.19 to 0.94 and from 0.20 to 0.98, respectively. Significant departures from Hardy–Weinberg equilibrium expectations occurred in four loci. These markers will be useful for the assessment of genetic diversity for this species.     

Low genetic diversity and high genetic differentiation in the critically endangered Omphalogramma souliei (Primulaceae): implications for its conservation

Omphalogramma souliei Franch. is an endangered perennial herb only distributed in alpine areas of SW China. ISSR markers were applied to determine the genetic variation and genetic structure of 60 individuals of three populations of O. souliei in NW Yunnan, China. The genetic diversity at the species level is low with P=42.5% (percentage of polymorphic bands) and Hsp=0.1762 (total genetic diversity). However, a high level of genetic differentiation among populations was detected based on different measures (Nei's genetic diversity analysis: Gst=0.6038; AMOVA analysis: Fst=0.6797). Low level of genetic diversity within populations and significant genetic differentiation among populations might be due to the mixed mating system in which xenogamy predominated and autogamy played an assistant role in O. souliei. The genetic drift due to small population size and limited current gene flow also resulted in significant genetic differentiation. The assessment of genetic variation and differentiation of the endangered species provides important information for conservation on a genetic basis. Conservation strategies for this rare endemic species are proposed.     

<Emphasis Type="Italic">Tiliqua rugosa</Emphasis> microsatellites: isolation via enrichment and characterisation of loci for multiplex PCR in <Emphasis Type="Italic">T. rugosa</Emphasis> and the endangered <Emphasis Type="Italic">T. adelaidensis</Emphasis>

We used an enrichment technique to isolate 18 novel di and tri microsatellites for the socially monogamous lizard Tiliqua rugosa. These loci were amplified in conjunction with previously described loci in two and three PCR multiplexes for T. rugosa and the endangered T. adelaidensis, respectively. The loci were highly polymorphic in both species, exhibiting between 2 and 32 alleles with observed heterozygosity ranging from 0.43 to 0.96. These markers will be useful for population-level analyses and can contribute to a genetic foundation for conservation strategies for the endangered T. adelaidensis.     

Genetic diversity of the endangered Chinese endemic herb Primulina tabacum (Gesneriaceae) revealed by amplified fragment length polymorphism (AFLP)

Primulina tabacum Hance, is a critically endangered perennial endemic to limestone area in South China. Genetic variability within and among four extant populations of this species was assessed using AFLP markers. We expected a low genetic diversity level of this narrowly distributed species, but our results revealed that a high level of genetic diversity remains, both at population level (55.5% of markers polymorphic, H _E = 0.220, I _S = 0.321), and at species level (P = 85.6% of markers polymorphic, H _E = 0.339, I _S = 0.495), probably resulting from its refugial history and/or breeding system. High levels of genetic differentiation among populations was apparent based on Nei’s genetic diversity analysis (G _st=0.350). The restricted gene flow between populations is a potential reason for the high genetic differentiation. The population genetic diversity of P. tabacum revealed here has clear implications for conservation and management. To maintain present levels of genetic diversity, in situ conservation of all populations is necessary.     

Development of microsatellite markers in Myotis sodalis and cross-species amplification in M. gricescens, M. leibii, M. lucifugus, and M. septentrionalis

The Indiana bat (Myotis sodalis) is a highly endangered vespertilionid bat whose distribution is associated with limestone caves in the eastern United States. We present nine new polymorphic nuclear microsatellite markers for Myotis sodalis developed using an enriched library method. A total of 62 M. sodalis from two populations were used to estimate genetic diversity parameters. In M. sodalis, the number of alleles observed for each locus ranged from 17 to 48 alleles and expected heterozygosity ranged from 0.894 to 0.973. The 9 microsatellite markers were also tested on M. gricescens, M. leibii, M. lucifugus, and M. septentrionalis. These polymorphic microsatellite markers provide a valuable tool for investigating the population genetics of these species and will provide important genetic data useful for the conservation and recovery of the endangered Indiana bat.     

Twenty polymorphic microsatellite markers in the Asiatic lion (Panthera leo persica)

The Asiatic lion (Panthera leo persica) is driven to a single habitat in Gir forests in India for its survival. In order to devise adequate conservation and management strategies for this critically endangered species, it is important to characterize its genetic diversity and understand its population structure. Here we report twenty microsatellite loci, in addition to seven reported earlier, from the genome of a pure Asiatic lion. The microsatellite loci described here will provide potentially useful markers for the assessment of genetic variability in the only existing wild population of the Asiatic lions and other big cat species.     

Microsatellite DNA primers for the endangered vermilion darter,Etheostoma chermocki,and cross‐species amplification in other darters (Percidae: Etheostoma)

The endangered vermilion darter (Etheostoma chermocki) is endemic to the Black Warrior River system in the Mobile Basin in Alabama. Restoration and conservation of this species require an understanding of its population genetic structure, which can be characterized using microsatellite DNA. Nine microsatellite loci were developed; eight loci were polymorphic. Although observed heterozygosity was lower than expected heterozygosity in most polymorphic loci, only one locus showed significant deviation from Hardy–Weinberg equilibrium. These nine markers were tested in an additional 24 species of Etheostoma and appear to have sufficient allelic variation to be useful in studies of population genetic structure.     

Six new polymorphic microsatellite loci isolated and characterized from the African savannah elephant genome

The African savannah elephant (Loxodonta africana) is a ‘keystone’ species that plays a vital role in regulating the dynamics of both plant and animal communities and yet it is endangered and its numbers have been reduced to approximately 500 000 across their entire continental range. Molecular genetic markers are important tools for providing genetic information useful in formulating effective management and conservation strategies for the surviving elephant populations. We describe the isolation and characterization of six new polymorphic microsatellite markers in the African savannah elephant and demonstrate that these loci can be PCR (polymerase chain reaction)‐multiplexed, a desirable attribute that saves costs in large‐scale microsatellite screening.