Highly polymorphic microsatellite markers for the highly polymorphic strawberry poison-dart frog and some of its congeners

Members of the poison-dart frog genus Oophaga, including the strawberry poison-dart frog (O. pumilio) display among the most striking examples of color polymorphism of any amphibians. We developed twelve novel microsatellite markers with di-, tri-, and tetra-nucleotide repeats for this genus. These loci are highly polymorphic with between 2 and 29 alleles (average = 14.7) and high heterozygosity (H _O = 0.704). These highly polymorphic markers should be useful for resolving fine-scale genetic differences between the different color morphs of these highly variable species, for investigations into the ecological importance of this variation, and for determining the effects of habitat loss and fragmentation on population persistence in these species.     

An ecological genetic delineation of local seed‐source provenance for ecological restoration

An increasingly important practical application of the analysis of spatial genetic structure within plant species is to help define the extent of local provenance seed collection zones that minimize negative impacts in ecological restoration programs. Here, we derive seed sourcing guidelines from a novel range‐wide assessment of spatial genetic structure of 24 populations of Banksia menziesii (Proteaceae), a widely distributed Western Australian tree of significance in local ecological restoration programs. An analysis of molecular variance (AMOVA) of 100 amplified fragment length polymorphism (AFLP) markers revealed significant genetic differentiation among populations (Φ_PT = 0.18). Pairwise population genetic dissimilarity was correlated with geographic distance, but not environmental distance derived from 15 climate variables, suggesting overall neutrality of these markers with regard to these climate variables. Nevertheless, Bayesian outlier analysis identified four markers potentially under selection, although these were not correlated with the climate variables. We calculated a global R‐statistic using analysis of similarities (ANOSIM) to test the statistical significance of population differentiation and to infer a threshold seed collection zone distance of ~60 km (all markers) and 100 km (outlier markers) when genetic distance was regressed against geographic distance. Population pairs separated by >60 km were, on average, twice as likely to be significantly genetically differentiated than population pairs separated by <60 km, suggesting that habitat‐matched sites within a 30‐km radius around a restoration site genetically defines a local provenance seed collection zone for B. menziesii. Our approach is a novel probability‐based practical solution for the delineation of a local seed collection zone to minimize negative genetic impacts in ecological restoration.     

Five hundred microsatellite loci for Peromyscus

Mice of the genus Peromyscus, including several endangered subspecies, occur throughout North America and have been important models for conservation research. We describe 526 primer pairs that amplify microsatellite DNA loci for Peromyscus maniculatus bairdii, 467 of which also amplify in Peromyscus polionotus subgriseus. For 12 of these loci, we report diversity data from a natural population. These markers will be an important resource for future genomic studies of Peromyscus evolution and mammalian conservation.     

Population genetics and conservation of the threatened southeastern beach mouse (Peromyscus polionotus niveiventris): subspecies and evolutionary units

We investigated genetic diversity within the southeastern beach mouse (SEBM-Peromyscus polionotus niveiventris) and also tested the hypothesis that the subspecies recognition of P.p. niveiventris, based on size and color differences, is congruent with this taxon representing a discrete evolutionary lineage. We used ten polymorphic microsatellite loci and mitochondrial cytochrome-b gene DNA sequences to investigate genetic diversity and population structure within the SEBM, and to determine the level of divergence between the SEBM and the nearest known inland subspecies of the oldfield mouse (Peromyscus polionotus rhoadsi). Moderate genetic distances were observed between the SEBM and the inland oldfield mouse based on microsatellite data, with F _ST values ranging from 0.11 to 0.22 between these taxa. Additionally, mitochondrial DNA haplotypes of the SEBM formed a distinct monophyletic group relative to haplotypes sampled from P. p. rhoadsi. Based on previous estimates of rates of mitochondrial DNA evolution in rodents, we inferred that Pleistocene sea-level fluctuations are likely responsible for the historical isolation of the SEBM lineage from mainland P. polionotus. Our data demonstrate the genetic distinctiveness of the SEBM, justifying the current subspecies designation for the SEBM and its continued protection under the United States Endangered Species Act. We classify the Cape Canaveral and Smyrna Dunes Park populations of SEBM as a single evolutionary significant unit. The two known extant allopatric populations of the SEBM showed some differentiation in microsatellite frequencies and were moderately reciprocally distinguishable based on assignment to distinct genetic clusters by a Bayesian admixture procedure. These results justify the classification of these two extant SEBM populations as distinct management units that should be independent targets of management and conservation attention.     

Spatial and temporal dynamics of the male effective population size in bumblebees (Hymenoptera: Apidae)

Eusociality and male haploidy of bumblebees (Bombus spp.) enhance the deleterious effects of population decline and aggravate the degeneration of population fitness compared to solitary and diploid species. The highly dispersive male sex may be the prime driver to connect otherwise isolated populations. We therefore studied the temporal and spatial structure of the male population of Bombus terrestris (Linnaeus 1758) and Bombus lapidarius (Linnaeus 1758) using microsatellite DNA markers. We found that the majority of the males in a 1000 m2 sampling area originated from colonies located outside of the workers foraging range, which was consistent with the genetic distances among colonies. The analyses of temporal population sub-structure based on both colony detection rate over time and the clustering software STRUCTURE consistently suggested one large and temporally unstructured male population. Our results indicate an extended male flight distance for both species. Though the range of queen dispersal remains to be studied, the effective size (N _e) of bumblebees is increased by extended male mating flight ranges (A _m ) exceeding worker foraging distance by factor 1.66 (A _m  = 69.75 km²) and 1.74 (A _m  = 13.41 km²), B. terrestris and B. lapidarius, respectively. Thus this behaviour may counteract genetic deprivation and its effects. All populations were genetically highly diverse and showed no signs of inbreeding. We discuss the implications of our findings in context of bumblebee population dynamics and conservation. We also highlight the effects and benefits of sampling both workers and males for population genetic studies.     

Mitochondrial diversification of the Peromyscus mexicanus species group in Nuclear Central America: biogeographic and taxonomic implications

The mountain mice of the Peromyscus mexicanus group currently encompass six known species; however, the limits between species remain uncertain, with two considered monotypic and the other four having multiple associated subspecific names. Based on the most comprehensive sampling of the group throughout its distribution in Nuclear Central America, we used data of the mitochondrial cytochrome b gene to assess its genetic diversity, phylogeny, and main biogeographic and diversification patterns. Our mitochondrial phylogeny only partially reflects the current taxonomy of the group, in agreement with some of the taxonomically recognized species. Specifically, our phylogenetic results show that the group is highly structured, including four main clades with genetic distances ranging from 11 to 8.6%. A remarkable level of differentiation is found at a more local level, defined as 15 different lineages with high nucleotide and haplotype diversity (π = 0.068, h = 0.99), and with divergence and genetic distance values (p‐uncorrected = 9.9–2.4%; K2P = 10.8–3.0%) similar to values observed between species within Peromyscus. Accordingly, we propose that the reference name P. mexicanus is polyphyletic and should be restricted to the mountains of central Mexico west of the Isthmus of Tehuantepec. We suggest to limit the other five recognized specific names to equal number of lineages, as monophyletic, and to revalidate three junior synonyms: Peromyscus salvadorensis, P. nicaraguae and P. tropicalis. The Isthmus of Tehuantepec, the Motagua‐Polochic‐Jocotán fault system, the Maya Highlands and the Honduras Depression are examples of geographic features that are likely associated with the differentiation of main lineages. Some other lineages may represent candidate species, hence the need to review the taxonomic status of the entire P. mexicanus group.     

Polymorphic microsatellites identified by cross-species amplifications in the European Coot Fulica atra

We tested the cross-amplification of 26 microsatellites developed for passerines and an additional three developed for Gallinula species in eight European Coots from two populations. Sixteen microsatellite markers successfully amplified, of which nine were polymorphic with 2–6 alleles (mean 3.7 alleles) and an expected heterozygosity (H _e) ranging from 0.375 to 0.805 (mean H _e = 0.589). On average, we found 2.22 alleles/locus and a mean H _e of 0.440 in one nest, and 2.56 alleles/locus and a mean H _e of 0.494 in the other one. These nine polymorphic markers could be of potential use in studies of genetic variability, population structure and reproductive strategy of European Coots.     

Polymorphic microsatellite DNA markers in black grouse (Tetrao tetrix)

Ten GATA microsatellite DNA markers were isolated and characterized from black grouse (Tetrao tetrix) using an enrichment cloning procedure. High levels of heterozygosity (mean H_O = 0.74 ± 0.026), and a large number of alleles (range = 5–16) were resolved in 70 individuals, indicating these markers will be useful for examining parentage, inbreeding and population structure in black grouse. No evidence for linkage disequilibrium or the presence of null alleles was found.     

Conservation genetics assessment and phylogenetic relationships of critically endangered Hucho bleekeri in China

Hucho bleekeri is a critically endangered salmonid fish found in the Yangtze River drainage in China. In this study, the genetic diversity of a small population (n = 43) was first assessed with partial mitochondrial DNA sequences (D‐loop region and a cytochrome b gene [CYTB] gene fragment) and 15 microsatellite markers. Low levels of nucleotide diversity (Pi) were demonstrated in the H. bleekeri population based on the two mitochondrial DNA markers. The number of haplotypes (h) and the haplotype diversity (Hd) in the D‐loop region (12 haplotypes and Hd = 0.8208) were higher than in the CYTB gene fragment (three haplotypes and Hd = 0.0941). The number of microsatellite alleles (Na) ranged from 2 to 13 in these individuals. The mean observed heterozygosity (Ho) and the expected heterozygosity (He) were 0.59719 and 0.44735, respectively. Analysis of molecular variance showed that the degree of differentiation in the population was low (F_ST = 0.04041) and the coefficient of inbreeding (F_IS) was negative, indicating no obvious evidence of inbreeding in this population. A demographic assessment suggested that this species expanded a long time ago, but has suffered great losses in recent years. A molecular phylogenetic analysis clearly indicated that H. bleekeri is not introgressed by Brachymystax lenok tsinlingensis. The baseline population genetic information supplied by this study will be vital in monitoring this highly threatened species.     

Isolation and characterization of eight microsatellite loci from <Emphasis Type="Italic">Lycorma delicatula</Emphasis> (White) (Hemiptera: Fulgoridae) for population genetic analysis in Korea

Lycorma delicatula (White) is native to China but is becoming an important insect pest in Korea. Polymorphic DNA markers like microsatellites are widely used for characterizing dispersal patterns and capacity of invasive insect pests which can contribute to designing effective management of the species. To facilitate such population genetic studies of L. delicatula in Korea, we isolated and characterized eight microsatellite loci for L. delicatula using a hybridization-biotin enrichment method. We further used these novel microsatellite loci to determine population genetic parameters for 33 L. delicatula specimens collected from Cheonan, South Korea where outbreaks of this species were first reported in Korea. The number of alleles per locus ranged from three to ten, with an average of 6.25. The mean expected (H _E) and observed heterozygosities (H _O) were 0.575 and 0.626, respectively. The eight loci showed no deviation from Hardy–Weinberg equilibrium according to the adjusted significance threshold (P = 0.00625), and there was no linkage disequilibrium between each pair of these eight markers. Bayesian cluster analysis using the program structure revealed no evidence of genetic structuring in L. delicatula samples from Cheonan. These new microsatellite markers will be widely applicable to future ecological genetic studies of L. delicatula, including assessment of the level of gene flow and genetic connectivity among populations that are necessary for effective management and monitoring of the species.     

Cross‐species amplification among peromyscines of new microsatellite DNA loci from the oldfield mouse (Peromyscus polionotus subgriseus)

We describe polymerase chain reaction (PCR) primers and conditions to amplify 11 microsatellite DNA loci isolated from the oldfield mouse (Peromyscus polionotus subgriseus). These were tested for amplification using nine species and subspecies maintained at the Peromyscus Genetic Stock Center, with an average success rate of 65% and two loci amplifying in all species. Polymorphism was tested within the P. polionotus subgriseus and the recently obtained P. maniculatus sonorensis colonies. P. p. subgriseus had modest numbers of alleles per locus (1–4), whereas P. m. sonorensis had many alleles per locus (5–10) and high expected heterozygosities (0.625–0.878).     

Influence of translocation strategy and mating system on the genetic structure of a newly established population of island ptarmigan

Island populations and populations established by reintroductions are prone to extinction, in part because they are vulnerable to deterministic and stochastic phenomena associated with geographic isolation and small population size. As population size declines, reduced genetic diversity can result in decreased fitness and reduced adaptive potential, which may hinder short- or long-term population viability. We used 32 microsatellite markers to investigate the conservation genetics of a newly established population of Evermann’s Rock Ptarmigan (Lagopus muta evermanni) at Agattu Island, in the western Aleutian Archipelago, Alaska. We found low genetic diversity (observed heterozygosity = 0.41, allelic richness = 2.2) and a small effective population size (N _e  = 28.6), but a relatively large N _e /N ratio = 0.55, which was attributed to multiple paternity in 80% of the broods and low reproductive skew among males (λ = 0.29). Moreover, successful breeding pairs were less related to each other than random male–female pairs. For conservation efforts based on reintroductions, a mating system with high rates of multiple paternity may facilitate retention of genetic diversity, thereby reducing the potential for inbreeding in small or isolated populations. Our results underscore the importance of quantifying genetic diversity and understanding the breeding behavior of translocated populations.     

Genetic variability, body characteristics and reproductive parameters of neighbouring rural and urban common kestrel (Falco tinnuculus) populations

We tested the genetic and ecological differences between neighbouring urban and rural populations of common kestrels (Falco tinnuculus) in southern Bohemia. The aims were to (1) assess the genetic variability of the studied kestrel populations using microsatellite markers, (2) check the genetic relatedness of individuals within the urbanization gradient, and (3) compare possible gradients of body characteristics and reproductive parameters on the urbanization gradient. The mean expected allelic polymorphism did not differ among the studied populations, which were not genetically separated (F _ST  = 0.0003, P = 0.781). Also, an individual assignment test did not show a separation of these populations. Urban kestrels that bred in the city centre were indicatively more related than others, and no relationship was found in the rural kestrel population. Kestrel females were heavier towards the city centre, but males did not show this relationship. Nest distance from the city centre had no significant effect on any of the tested reproductive parameters. Our results do not support the notion of genetic differentiation between rural and urban kestrels, but revealed trends in body characteristics and genetic relatedness along the urbanization gradient.     

Speciation genomics and a role for the Z chromosome in the early stages of divergence between Mexican ducks and mallards

Speciation is a continuous and dynamic process, and studying organisms during the early stages of this process can aid in identifying speciation mechanisms. The mallard (Anas platyrhynchos) and Mexican duck (A. [p.] diazi) are two recently diverged taxa with a history of hybridization and controversial taxonomy. To understand their evolutionary history, we conducted genomic scans to characterize patterns of genetic diversity and divergence across the mitochondrial DNA (mtDNA) control region, 3523 autosomal loci and 172 Z‐linked sex chromosome loci. Between the two taxa, Z‐linked loci (Φ_ST = 0.088) were 5.2 times more differentiated than autosomal DNA (Φ_ST = 0.017) but comparable to mtDNA (Φ_ST = 0.092). This elevated Z differentiation deviated from neutral expectations inferred from simulated data that incorporated demographic history and differences in effective population sizes between marker types. Furthermore, 3% of Z‐linked loci, compared to <0.1% of autosomal loci, were detected as outlier loci under divergent selection with elevated relative (Φ_ST) and absolute (d_XY) estimates of divergence. In contrast, the ratio of Z‐linked and autosomal differentiation among the seven Mexican duck sampling locations was close to 1:1 (Φ_ST = 0.018 for both markers). We conclude that between mallards and Mexican ducks, divergence at autosomal markers is largely neutral, whereas greater divergence on the Z chromosome (or some portions thereof) is likely the product of selection that has been important in speciation. Our results contribute to a growing body of literature indicating elevated divergence on the Z chromosome and its likely importance in avian speciation.     

Limited cross‐species microsatellite amplification and the isolation and characterization of new microsatellite markers for the greater stick‐nest rat (Leporillus conditor)

The greater stick‐nest rat (Leporillus conditor) is a conilurid rodent whose recent history provides an opportunity to examine genetic changes in reintroduced populations. We trialled 63 known microsatellite primers from Rattus rattus and Mus musculus in L. conditor. Three primer pairs produced polymorphic loci (number of alleles = 2–3, mean, H_E = 0.42). Subsequently, we isolated and characterized 12 novel polymorphic microsatellites (mean number of alleles = 5–16, mean H_E = 0.76) from L. conditor from genomic libraries for use in population genetic studies.     

Tandem repeat markers for population genetic studies of the protected California tiger salamander (<Emphasis Type="Italic">Ambystoma californiense</Emphasis>)

Habitat loss is the single greatest threat to persistence of the critically threatened California tiger salamander (Ambystoma californiense). To aid management plans that designate critical habitat for this species, I developed and characterized 21 tetranucleotide microsatellite markers using two native populations in Santa Barbara and Alameda Counties. Allelic variation and average heterozygosities were lower in the endangered Santa Barbara population (allele range 1–4, mean 2.4; H _O = 0.308 H _E = 0.288) compared with the threatened Alameda population (allele range 2–10, mean 6.7; H _O = 0.712, H _E = 0.722). In-depth population studies using these markers will provide vital information for plans to assign critical habitat that optimize gene flow among breeding populations, as well as for identifying non-native hybrid genotypes that threaten native A. californiense stocks. Beyond the conservation goals for A. californiense, the close phylogenetic relationships within the tiger salamander complex also suggest a broad utility for population studies using these markers.     

Genetic structure of the Painted Bunting and its implications for conservation of migratory populations

The Painted Bunting Passerina ciris is a Neotropical songbird which breeds primarily in the USA during the summer and migrates to Mexico, Central America, southern Florida and the Caribbean over the winter. Male Painted Buntings are brightly coloured, which makes them highly sought after as pets, particularly in Mexico, Central America and Europe. We used short sequence repeats (microsatellite DNA) to investigate the population genetic structure of the Painted Bunting and its implications in conservation management of migratory populations. We found a detectable level of population differentiation as revealed by pairwise F_ST and R_ST comparisons and Bayesian clustering analyses, with strong support for differentiation between eastern and western Painted Buntings (e.g. Oklahoma and Georgia F_ST = 0.1; P = 0.005; R_ST = 0.18; P = 0.04) in accordance with previous mitochondrial DNA analysis. We recovered additional support for two sub‐groups within the western clade. While linking migrant songbirds captured outside of the USA to their breeding populations remains a challenge, we show that natural levels of population genetic differentiation can be detected via microsatellite DNA markers and exploited in migratory connectivity studies. We also demonstrate the potential utility of our low‐cost markers for population identification of birds recovered from the pet trade by screening a small subset of samples (n = 5) collected as part of wildlife tracking. We discuss the implications of our results for future efforts to understand patterns of population decline in Painted Buntings more generally, as well as how we might expand this methodology to combat illegal pet‐trade activity in this and other songbird species.     

Evaluation of genetic introgression from domesticated pigs into the Ryukyu wild boar population on Iriomote Island in Japan

We evaluated genetic introgression from domesticated pigs into the Ryukyu wild boar (RWB) population on Iriomote Island based on their genetic structure and diversity. We used a combination of mitochondrial DNA D‐loop region (596 bp) polymorphisms and 23 microsatellite markers. RWBs (n = 130) were collected from 18 locations on Iriomote Island and compared with 66 reference samples of European and Asian domestic pigs. We identified six distinct haplotypes, involving 22 single nucleotide polymorphisms (including one insertion) in the RWB population. The phylogenetic tree had two branches: the RWB group and domestic lineage. Fourteen of 130 RWBs (10.8%) belonged to the European domestic lineage, including 11 RWBs from the Panari Islands, northwest of Iriomote Main Island (IMI). The heterozygosity values, total number of alleles, number of effective alleles and polymorphism information content of the RWB groups were lower than those of the European domestic groups. The RWB population on IMI had a lower heterozygous deficiency index (F_IS = 0.059) than did the other populations, which indicates that this population was more inbred. There was a large genetic distance (F_ST = 0.560) between RWBs on IMI and the Meishan populations. Structure analysis using the 23 microsatellite markers revealed that 16 RWBs had an admixture pattern between RWB and domesticated pig breeds. These results suggest that gene flow may have occurred from domestic pigs to RWBs and demonstrate that there was low genetic variation in the IMI population.     

Fine genetic mapping of greenbug aphid-resistance gene <Emphasis Type="Italic">Gb3</Emphasis> in <Emphasis Type="Italic">Aegilops tauschii</Emphasis>

The greenbug, Schizaphis graminum (Rondani), is an important aphid pest of small grain crops especially wheat (Triticum aestivum L., 2n = 6x = 42, genomes AABBDD) in many parts of the world. The greenbug-resistance gene Gb3 originated from Aegilops tauschii Coss. (2n = 2x = 14, genome D^tD^t) has shown consistent and durable resistance against prevailing greenbug biotypes in wheat fields. We previously mapped Gb3 in a recombination-rich, telomeric bin of wheat chromosome arm 7DL. In this study, high-resolution genetic mapping was carried out using an F_2:3 segregating population derived from two Ae. tauschii accessions, the resistant PI 268210 (original donor of Gb3 in the hexaploid wheat germplasm line ‘Largo’) and susceptible AL8/78. Molecular markers were developed by exploring bin-mapped wheat RFLPs, SSRs, ESTs and the Ae. tauschii physical map (BAC contigs). Wheat EST and Ae. tauschii BAC end sequences located in the deletion bin 7DL3-0.82–1.00 were used to design STS (sequence tagged site) or CAPS (Cleaved Amplified Polymorphic Sequence) markers. Forty-five PCR-based markers were developed and mapped to the chromosomal region spanning the Gb3 locus. The greenbug-resistance gene Gb3 now was delimited in an interval of 1.1 cM by two molecular markers (HI067J6-R and HI009B3-R). This localized high-resolution genetic map with markers closely linked to Gb3 lays a solid foundation for map based cloning of Gb3 and marker-assisted selection of this gene in wheat breeding.