PERMANENT GENETIC RESOURCES: Isolation of microsatellite loci from the endangered plant Sibara filifolia (Brassicaceae)

Sibara filifolia (Brassicaceae) is a federally endangered annual herb found on two of the California Channel Islands. Previous studies based on allozymes revealed little genetic variability on San Clemente Island. Nine polymorphic microsatellite loci were isolated from individuals on San Clemente Island. We found low levels of allelic variation (mean N_A = 2.3), with seven loci exhibiting significant deviations from Hardy–Weinberg equilibrium (P < 0.01) and 10 pairs of loci exhibiting significant linkage disequilibrium (P < 0.01). Most of the observed variability (mean H_O = 0.003) occurred among populations or in rare homozygous individuals.     

Isolation and characterization of variable microsatellite loci in Lilium philadelphicum (Liliaceae)

Here we describe the characterization of six polymorphic microsatellite loci for Lilium philadelphicum (Liliaceae). Polymorphism levels ranged from 7 to 30 alleles per locus with a mean number of 15 alleles per locus. Observed heterozygosity (H_O) ranged from 0.17 to 0.82 and did not deviate from Hardy–Weinberg expectations in the three western Montana populations included in the analysis. These loci are proving useful in studying gene flow between populations of this species distributed across its range in North America.     

Sex‐biased dispersal and high levels of gene flow among local populations in the argentine boa constrictor,Boa constrictor occidentalis

Abstract The knowledge of dispersal is essential to understand the ecology of any species, since population dynamics, spatial distribution and genetic structure are closely tied to patterns of movement. In this paper we estimate dispersal patterns in natural populations of the endangered snake Boa constrictor occidentalis (Serpentes, Boidae), using allozymes as genetic markers. Blood samples were obtained from a total of 120 adult individuals of nine localities from two areas, Sobremonte and Pocho, in Argentina. Only four out of a total of 24 loci were polymorphic: 6‐Pgdh‐1, Cat‐1, Ldh and Hp. The values of expected and observed mean heterozygosities, the percentage of polymorphic loci and mean number of alleles per locus for each population confirm the low levels of genetic variability in this snake. F_ST and N_em mean values were 0.0089 and 46.1 for Sobremonte and 0.0379 and 7.46 for Pocho, indicating important levels of gene flow. A comparison of F_ST between genders in both areas suggests a male‐biased dispersal, which could be explained by the characteristics of the mating system: males carry out an active mate search of receptive females, and philopatry could be selected in females due to the benefits of the familiarity with the natal area in the use of local resources.     

Low genetic differentiation among populations ofArabis serrata (Brassicaceae) along an altitudinal gradient

We investigated the extent of genetic differentiation among populations of fujihatazao,Arabis serrata along an altitudinal gradient at Mt. Fuji in Shizuoka Prefecture. This species is a perennial plant, widely distributed in Japan forming small isolated populations. However, at Mt. Fuji, this species constitutes a large population distributed from 1440 to 2400 m altitude. A total of 411 individuals were sampled from ten subpopulations. Eighteen loci were detected on eleven enzyme systems. Eleven loci were monomorphic and seven loci were polymorphic with a mean of 2.11 alleles per loci. Nei's genetic distance (mean 0.01) and genetic identity (mean 0.968) were very similar among populations indicating a low genetic differentiation. The total genetic diversity (H _T ) estimated for the polymorphic loci was, in average, 0.396. The mean gene differentiation (G_ST=0.091) was very low. Gene frequency of seven polymorphic loci was analyzed by spatial autocorrelation methods based on Moran's indexes. Only Pgi-3 exhibited a significant negative autocorrelation (−0.160;P<0.05); other loci values ranged from −0.134 to 0.027. Gene flow estimated by indirect methods varied between genes but most of the values were high (meanNm=20.8) suggesting that subpopulations at different altitudes are probably connected. Despite plants at different altitudes present different ecological traits (e.g., differences in phenology, growth and reproductive traits), subpopulations ofA. serrata are still low differentiated, at least for the loci studied. This may be explained by the recent origin of some habitats (e.g., second crater and surrounded areas) in this locality.     

Isolation and characterization of polymorphic microsatellite loci from Tetratheca ericifolia (Elaeocarpaceae)

We identified 11 informative microsatellite loci in Tetratheca ericifolia from an (AG)_n‐enriched library. Using these loci on 32 individuals from two populations (16 individuals from each), we detected an average of 11.3 alleles per locus (range of five to 21, average expected heterozygosity of 0.728), of which 56% were unique to one population or the other. All loci were amplifiable in seven to 12 of a further 12 species of Tetratheca under the same reaction conditions. The markers will be useful tools for evolutionary studies of this Australian endemic group.     

Development of polymorphic microsatellite loci in the hermaphroditic freshwater snails Drepanotrema surinamense and Drepanotrema depressissimum

We characterized new variable microsatellites in two congeneric species of hermaphroditic freshwater snails (Drepanotrema depressissimum and D. surinamense), as well as conditions for multiplexing and simultaneously genotyping sets of loci. D. depressissimum had high mean gene diversity (> 0.8) and large number of alleles (= 10.9) per population. Most loci and populations were at Hardy–Weinberg equilibrium. The F_ST estimates were low among lesser Antilles populations and larger with a Venezuelan population. Far less diversity was found in D. surinamense with mean number of alleles and gene diversity per population of 2.8 and 0.34, respectively. Very few heterozygous individuals were observed. The most likely explanation is a high selfing rate (> 0.825) in this species. Unsurprisingly, the F_ST estimates among populations were much higher than in D. depressissimum. Cross‐species amplification in three congeneric species was on the whole unsuccessful.     

Cross‐species amplification of microsatellite loci in Aquilegia and Semiaquilegia (Ranunculaceae)

We developed 16 microsatellite loci from an F₂ hybrid between Aquilegia formosa and Aquilegia pubescens. In samples of 28 individuals, we found an average of 14 alleles per locus from each parental species. We tested these loci for cross‐amplification in 10 additional species of Aquilegia and found that all 16 loci amplified in other North American species and 12 consistently amplified in European or Asian species. Nine loci amplified in the sister species to Aquilegia, Semiaquilegia adoxoides. The success of cross‐species amplification suggests that these microsatellites should prove useful for studies in a broad range of Aquilegia species.     

Identification of microsatellite loci in Collinsia verna (Veronicaceae)

We developed eight polymorphic microsatellite loci for Collinsia verna (Veronicaceae). In a sample of 18–35 individuals from a single population, we found two to 15 alleles per locus (mean 8.3). We also tested these loci for cross‐amplification in all 22 species in the tribe Collinseae. Overall, more than half the species in the tribe amplified one microsatellite while three species most closely related to C. verna (Collinsia violacea, Collinsia parviflora and Collinsia grandiflora) amplified multiple microsatellite loci. These microsatellite loci will be used in future studies of mating system in this tribe and other quantitative genetic and population genetic studies.     

Polymorphic microsatellite loci in the Coffee Berry Borer,Hypothenemus hampei (Coleoptera,Scolytidae)

Microsatellite loci were isolated from the coffee berry borer, Hypothenemus hampei, which is regarded as the major pest in coffee cultures. Seven polymorphic loci were obtained from an enriched genomic library. A low to moderate genetic diversity was observed per locus, with an observed number of alleles ranging from two to five in the 39 unrelated individuals sampled from Ethiopia. A clear deficit of heterozygotes within the population (mean heterozygosities, H_O = 0.10/H_E = 0.50) and an extreme inbreeding (F_IS, 0.70–1.00) were demonstrated. Cross‐species amplifications showed that some of the markers could be useful in two closely related Hypothenemus species.     

Isolation and characterization of microsatellites in Chinese soft‐shelled turtle,Pelodiscus sinensis

Fifteen polymorphic microsatellite loci were developed for the Chinese soft‐shelled turtle (Pelodiscus sinensis) from the (GT)_n microsatellite‐enriched genomic library, using the fast isolation by amplified fragment length polymorphism of sequences containing repeats protocol. The polymorphism of all 15 loci ranged from two to seven alleles with observed heterozygosities ranging from 0.03 to 0.98 (mean 0.43) in one population of 40 individuals. These novel loci will be helpful for understanding the population structure at genetic level and marker‐assisted breeding of this vulnerable species.     

Population genomics of local adaptation versus speciation in coral reef fishes (Hypoplectrus spp,Serranidae)

Are the population genomic patterns underlying local adaptation and the early stages of speciation similar? Addressing this question requires a system in which (i) local adaptation and the early stages of speciation can be clearly identified and distinguished, (ii) the amount of genetic divergence driven by the two processes is similar, and (iii) comparisons can be repeated both taxonomically (for local adaptation) and geographically (for speciation). Here, we report just such a situation in the hamlets (Hypoplectrus spp), brightly colored reef fishes from the wider Caribbean. Close to 100,000 SNPs genotyped in 126 individuals from three sympatric species sampled in three repeated populations provide genome‐wide levels of divergence that are comparable among allopatric populations (F_st estimate = 0.0042) and sympatric species (F_st estimate = 0.0038). Population genetic, clustering, and phylogenetic analyses reveal very similar patterns for local adaptation and speciation, with a large fraction of the genome undifferentiated (F_st estimate ≈ 0), a very small proportion of F_st outlier loci (0.05–0.07%), and remarkably few repeated outliers (1–3). Nevertheless, different loci appear to be involved in the two processes in Hypoplectrus, with only 7% of the most differentiated SNPs and outliers shared between populations and species comparisons. In particular, a tropomyosin (Tpm4) and a previously identified hox (HoxCa) locus emerge as candidate loci (repeated outliers) for local adaptation and speciation, respectively. We conclude that marine populations may be locally adapted notwithstanding shallow levels of genetic divergence, and that from a population genomic perspective, this process does not appear to differ fundamentally from the early stages of speciation.     

Development and characterization of microsatellite markers from the yellow passion fruit (Passiflora edulis f. flavicarpa)

Here we described the development of the first set of Passiflora microsatellite loci isolated from an enriched genomic library. A sample of 43 individuals from 12 accessions of the yellow passion fruit was used to characterize those loci, which revealed up to 20 alleles per locus. Two loci were monomorphic. The observed (H_O) and expected (H_E) heterozygosities were very similar, as expected for a self‐incompatible species. Allelic diversity (H_T) was 0.444. This set of markers will permit genetic structure analyses of cultivated and wild populations of Passiflora, and contribute for integrating genetic maps based on dominant markers, as they can provide bridge alleles.     

Genetic diversity of the endangered endemic Agave victoriae-reginae (Agavaceae) in the Chihuahuan Desert

Long-lived perennials are a species-rich, ecologically important component of the North American deserts, yet we know little about their genetic structure, information important for their conservation. Agave victoriae-reginae is an endemic of the Chihuahuan Desert of northern Mexico that is endangered by collection for the ornamental trade. We examined levels and patterns of variation at ten polymorphic allozyme loci in ten populations representing the range of the species. Levels of genetic variation (mean H_e= 0.335) and differentiation (mean F_ST = 0.236) were high. Phenetic clustering suggested the existence of at least three distinct groups of populations. If this pattern of variation is representative of other long-lived desert perennials, it may explain the species richness of this group and will pose a real challenge to gene conservation efforts.     

Genetic variation and hybridization in SwedishSchoenus (Cyperaceae)

Schoenus ferrugineus andS. nigricans have restricted distributions in Sweden and are almost exclusively confined to calcareous fen habitats. AtS. nigricans sites,S. ferrugineus is usually also present, and hybrids are frequently found. In this report, I used allozymes to estimate the amount of gene flow between the two species, and to compare the partitioning of genetic diversity in each of them. Thirteen loci were analysed at eight different enzyme systems. Seven loci were variable between or within the species. The two species had completely different alleles at two of the seven variable loci, whereas there was overlap at five loci. In all, 22 different alleles were found. Six of these alleles were confined toS. nigricans, and five alleles were confined toS. ferrugineus. Nei's genetic identity was 0.55.—InS. ferrugineus, three loci (23%) were polymorphic, and the average number of alleles per polymorphic locus was 2.0 (each polymorphic locus had two alleles). InS. nigricans, three loci (23%) were polymorphic, and the average number of alleles per polymorphic locus was 2.3.—The proportion of genetic diversity due to variation among sites (G _ST) was fairly similar in the two species, mean over loci = 0.12 inS. ferrugineus and 0.15 inS. nigricans. However, the proportion of genetic diversity due to variation among individuals within sites (G _IS) differed markedly between the two species, mean over loci = 0.54 inS. ferrugineus and 0.17 inS. nigricans. Accordingly, there was a much higher individual heterozygosity inS. nigricans than inS. ferrugineus. — Most hybrids were interpreted as F₁ hybrids. However, a small proportion, 0.5–1.6 %, were F_n hybrids or back-crosses.—On the Swedish mainland, all former occurrences ofS. nigricans are extinct, but viable hybrids are still present at a few sites in southernmost Sweden.     

Characterization of 10 trinucleotide microsatellite loci in the Critically Endangered Pyrenean yam Borderea chouardii (Dioscoreaceae)

The low levels of allozymic variability found in the Critically Endangered Borderea chouardii prompted us to develop microsatellite markers to assess the genetic variability and population structure for the adequate conservation management of this species. A (CTT)_n‐enriched partial genomic library was constructed. Ten polymorphic microsatellite loci were isolated from it, rendering 51 alleles in 47 individuals analysed. The allelic pattern observed for all of the loci with more than two alleles suggests that B. chouardii is tetraploid.     

Characterization of 12 polymorphic microsatellite loci in the Japanese bush warbler Cettia diphone

Japanese bush warblers, Cettia diphone, are a common species in Japan and have a polygynous breeding system. In the breeding ground some males have their own territories with more than one female. Male floaters are also not uncommon in the breeding ground. To understand breeding strategy in this species, exact parentage should be elucidated. In order to obtain a tool for this purpose, we isolated 34 microsatellite loci from a genomic library in this species and developed primers for 12 loci. These primers were tested in the Japanese bush warbler and successfully amplified. In analyses of 49 unrelated individuals, allelic numbers ranged from two to 22, and observed heterozygosity (H_O) ranged from 0.27 to 0.854 except for two loci (Cdi29 and Cdi35a) with H_O < 0.1.     

Low Levels of Genetic Diversity within Populations of Hosta clausa (Liliaceae)

Abstract Genetic diversity of Korean populations in Hosta clausa was investigated using starch gel electrophoresis. Hosta clausa is widespread, grows only along streamsides, and has both sexual and asexual reproduction. Populations of the species are small and isolated. Thirty-two percent of the loci examined were polymorphic, and mean genetic diversity within populations (He_p=0.082) was lower than mean estimates for species with very similar life history characteristics (0.131), particularly for its congener H. yingeri (0.250). The mean number of multilocus genotypes per population was 8.7, and genotypic diversity index (D_G) was 0.84. Significant differences in allele frequencies among populations were found in all seven polymorphic loci (P < 0.001). About one-fifth of the total allozyme variation was among populations (G_ST=0.192). Indirect estimate of the number of migrants per generation (Nm=0.48, calculated from mean G_ST) and nine private alleles found indicate that gene movement among populations was low. The low levels of genetic diversity within populations and the relatively high levels of genetic diversity among populations suggest that strong moist habitat preferences, clonal reproduction, low level of gene flow among populations, genetic drift, and historical events may have played roles in the genetic structuring of the species.     

Evolutionary implications of allozyme and RAPD variation in diploid populations of dioecious buffalograss Buchloë dactyloides

Buffalograss, Buchloë dactyloides, is widely distributed throughout the Great Plains of North America, where it is an important species for rangeland forage and soil conservation. The species consists of two widespread polyploid races, with narrowly endemic diploid populations known from two regions: central Mexico and Gulf Coast Texas. We describe and compare the patterns of allozyme and RAPD variation in the two diploid races, using a set of 48 individuals from Texas and Mexico (four population samples of 12 individuals each). Twelve of 22 allozyme loci were polymorphic, exhibiting 35 alleles, while seven 10-mer RAPD primers revealed 98 polymorphic bands. Strong regional differences were detected in the extent of allozyme polymorphism: Mexican populations exhibited more internal gene diversity (H_e= 0.20, 0.19) than did the Texan populations (H_e= 0.08, 0.06), although the number of RAPD bands in Texas (n= 62) was only marginally smaller than in Mexico (n= 68). F-statistics for the allozyme data, averaged over loci, revealed strong regional differentiation (mean F_RT=+ 0.30), as well as some differentiation among populations within regions (mean F_PR=+ 0.09). In order to describe and compare the partitioning of genetic variation for multiple allozyme and RAPD loci, we performed an Analysis of Molecular Variance (AMOVA). AMOVA for both allozyme and RAPD data revealed similar qualitative patterns: large regional differences and smaller (but significant) population differences within regions. RAPDs revealed greater variation among regions (58.4% of total variance) than allozymes (45.2%), but less variation among individuals within populations (31.9% for RAPDs vs. 45.2% for allozymes); the proportion of genetic variance among populations within regions was similar (9.7% for RAPDs vs. 9.6% for allozymes). Despite this large-scale concordance of allozyme and RAPD variation patterns, multiple correlation Mantel techniques revealed that the correlations were low on an individual by individual basis. Our findings of strong regional differences among the diploid races will facilitate further study of polyploid evolution in buffalograss.     

Patterns of within and between-colony microsatellite variation in the endangered Vancouver Island marmot (Marmota vancouverensis): implications for conservation

Among the 14 extant species of the genus Marmota the Vancouver Island marmot (Marmota vancouverensis) is the most endangered. In 2007 as few as 85 individuals were left in the wild, with an additional 162 individuals maintained in captivity. To facilitate genetic monitoring of both wild and captive populations, polymorphic genetic markers were identified. Thirty-three different microsatellite loci were tested for amplification and variability in ≥30 wild-born individuals. Only 11 of these loci proved to be polymorphic and were subsequently analysed in 105 samples collected from wild Vancouver Island marmots. The average number of alleles (A) at those 11 loci was only 2.1, and the intraspecific variation (H _E between 8 and 23% within colonies) was low compared to other marmot species. Variation within the small and geographically isolated Mt. Washington colony was particularly low (A = 1.3, H _E = 0.08). Genetic distances between the Mt. Washington colony (11 individuals) and those of the Nanaimo Lakes region (94 individuals) on southern Vancouver Island were large (D values ranging from 0.42 to 0.50), while genetic distances among colonies within the latter area were much smaller (D values from 0.01 to 0.13). Given the low within-population genetic variation, and the resulting risk of inbreeding depression at Mt. Washington, we support the decision to maximize overall genetic variation of the species by crossbreeding marmots from the two different areas despite the possibility of local adaptation.