Isolation and characterization of microsatellite loci in Amazonian red‐handed howlers Alouatta belzebul (Primates,Plathyrrini)

Ten highly polymorphic microsatellite loci are described for Amazonian red‐handed howlers (Alouatta belzebul), an endemic Brazilian primate species subject to intense hunting pressure. The number of alleles observed in 30 individuals ranged from nine to 20, and observed heterozygosity varied from 0.35 to 0.93. No linkage associations were evident from pairwise comparisons of loci. These microsatellites offer a powerful tool for fine‐scale studies of genetic structure in both captive colonies and wild populations of red‐handed howlers.     

Polymorphic microsatellite loci for population studies of the razor clam, <Emphasis Type="Italic">Sinonovacula constricta</Emphasis>

The razor clam (Sinonovacula constricta) is an important aquacultured bivalve in China. The natural populations of this species are decreasing quickly. To facilitate studies on genetic diversity and population structure of wild populations, microsatellites were isolated from a CA enriched genomic library. Eight microsatellite loci were polymorphic in 30 individuals from Chongming in Shanghai, China. The number of alleles per polymorphic locus varied from 6 to 13 and the values of observed heterozygosity and expected heterozygosity ranged from 0.350 to 1.000 and from 0.602 to 0.902, respectively. These microsatellites are being used in studying population differentiation and genetic diversity for effective conservation and management genetic resources of S. constricta.     

Microsatellite loci in eelgrass Zostera marina reveal marked polymorphism within and among populations

Using an enriched genomic library, we developed seven (CT)n/(GA)n microsatellite loci for eelgrass Zostera marina L. Enrichment is described and highly recommended for genomes in which microsatellites are rare, such as in many plants. A test for polymorphism was performed on individuals from three geographically separated populations (N = 15/population) and revealed considerable genetic variation. The number of alleles per locus varied between five and 11 and the observed heterozygosities for single loci ranged from 0.16 to 0.81 within populations. Mean allele lengths were markedly different among populations, indicating that the identified loci will be useful in studying population structure in Z. marina. As the frequency of the most abundant multilocus genotype within populations was always < 1%, these loci have sufficient resolving power to address clone size in predominantly vegetatively reproducing populations.     

Isolation and characterization of microsatellite marker loci in the Wagner’s mustached bat <Emphasis Type="BoldItalic">Pteronotus psilotis</Emphasis> (Chiroptera: Mormoopidae) and cross-amplification in other related species

Pteronotus psilotis, a mormoopid bat, is an insectivorous, gregarious and strict cave-dwelling species that is found areas between the sea level and an elevation of about 1000 masl. This species is present in diverse habitats ranging from rain forest to dry deciduous forest. Nine microsatellite loci were developed for Wagner’s mustached bat, Pteronotus psilotis using the next-generation sequencing approach, and their utility for population genetics studies was assessed. All loci were polymorphic (7–15 alleles) and characterized in 30 individuals from three P. psilotis populations, with the levels of observed and expected heterozygosity ranging from 0.280 to 0.867 and 0.584 to 0.842, respectively. One locus showed significant departures from Hardy–Weinberg expectations after Bonferroni correction. Cross-amplification in 11 other bat species was tested, for which eight microsatellites were successfully amplified, and of these seven were polymorphic. The development of these new microsatellite loci will contribute to investigations of genetic population structure, genetic diversity and gene flow in P. psilotis populations, as well as in other closely related bat species.     

Isolation and characterization of ten polymorphic microsatellite loci in Ixodes arboricola, and cross-amplification in three other Ixodes species

We characterized ten polymorphic microsatellite loci from the tree-hole tick, Ixodes arboricola. Loci were screened in 11–18 individuals from three Belgian populations and five to ten alleles were found at each locus. Seven loci did not show deviations from Hardy–Weinberg equilibrium conditions and there were no indications for null alleles at these loci. The three other loci showed significant heterozygote deficiencies in at least one population, and a high potential for the occurrence of null alleles. We observed no effect of potential host DNA on the scoring of the microsatellites. Cross-amplification of the microsatellites was tested in eight specimens of three congeneric species: I. ricinus, I. hexagonus and I. frontalis. Depending on the species, six or seven of the loci were amplified in ≥4 of the 8 specimens and were polymorphic in each of these species (except for Ixaf 11 in I. frontalis and I. ricinus). These loci thus provide a tool for population genetic analysis of I. arboricola. The suitability of these markers needs to be further investigated in its congeners.     

Evidence of population bottleneck in Astragalus michauxii (Fabaceae), a narrow endemic of the southeastern United States

Genetic factors such as decreased genetic diversity and increased homozygosity can have detrimental effects on rare species, and may ultimately limit potential adaptation and exacerbate population declines. The Gulf and Atlantic Coastal Plain physiographic region has the second highest level of endemism in the continental USA, but habitat fragmentation and land use changes have resulted in catastrophic population declines for many species. Astragalus michauxii (Fabaceae) is an herbaceous plant endemic to the region that is considered vulnerable to extinction, with populations generally consisting of fewer than 20 individuals. We developed eight polymorphic microsatellites and genotyped 355 individuals from 24 populations. We characterized the population genetic diversity and structure, tested for evidence of past bottlenecks, and identified evidence of contemporary gene flow between populations. The mean ratios of the number of alleles to the allelic range (M ratio) across loci for A. michauxii populations were well below the threshold of 0.68 identified as indicative of a past genetic bottleneck. Genetic diversity estimates were similar across regions and populations, and comparable to other long-lived perennial species. Within-population genetic variation accounted for 92 % of the total genetic variation found in the species. Finally, there is evidence for contemporary gene flow among the populations in North Carolina. Although genetic factors can threaten rare species, maintaining habitats through prescribed burning, in concert with other interventions such as population augmentation or (re)introduction, are likely most critical to the long term survival of A. michauxii.     

Isolation and characterization of 18 polymorphic microsatellite markers for the “Female Ginseng” Angelica sinensis (Apiaceae) and cross-species amplification

Angelica sinensis (Apiaceae) is an endangered alpine herb that is widely used as a medicinal plant in traditional Chinese medicine (TCM). Wild populations of A. sinensis have become quite rare in China. Thus, population genetics studies of this species are urgently needed for its effective conservation and sustainable use. However, to date, no microsatellite loci have been isolated in A. sinensis. To address this issue, we isolated 18 polymorphic loci and genotyped 120 individuals collected from 6 populations. The number of alleles per locus ranged from 1.2 to 5.5, and the average was 2.4. The observed and expected heterozygosity per locus for a population varied, respectively, from 0.000 to 0.983 (averaged at 0.198) and from 0.066 to 0.661 (averaged at 0.333). Deviation from the Hardy–Weinberg equilibrium (p < 0.01) was observed for 4 to 14 loci in various populations. These microsatellite markers were cross-amplified in 10 species affinis, and 7 loci were successfully amplified in all species. These microsatellite markers are useful for genetic studies, the conservation management of A. sinensis, and identification of A. sinensis.     

Isolation and characterization of microsatellites in European alpine marmots (Marmota marmota)

For future investigations of the mating system of a highly social mammal (Marmota marmota), we identified 16 new microsatellites using an enrichment protocol. Five loci were revealed to be polymorphic. The polymorphism was rather low (two to six alleles among 24 individuals). However, these markers, added to the other six published microsatellites for M. marmota and Spermophilus citellus, will help to assess dispersal patterns and test for genetic monogamy in alpine marmots from the European Alps.     

Case study of microsatellite polymorphism of European perch in selected commercially important lakes of Latvia

Polymorphism of selected microsatellite markers was described for European perch populations of inland Lake Kala and costal Lake Babites in Latvia. The data consisted of ten microsatellite loci analysed for 90 individuals from the Lake Babites (n?=?45) and the Lake Kala (n?=?45). Both lakes differ in water area, connection with the Gulf of Riga (Baltic Sea), lake type, and fish species. In the Lake Kala population, the average number of alleles per locus varied from 4 to 15 and in the population of Lake Babites from 4 to 12. Low but significant genetic differentiation was detected between populations (F_ST?=?0.046). Observed and expected heterozygosity in both lakes was similar (Kala: H_o?=?0.680 and H_e?=?0.816; Babites: H_o?=?0.693 and H_e?=?0.815). The number of unique alleles per locus was 2.3 in Lake Kala and 0.6 in Lake Babites. The present study showed relatively high polymorphism of analysed microsatellite markers.     

Isolation and characterization of 11 microsatellite primers for a temperate reef fish, the California sheephead (Semicossyphus pulcher)

Eleven microsatellites were characterized for Semicossyphus pulcher (California sheephead) using an enrichment protocol. The number of alleles varied from three to 14 for a sample of 40 individuals from two populations. Expected heterozygosities ranged from 0.311 to 0.891. All loci but one were in Hardy-Weinberg equilibrium. No evidence for linkage disequilibrium was observed. These polymorphic microsatellites will be useful for genetic diversity and connectivity analyses of S. pulcher.     

Isolated populations of a rare alpine plant show high genetic diversity and considerable population differentiation

Background and Aims

Gene flow and genetic variability within and among alpine plant populations can be greatly influenced by the steep environmental gradients and heterogeneous topography of alpine landscapes. In this study, the effects are examined of natural isolation of alpine habitats on genetic diversity and geographic structure in populations of C. thyrsoides, a rare and isolated European Alpine monocarpic perennial with limited seed dispersal capacity.

Methods

Molecular diversity was analysed for 736 individuals from 32 populations in the Swiss Alps and adjacent Jura mountains using five polymorphic microsatellite loci. Pollen flow was estimated using pollen grain-sized fluorescent powder. In addition, individual-based Bayesian approaches were applied to examine population structure.

Key Results

High within-population genetic diversity (H_E = 0·76) and a relatively low inbreeding coefficient (F_IS = 0·022) were found. Genetic differentiation among populations measured with a standardized measure was considerable (G′_ST = 0·53). A significant isolation-by-distance relationship was found (r = 0·62, P < 0·001) and a significant geographic sub-structure, coinciding with proposed postglacial migration patterns. Altitudinal location and size of populations did not influence molecular variation. Direct measures of pollen flow revealed that insect-mediated pollen dispersal was restricted to short distances within a population.

Conclusions

The natural isolation of suitable habitats for C. thyrsoides restricts gene flow among the populations as expected for a monocarpic species with very limited seed dispersal capacities. The observed high within-population genetic diversity in this rare monocarpic perennial is best explained by its outcrossing behaviour, long-lived individuals and overlapping generations. Despite the high within-population genetic diversity, the considerable genetic differentiation and the clear western–eastern differentiation in this species merits consideration in future conservation efforts.Key words: Alpine plant, Campanula thyrsoides, genetic diversity, gene flow, genetic differentiation, glacial history, G′_ST, habitat isolation, microsatellites, monocarpy, SSR     

Genetic diversity of common carp from two largest Chinese lakes and the Yangtze River revealed by microsatellite markers

Six polymorphic microsatellites (eight loci) were used to study the genetic diversity and population structure of common carp from Dongting Lake (DTC), Poyang Lake (PYC), and the Yangtze River (YZC) in China. The gene diversity was high among populations with values close to 1. The number of alleles per locus ranged from 2 to 11, and the average number of alleles among 3 populations ranged from 6.5 to 7.9. The mean observed (H _O) and expected (H _E) heterozygosity ranged from 0.4888 to 0.5162 and from 0.7679 to 0.7708, respectively. Significant deviations from Hardy–Weinberg Equilibrium expectation were found at majority of the loci and in all three populations in which heterozygote deficits were apparent. The analysis of molecular variance (AMOVA) indicated that the percent of variance among populations and within populations were 3.03 and 96.97, respectively. The Fst values between populations indicated that there were significant genetic differentiations for the common carp populations from the Yangtze River and two largest Chinese freshwater lakes. The factors that may result in genetic divergence and significant reduction of the observed heterozygosity were discussed.     

Monitoring reveals two genetically distinct brown trout populations remaining in stable sympatry over 20 years in tiny mountain lakes

Detecting population subdivision when apparent barriers to gene flow are lacking is important in evolutionary and conservation biology. Recent research indicates that intraspecific population complexity can be crucial for maintaining a species′ evolutionary potential, productivity, and ecological role. We monitored the genetic relationships at 14 allozyme loci among ~4,000 brown trout (Salmo trutta) collected during 19 years from two small interconnected mountain lakes (0.10 and 0.17 km², respectively) in central Sweden. There were no allele frequency differences between the lakes. However, heterozygote deficiencies within lakes became obvious after a few years of monitoring. Detailed analyses were then carried out without a priori grouping of samples, revealing unexpected differentiation patterns: (i) the same two genetically distinct (F _ST ≥ 0.10) populations occur sympatrically at about equal frequencies within both lakes, (ii) the genetic subdivision is not coupled with apparent phenotypical dichotomies, (iii) this cryptic structure remains stable over the two decades monitored, and (iv) the point estimates of effective population size are c. 120 and 190, respectively, indicating that genetic drift is important in this system. A subsample of 382 fish was also analyzed for seven microsatellites. The genetic pattern does not follow that of the allozymes, and in this subsample the presence of multiple populations would have gone undetected if only scoring microsatellites. Sympatric populations may be more common than anticipated, but difficult to detect when individuals cannot be grouped appropriately, or when markers or sample sizes are insufficient to provide adequate statistical power with approaches not requiring prior grouping.     

Microsatellite markers for Lycium ruthenicum (Solananeae)

We developed microsatellite markers in Lycium ruthenicum, a desert plant widely distributed in northwestern China. In order to investigate its population genetic structure, genetic diversity, and its evolutionary history, we have isolated 11 novel microsatellite loci primers and characterized them in 24 individuals from 3 populations of L. ruthenicum using the combined biotin capture technique. For these microsatellites, one to seven alleles per locus were identified. The observed heterozygosities ranged from 0 to 0.958, meanwhile the expected heterozygosities ranged from 0 to 0.841. These microsatellite markers could be first useful for population level studies like genetic diversity and structure in this species.     

Gene polymorphisms for elucidating the genetic structure of the heavy-metal hyperaccumulating trait in Thlaspi caerulescens and their cross-genera amplification in Brassicaceae

Genetic polymorphism was investigated in Thlaspi caerulescens J. & C. Presl at 15 gene regions, of which seven have been identified to putatively play a role in heavy-metal tolerance or hyperaccumulation. Single nucleotide and length polymorphisms were assessed at four cleaved amplified polymorphic sequences (CAPS) and 11 simple sequence repeat (microsatellite) loci, respectively. The utility of these loci for genetic studies in T. caerulescens was measured among seven natural populations (135 individuals). Fourteen loci rendered polymorphism, and the number of alleles per locus varied from 2 to 5 and 1 to 27 for CAPS and microsatellites, respectively. Up to 12 alleles per locus were detected in a population. The global observed heterozygosity per population varied between 0.01 and 0.31. Additionally, cross-species/genera amplification of loci was investigated on eight other Brassicaceae (five individuals per population). Overall, 70% of the cross-species/genera amplifications were successful, and among them, more than 40% provided intraspecific polymorphisms within a single population. This indicates that such markers may, as well, allow comparative population genetic or mapping studies between and within several Brassicaceae, particularly for genes involved in traits such as heavy-metal tolerance and/or hyperaccumulation.     

Isolation and characterization of microsatellite loci in Bemisia tabaci

Bemisia tabaci (Hemiptera: Aleyrodidae) is a haplo‐diploid species with a global distribution demonstrating strong geographical structure with eight recognizable genetic groups. Fifteen microsatellite loci (335 alleles, 6–44 alleles per locus) were derived from four of the eight groups and were then screened across 33 populations. These loci clearly differentiate the populations. The microsatellites amplified best in individuals from genetic groups rep‐resenting the Mediterranean, Middle East, Asia (three groups) and Australasia/Oceania and amplified less well with populations from sub‐Saharan Africa and the New World. This differential amplification pattern is a direct result of the relatedness to the microsatellite source material.     

Development and multiplex PCR amplification of microsatellite markers in the commercial clam Venerupis rhomboides (Mollusca: Bivalvia)

Venerupis rhomboides is a commercial clam whose production could be enhanced through effective management of natural and hatchery stocks. This study provides the first panel of microsatellite markers for the exploitation of this species according to genetic criteria. A total of 22 polymorphic microsatellite loci were isolated and characterized from two genomic libraries enriched for different motifs. The number of alleles per locus ranged from 2 to 14 in a sample of 20 clams from Spain, and the observed and expected heterozygosity from 0 to 0.95 and 0.05–0.901, respectively. Sixteen loci were in agreement with Hardy–Weinberg equilibrium after sequential Bonferroni correction and linkage disequilibrium between loci pairs was not detected. To reduce the cost of the genotyping process, tri- and pentaplex PCRs, amplifying a total of 13 microsatellites loci were optimized. The microsatellites developed here represent the first nuclear markers described in V. rhomboides and will be useful tools for genetic studies involving assessment of genetic variation and population structure of natural and cultivated populations, assignment testing, construction of genetic linkage maps and dissection of production traits.     

Genetic diversity and population structure of the endangered alpine quillwort Isoetes hypsophila (Isoetaceae) revealed by SSR analysis

Isoetes hypsophila Hand.-Mazz. (Isoetaceae) is an endangered alpine quillwort endemic to the Qinghai-Tibetan Plateau (QTP). A total of 247 individuals of I. hypsophila from 11 populations were analyzed using ten pairs simple sequence repeats (SSR) markers. Seventy-seven alleles were recorded, with an average of 7.7 alleles per locus (ranging from 3 to 12) and the expected heterozygosity (H_E) ranged from 0.392 to 0.536 (average 0.471). Clear population structure among I. hypsophila populations was found and the proportion of genetic differentiation among populations accounted for about 20% of total genetic diversity (F_ST = 0.178; probability of allelic co-ancestory F_coal = 0.238). This genetic structure was probably due to limited gene flow hindered by complex terrains, bottlenecks resulting from water fluctuation in habitats, and repeated population retreat and recolonization during the ice ages. In conservation, four management units corresponding to four geographic regions were suggested, and the populations from different management units with higher genetic variation should be the priority targets of both in situ and ex situ conservation.     

Isolation and characterization of 19 polymorphic microsatellite loci from Neosalanx taihuensis,a rapidly invasive and adaptative species

Neosalanx taihuensis can very rapidly adapt to new environmental conditions and quickly becomes the dominant species when introduced to lakes and reservoirs. To better understand the genetic mechanism responsible for this remarkable ability to invade and adapt to diverse ecosystems, 19 polymorphic microsatellite loci were isolated from the genomic DNA of N. taihuensis. These microsatellite loci were identified from 24 individuals from an invasive population sampled in Fuxian Lake, Yunnan Plateau, Southwest China. The number of alleles per locus ranged from 2 to 14. Observed and expected heterozygosities ranged from 0.0417 to 0.8636 and from 0.3112 to 0.9202, respectively. The polymorphism information content (PIC) varied from 0.2583 to 0.8928. These microsatellite loci will be essential in performing molecular analyses of genetic diversity and genetic structure in the future. This will ultimately facilitate better understanding of the genetic mechanisms that allow N. taihuensis to successfully invade and adapt to new ecosystems.     

Genetic variation in the major histocompatibility complex of the European brown hare (Lepus europaeus) across distinct phylogeographic areas

The major histocompatibility complex is one of the best studied systems in vertebrates providing evidence for the long-term action of selection. Here, we examined the intra- and inter-population genetic diversity of the MHC class II DRB locus in European brown hare (Lepus europaeus) and correlated the results with genetic variability already estimated from the MHC DQA locus and from maternally (mitochondrial DNA (mtDNA)) and biparentally (allozymes, microsatellites) inherited loci. L. europaeus showed remarkable genetic polymorphism in both DQA and DRB1 loci. The Anatolian populations exhibited the highest genetic polymorphism for both loci. Balancing selection has established increased variability in the European populations despite the founder effects after the last glaciation. Different evolutionary rates were traced for DRB1 and DQA loci, as evidenced by the higher number of common DRB1 than DQA alleles and the greater differences between DRB1 alleles with common origin in comparison with DQA alleles. The high number of rare alleles with low frequencies detected implies that frequency-dependent selection drives MHC evolution in the brown hare through the advantage of rare alleles. Both loci were under the influence of positive selection within the peptide-binding region. The functional polymorphism, recorded as amino acid substitutions within the binding pockets, fell also within distinct geographic patterns, yet it was much narrower than the genetic polymorphism. We hypothesize that certain structural and functional characteristics of the binding pockets set limitations to the actual shape of genetic polymorphism in MHC.