New polymorphic microsatellite loci for the Macedonian crested newt, Triturus macedonicus, and cross-priming testing in four other crested newt species

Eleven microsatellites have been characterized for Triturus macedonicus. Nine loci showed different variation patterns in a sample of 40 individuals from a single breeding pond in Zagori province (Greece), with an average number of 4.2 alleles per locus and an expected heterozygosity ranging from 0.1199 to 0.8079. Distinct cross-priming amplification rates were recovered on four additional crested newt species. Two monomorphic T. macedonicus loci were polymorphic in other Triturus species. The microsatellites developed herein could be a useful intraspecific genetic tool to undertake fine-scale population genetic analyses as well as in the study of contact zones between crested newt species.     

Genetic dissimilarity predicts paternity in the smooth newt (Lissotriton vulgaris)

Under sperm competition, paternity is apportioned by polyandrous females according to the order of matings and the genetic quality of the inseminating males. In order to distinguish between these two effects, we sequentially paired 12 female smooth newts (Lissotriton vulgaris) with each of two males and, where possible, repeated the same procedure in reverse order of the identical males after assumed sperm depletion. For a total of 578 offspring, amplified fragment length polymorphisms genetic markers revealed multiple paternities in all matings, without significant first- or second-male sperm precedence. The paternity share of individual males was transitive across the two trials with male order switch, and successful males had a significantly higher genetic dissimilarity to the female than expected by chance. We argue that patterns of paternity in natural newt populations are determined through a combination of good genes and relatedness.     

Fifteen polymorphic microsatellite markers for the giant spiny frog, Paa spinosa

We characterized 15 polymorphic microsatellite loci for the giant spiny frog, Paa spinosa. These loci were highly polymorphic when screened in 38 individuals from two different populations, with nine to 23 alleles per locus. The range of observed and expected heterozygosities was 0.231-0.916 and 0.296-0.944, respectively. These polymorphic loci will be useful for assessing genetic diversity, population structure, gene flow, population assignment and determining paternity in the giant spiny frog.     

Characterization of microsatellite loci for a barrenwort species (Epimedium diphyllum,Berberidaceae)

Microsatellites were identified and characterized from Epimedium diphyllum, a species of barrenworts, both attractive garden plants and valuable medicinal plants. Some Japanese species of Epimedium are threatened with extinction, and are listed in the Red Data Book of plants in Japan. Natural hybrid zones also have been reported among some taxa of Japanese Epimedium. We developed eight polymorphic microsatellite primers for population genetic analyses of E. diphyllum. The numbers of alleles per locus ranged from 10 to 20, with observed levels of heterozygosity between 0.85 and 1.00. These primer sets yielded amplification in the other three Japanese Epimedium. These markers will be valuable for conservation genetics, evolutionary biology, pharmacognostic study, and horticultural study of Epimedium.     

Isolation and variability of polymorphic microsatellite loci in Aedes aegypti,the vector of dengue viruses

We isolated five microsatellite sequences from an enriched‐(CAA)_n library of 5000 recombinant clones in Aedes aegypti. Two polymorphic microsatellites from our library and four from other sequence databases were tested: we investigated their polymorphism and Mendelian inheritance in mosquito populations. Our results indicate that trinucleotide repeat markers could be used to differentiate Ae. aegypti populations, making them valuable tools for the study of population genetic structure.     

Development and characterization of polymorphic microsatellite loci in the endangered Miami blue butterfly (Cyclargus thomasi bethunebakeri)

The Miami blue butterfly (Cyclargus thomasi bethunebakeri) is a state-endangered taxon in Florida and a candidate for federal listing. Here we report 12 polymorphic microsatellite loci appropriate for use in population and conservation studies. We genotyped 114 individuals sampled from a metapopulation in the lower Florida Keys over a 2-year period (2005-2006). These results show 4-14 alleles per locus, and ranges of observed and expected heterozygosities are 0.02679-0.79630 and 0.06154-0.69565, respectively. Large deviations from Hardy-Weinberg equilibrium (HWE) are observed across the whole sample set. When a single breeding population is analysed alone, seven of the loci are in HWE.     

Past forest cover explains current genetic differentiation in the Carpathian newt (Lissotriton montandoni), but not in the smooth newt (L. vulgaris)

Aim

Current genetic variation and differentiation are expected to reflect the effects of past rather than present landscapes due to time lags, that is, the time necessary for genetic diversity to reach equilibrium and reflect demography. Time lags can affect our ability to infer landscape use and model connectivity and also obscure the genetic consequences of recent landscape changes. In this work, we test whether past forest cover better explains contemporary patterns of genetic differentiation in two closely related but ecologically distinct newt species—Lissotriton montandoni and L. vulgaris.

Location

Carpathian Mountains and foothills.

Methods

Genetic differentiation between populations was related to landscape resistance optimized with tools from landscape genetics, for multiple timeframes, using forest-cover data from 1963 to 2015. Analyses were conducted for pairs of populations at distances from 1 to 50 km.

Results

We find evidence for a time lag in L. montandoni, with forest cover from 40 years ago (ca. 10 newt generations) better explaining current genetic differentiation. In L. vulgaris, current genetic differentiation was better predicted by present land-cover models with lower resistance given to open forests. This result may reflect the generalist ecology of L. vulgaris, its lower effective population sizes and exposure to habitat destruction and fragmentation.

Main Conclusions

Our study provides evidence for time lags in L. montandoni, showing that the genetic consequences of landscape change for some species are not yet evident. Our findings highlight the interspecific variation in time lag prevalence and demonstrate that current patterns of genetic differentiation should be interpreted in the context of historical landscape changes.     

Isolation and characterization of 30 polymorphic microsatellite loci from the mycophagous fly Drosophila innubila

Drosophila innubila is a mushroom-feeding member of the quinaria group, found in the woodlands and forests of the 'sky islands' in Arizona and New Mexico and extending south into central Mexico. Here, we describe and characterize 30 polymorphic microsatellite loci from D. innubila collected in the Chiricahua Mountains in Arizona. The number of alleles ranged from three to 21, and observed heterozygosity ranged from 0.0513 to 0.9737. Six loci were putatively X-linked, six departed from Hardy-Weinberg equilibrium, seven had evidence of null alleles, and six showed evidence of linkage disequilibrium. These markers will be useful for examining population structure of D. innubila and its association with male-killing Wolbachia.     

Characterization of polymorphic microsatellite loci in the aphid species Macrosiphum euphorbiae (Hemiptera: Aphididae)

Fourteen microsatellite loci were isolated and characterized in the potato aphid, Macrosiphum euphorbiae Thomas, by screening a genomic library with the oligonucleotide probes (GA)₁₀ (GT)₁₀ and (GATA)₄. Allelic diversity was estimated in samples collected from potato fields in Tunisia. Ten loci displayed polymorphism that ranged from two to four alleles per locus and the observed heterozygosity ranged from zero to one. These markers could be used to study the population genetic structure of this polyphagous aphid species.     

Isolation and characterization of polymorphic microsatellite loci in the olive flounder (Paralichthys olivaceus)

We described the isolation and characterization of 27 new microsatellite loci from olive flounder, Paralichthys olivaceus. All loci were found to be polymorphic, and had between five and 22 alleles with observed heterozygosity ranging from 0.161 to 1.0 in 31 individuals examined. These micorsatellite makers are likely to be useful for studies of genome mapping, mating systems and population genetics in this species.     

New microsatellite loci for the endangered scalloped hammerhead shark, Sphyrna lewini

We isolated 15 microsatellite markers for the scalloped hammerhead shark, Sphyrna lewini. Loci were tested on 80 specimens of S. lewini from four Eastern Pacific samples. The number of alleles per locus ranged from 6 to 31 (mean = 14). Observed and expected levels of heterozygosity per locus ranged from 0.39 to 0.91 (mean = 0.70) and from 0.54 to 0.90 (mean = 0.76), respectively. No pairs of loci were in gametic disequilibrium after Bonferroni correction of α. One locus showed significantly lower heterozygosity than expected under Hardy–Weinberg proportions in two populations, possibly caused by null alleles.     

Identification and characterization of 21 polymorphic microsatellite loci from the mycophagous fly Drosophila neotestacea

Drosophila neotestacea is a mushroom‐feeding fly that is common in the temperate and boreal forests of North America. Here I describe the isolation and characterization of 21 polymorphic microsatellite loci from the D. neotestacea genome. In a sample of flies from Rochester, New York, the expected and observed heterozygosities ranged from 0.344 to 1.000 and from 0.384 to 0.923, respectively. Of the 21 markers, six were likely X‐linked, seven showed a departure from Hardy–Weinberg equilibrium, and none were found to be in linkage disequilibrium. These loci will facilitate future ecological and population genetic studies of D. neotestacea.     

Isolation and characterization of six polymorphic microsatellite loci in South African hares (Lepus saxatilis F. Cuvier, 1823 and Lepus capensis Linnaeus, 1758)

We have developed six novel, polymorphic microsatellite markers for the scrub hare, Lepus saxatilis. These markers have been tested for 150 scrub hares and 67 Cape hares from different localities across southern Africa. All six loci amplified consistently well and most were highly polymorphic. Four loci amplified consistently in five different leporid genera. The microsatellites presented here will be useful for phylogeographical studies in the scrub hare and other leporidae.     

Homoplasy and mutation model at microsatellite loci and their consequences for population genetics analysis

Homoplasy has recently attracted the attention of population geneticists, as a consequence of the popularity of highly variable stepwise mutating markers such as microsatellites. Microsatellite alleles generally refer to DNA fragments of different size (electromorphs). Electromorphs are identical in state (i.e. have identical size), but are not necessarily identical by descent due to convergent mutation(s). Homoplasy occurring at microsatellites is thus referred to as size homoplasy. Using new analytical developments and computer simulations, we first evaluate the effect of the mutation rate, the mutation model, the effective population size and the time of divergence between populations on size homoplasy at the within and between population levels. We then review the few experimental studies that used various molecular techniques to detect size homoplasious events at some microsatellite loci. The relationship between this molecularly accessible size homoplasy size and the actual amount of size homoplasy is not trivial, the former being considerably influenced by the molecular structure of microsatellite core sequences. In a third section, we show that homoplasy at microsatellite electromorphs does not represent a significant problem for many types of population genetics analyses realized by molecular ecologists, the large amount of variability at microsatellite loci often compensating for their homoplasious evolution. The situations where size homoplasy may be more problematic involve high mutation rates and large population sizes together with strong allele size constraints.     

Isolation and characterization of polymorphic microsatellite loci for the northern flicker (Colaptes auratus; Aves)

To investigate the mating system of northern flickers (Colaptes auratus), we developed primers for 14 microsatellite loci and screened them in 68 unrelated adults and their offspring. All markers were highly polymorphic with 9 to +36 alleles per locus. One marker was Z‐chromosome linked; one marker exceeded the size standard range and could not be analysed further. We checked the other 12 markers for Mendelian inheritance in 36 broods for which the social parents were known. Seven markers showed evidence for the presence of null alleles, and three of those showed significant deviation from Hardy–Weinberg equilibrium. Markers were generally unlinked.     

Isolation and characterization of polymorphic microsatellite loci in the boll weevil,Anthonomus grandis Boheman (Coleoptera: Curculionidae)

The boll weevil (Anthonomus grandis Boheman) is a major insect pest of cotton in North America. Dispersal activity poses a threat to ongoing eradication efforts in the US, but little is known about the frequency of long‐distance migration. Nuclear molecular markers are needed to assess gene flow in relation to geographical distance. A biotin‐enrichment strategy was employed to develop microsatellite markers for the boll weevil. Of 23 loci isolated, 14 were polymorphic with three to 10 alleles per locus. Twelve of the polymorphic loci showed Mendelian inheritance and are likely to be useful in population genetics studies.     

Characterization of 12 polymorphic microsatellite markers for Georgia false indigo (Amorpha georgiana Wilbur var. georgiana), an endangered species, and their utility in other dwarf Amorpha L. species

In order to facilitate the addition of a genetic component to conservation management plans for Georgia false indigo (Amorpha georgiana var. georgiana), a rare legume of the southeastern USA, 12 polymorphic microsatellite markers were developed. No gametic disequilibrium was detected among locus pairs, but observations for five of the loci significantly deviated from expected Hardy-Weinberg proportions. Cross-species testing was successful and demonstrated the utility of the majority of the markers in congeners Amorpha georgiana var. confusa and Amorpha herbacea. The results also suggested that A. georgiana var. confusa is tetraploid rather than diploid.     

Characterization of the complete mitochondrial genome and a set of polymorphic microsatellite markers through next-generation sequencing for the brown brocket deer Mazama gouazoubira

The complete mitochondrial genome of the brown brocket deer Mazama gouazoubira and a set of polymorphic microsatellite markers were identified by 454-pyrosequencing. De novo genome assembly recovered 98% of the mitochondrial genome with a mean coverage of 9-fold. The mitogenome consisted of 16,356 base pairs that included 13 protein-coding genes, two ribosomal subunit genes, 22 transfer RNAs and the control region, as found in other deer. The genetic divergence between the mitogenome described here and a previously published report was ∼0.5%, with the control region and ND5 gene showing the highest intraspecific variation. Seven polymorphic loci were characterized using 15 unrelated individuals; there was moderate genetic variation across most loci (mean of 5.6 alleles/locus, mean expected heterozygosity = 0.70), with only one locus deviating significantly from Hardy-Weinberg equilibrium, probably because of null alleles. Marker independence was confirmed with tests for linkage disequilibrium. The genetic variation of the mitogenome and characterization of microsatellite markers will provide useful tools for assessing the phylogeography and population genetic patterns in M. gouazoubira, particularly in the context of habitat fragmentation in South America.     

New polymorphic microsatellite loci for population studies in the European anchovy, Engraulis encrasicolus (L.)

Eleven microsatellite loci were developed in the European anchovy, Engraulis encrasicolus, and tested in samples from two geographically distant populations (Atlantic and Mediterranean Sea). Number of alleles ranged from eight to 28 and observed heterozygosity from 0.440 to 0.920. There was no evidence of linkage disequilibrium, although two loci are indeed linked. All loci were in Hardy-Weinberg equilibrium, except for one locus in the Atlantic and two loci in the Mediterranean sample. These three loci plus two more showed evidence for null alleles.