Characterization of microsatellite loci isolated from the blacktip shark and their utility in requiem and hammerhead sharks

We isolated and characterized 16 microsatellite loci from the blacktip shark, Carcharhinus limbatus, and tested cross‐species amplification in 11 Carcharhinus species and five additional shark genera. Thirty‐six (1.6%) and 180 (48%) colonies were positive for dinucleotide repeat motifs from unenriched and enriched libraries, respectively. Heterozygosities of polymorphic loci ranged from 0.04 to 0.96 with two to 22 alleles per locus. Amplification products were observed at nine to 13 loci (five to 11 of which where polymorphic) in 10 Carcharhinus species. Several loci were also polymorphic in each of the additional genera examined.     

Polymorphic microsatellite markers for the gliding marsupials Petaurus australis and Petaurus breviceps

Habitat destruction is causing population decline of many hollow dependent species such as gliding marsupials of the Family Petauridae. Three petaurid species are now listed in some Australian states as either threatened, rare or vulnerable, precipitating a need for information on their basic biology and population structure. We isolated and characterized three polymorphic microsatellite loci from the yellow‐bellied glider (Petaurus australis) and six polymorphic microsatellite loci from the sugar glider (P. breviceps). Per‐locus heterozygosities range from 42%–92%, and cross‐species amplification studies show that between five and seven loci are polymorphic in the two target species as well as a related species P. norfolcensis.     

Cross-species amplification of 44 microsatellite loci developed for Chaetodon trifascialis, C. lunulatus and C. vagabundus in 22 related butterflyfish species

Forty‐four microsatellite primers developed for three species of butterflyfish were cross‐tested against 22 related confamilial species. Amplification success and cross‐species transferability of these markers were moderately high. Between 24 and 37 loci were amplified successfully in each species, with a mean success rate per species of 71.7% (± 1.8 SE). Rates of amplification success were comparable among primers designed for the three source species, ranging from a mean success rate per species of 16.9 loci (± 0.8 SE) for Chaetodon trifascialis source loci to 13.7 loci (± 1.5 SE) for C. vagabundus source loci. Polymorphism rates were high (76.1%± 3.1 SE of all successfully amplified loci), and 10 loci were polymorphic in all successfully amplified species (Tri14, B11, C5, D3, D113, D6, D117, D120, D111, D118). The number of alleles per polymorphic locus ranged from 2 to 8, and the average number of alleles across all polymorphic loci and all species was 3.6 (± 0.07 SE). Polymorphism rates were higher overall in primers designed for C. vagabundus (89.9%± 3.9 SE). Overall cross‐testing success was lowest for Heniochus chrysostomus, the most phylogenetically divergent species. The significant cross‐testing reported here provides a valuable resource that will enable population genetics studies to be undertaken on a range of butterflyfishes without the need for expensive and time‐consuming de novo microsatellite development.     

Using gap‐crossing capacity to evaluate functional connectivity of two Atlantic rainforest birds and their response to fragmentation

Abstract One of the main consequences of habitat loss and fragmentation is the increase in patch isolation and the consequent decrease in landscape connectivity. In this context, species persistence depends on their responses to this new landscape configuration, particularly on their capacity to move through the interhabitat matrix. Here, we aimed first to determine gap‐crossing probabilities related to different gap widths for two forest birds (Thamnophilus caerulescens, Thamnophilidae; and Basileuterus culicivorus, Parulidae) from the Brazilian Atlantic rainforest. These values were defined with a playback technique and then used in analyses based on graph theory to determine functional connections among forest patches. Both species were capable of crossing forest gaps between patches, and these movements were related to gap width. The probability of crossing 40 m gaps was 50% for both species. This probability falls to 10% when the gaps are 60 m (for B. culicivorus) or 80 m (for T. caerulescens). Actually, birds responded to stimulation about two times more distant inside forest trials (control) than in gap‐crossing trials. Models that included gap‐crossing capacity improved the explanatory power of species abundance variation in comparison to strictly structural models based merely on patch area and distance measurements. These results highlighted that even very simple functional connectivity measurements related to gap‐crossing capacity can improve the understanding of the effect of habitat fragmentation on bird occurrence and abundance.     

Characterization of comparative genome‐derived simple sequence repeats for acanthopterygian fishes

Simple sequence repeats (SSRs) have become one of the most popular molecular markers for population genetic studies. The application of SSR markers has often been limited to source species because SSR loci are too labile to be maintained in even closely related species. However, a few extremely conserved SSR loci have been reported. Here, we tested for the presence of conserved SSR loci in acanthopterygian fishes, which include over 14 000 species, by comparing the genome sequences of four acanthopterygian fishes. We also examined the comparative genome‐derived SSRs (CG‐SSRs) for their transferability across acanthopterygian fishes and their applicability to population genetic analysis. Forty‐six SSR loci with conserved flanking regions were detected and examined for their transferability among seven nonacanthopterygian and 27 acanthopterygian fishes. The PCR amplification success rate in nonacanthopterygian fishes was low, ranging from 2.2% to 21.7%, except for Lophius litulon (Lophiiformes; 80.4%). Conversely, the rate in most acanthopterygian fishes exceeded 70.0%. Sequencing of these 46 loci revealed the presence of SSRs suitable for scoring while fragment analysis of 20 loci revealed polymorphisms in most of the acanthopterygian fishes. Population genetic analysis of Cottus pollux (Scorpaeniformes) and Sphaeramia orbicularis (Perciformes) using CG‐SSRs showed that these populations did not deviate from linkage equilibrium or Hardy–Weinberg equilibrium. Furthermore, almost no loci showed evidence of null alleles, suggesting that CG‐SSRs have strong resolving power for population genetic analysis. Our findings will facilitate the use of these markers in species in which markers remain to be identified.     

Balancing selection maintains sex determining alleles in multiple‐locus complementary sex determination

Hymenopteran species in which sex is determined through a haplo‐diploid mechanism known as complementary sex determination (CSD) are vulnerable to a unique form of inbreeding depression. Diploids heterozygous at one or more CSD loci develop into females but diploids homozygous at all loci develop into diploid males, which are generally sterile or inviable. Species with multiple polymorphic CSD loci (ml‐CSD) may have lower rates of diploid male production than species with a single CSD locus (sl‐CSD), but it is not clear if polymorphism is consistently maintained at all loci. Here, we assess the rate of diploid male production in a population of Cotesia rubecula, a two‐locus CSD parasitoid wasp species, approximately 20 years after the population was introduced for biological control. We show that diploid male production dropped from 8–13% in 2005 and 2006 to 3–4% by 2015. We also show from experimental crosses that the population maintained polymorphism at both CSD loci in 2015. We use theory and simulations to show that balancing selection on all CSD alleles promotes polymorphism at several loci in ml‐CSD populations. Our study supports the hypothesis that ml‐CSD populations have lower diploid male production and are more likely to persist than comparable sl‐CSD populations.     

Characterization of six microsatellite DNA loci for Sorex arizonae

Sorex arizonae is a rare species that occupies a narrow range of habitat types in several mountain ranges of New Mexico, Arizona and Northern Mexico. Here we identify and characterize six microsatellite loci for this species. We screened 63 individuals from four different localities from New Mexico and Arizona to analyse genetic variability. Alleles ranged from three to 16. Heterozygosity ranged from 40% to 78%. Most polymorphic loci were in Hardy–Weinberg equilibrium with the exception of one locus. Primers appear to have reasonable cross‐species applicability as five loci amplified in another shrew species (Sorex monticolus).     

Genomic characterization of sex‐identification markers in Sebastes carnatus and Sebastes chrysomelas rockfishes

Fish have evolved a variety of sex‐determining (SD) systems including male heterogamy (XY), female heterogamy (ZW) and environmental SD. Little is known about SD mechanisms of Sebastes rockfishes, a highly speciose genus of importance to evolutionary and conservation biology. Here, we characterize the sex determination system in the sympatrically distributed sister species Sebastes chrysomelas and Sebastes carnatus. To identify sex‐specific genotypic markers, double digest restriction site – associated DNA sequencing (ddRAD‐seq) of genomic DNA from 40 sexed individuals of both species was performed. Loci were filtered for presence in all of the individuals of one sex, absence in the other sex and no heterozygosity. Of the 74 965 loci present in all males, 33 male‐specific loci met the criteria in at least one species and 17 in both. Conversely, no female‐specific loci were detected, together providing evidence of an XY sex determination system in both species. When aligned to a draft reference genome from Sebastes aleutianus, 26 sex‐specific loci were interspersed among 1168 loci that were identical between sexes. The nascent Y chromosome averaged 5% divergence from the X chromosome and mapped to reference Sebastes genome scaffolds totalling 6.9Mbp in length. These scaffolds aligned to a single chromosome in three model fish genomes. Read coverage differences were also detected between sex‐specific and autosomal loci. A PCR‐RFLP assay validated the bioinformatic results and correctly identified sex of five additional individuals of known sex. A sex‐determining gene in other teleosts gonadal soma‐derived factor (gsdf) was present in the model fish chromosomes that spanned our sex‐specific markers.     

Characterization of microsatellite loci developed for song wrens Cyphorhinus phaeocephalus

We developed six microsatellite loci for song wrens (Cyphorhinus phaeocephalus). Polymorphism ranged from four to 12 alleles with heterozygosities ranging from 21 to 90%. Three of the six loci deviated significantly from the Hardy–Weinberg equilibrium suggesting the presence of one or more null alleles. Cross‐species amplification indicated that these loci will have limited utility in other wren species.     

Microsatellite primers for the African mole‐rat genus Cryptomys and cross‐species amplification within the family Bathyergidae

We report 11 microsatellite loci for the African mole‐rat genus Cryptomys (Rodentia: Bathyergidae), isolated from the Damaraland mole‐rat (Cryptomys damarensis) and the Common mole‐rat (C. hottentotus hottentotus). All loci are highly polymorphic in the source species, with between six and 13 alleles and observed heterozygosity ranging from 0.54 to 0.86. Two C. damarensis loci (DMR1 and 6) may be located on the X‐chromosome. Cross‐species amplification was investigated in 10 Bathyergid species, representative of the five genera. The majority of loci amplified polymorphic product in all Cryptomys species tested. Amplification was also widespread in the other genera, except in Heterocephalus.     

Development of microsatellite markers in Acer capillipes

Acer capillipes is an insect‐pollinated tree species that grows in temperate regions of Japan. We isolated seven polymorphic microsatellite loci from this species using a dual‐suppression polymerase chain reaction (PCR) technique. The number of alleles per locus ranged from two to 10, and the expected heterozygosity ranged from 0.042 to 0.828. Cross‐species amplification from 14 other Acer species was successful for the majority of the isolated loci, suggesting that these loci may be useful for the characterization of other maple species.     

What remains from a 454 run: estimation of success rates of microsatellite loci development in selected newt species (Calotriton asper,Lissotriton helveticus,and Triturus cristatus) and comparison with Illumina‐based approaches

The development of microsatellite loci has become more efficient using next‐generation sequencing (NGS) approaches, and many studies imply that the amount of applicable loci is large. However, few studies have sought to quantify the number of loci that are retained for use out of the thousands of sequence reads initially obtained. We analyzed the success rate of microsatellite loci development for three amphibian species using a 454 NGS approach on tetra‐nucleotide motif‐enriched species‐specific libraries. The number of sequence reads obtained differed strongly between species and ranged from 19,562 for Triturus cristatus to 55,626 for Lissotriton helveticus, with 52,075 reads obtained for Calotriton asper. PHOBOS was used to identify sequences with tetra‐nucleotide repeat motifs with a minimum repeat number of ten and high quality primer binding sites. Of 107 sequences for T. cristatus, 316 for C. asper and 319 for L. helveticus, we tested the amplification success, polymorphism, and degree of heterozygosity for 41 primer combinations each for C. asper and T. cristatus, and 22 for L. helveticus. We found 11 polymorphic loci for T. cristatus, 20 loci for C. asper, and 15 loci for L. helveticus. Extrapolated, the number of potentially amplifiable loci (PALs) resulted in estimated species‐specific success rates of 0.15% (T. cristatus), 0.30% (C. asper), and 0.39% (L. helveticus). Compared with representative Illumina NGS approaches, our applied 454‐sequencing approach on specifically enriched sublibraries proved to be quite competitive in terms of success rates and number of finally applicable loci.     

Novel microsatellite loci for the study of the black‐capped vireo (Vireo atricapillus)

We identified 14 novel polymorphic microsatellite loci in the black‐capped vireo (Vireo atricapillus). We also attempted to amplify and genotype these loci in other Vireo species, including the white‐eyed vireo (Vireo griseus), red‐eyed vireo (Vireo olivaceus), and blue‐headed vireo (Vireo solitarius). In 33 genotyped black‐capped vireos from two locations, total alleles ranged from six to 20, with observed heterozygosity ranging from 0.58 to 0.91 and expected heterozygosity from 0.65 to 0.93. Two loci had detectable levels of null alleles. Many of the loci were able to be amplified in the related Vireo species.     

PCR primers for polymorphic microsatellite loci in the plant‐ant Azteca ulei cordiae (Formicidae: Dolichoderinae)

Twelve microsatellite loci were isolated from the Peruvian tropical plant‐ant, Azteca ulei cordiae (Hymenoptera: Dolichoderinae). High levels of within‐population variation were observed at most loci, with number of alleles ranging from four to 18, and heterozygosity from 0.118 to 1 per population sample. Cross‐species amplification of these loci was also successfully tested in several other species of the same ant genus Azteca.     

Microsatellite loci for the southern pine beetle (Dendroctonus frontalis) and cross‐species amplification in Dendroctonus

We developed microsatellite loci for the southern pine beetle (Dendroctonus frontalis). Twelve microsatellite loci were identified. Eight loci were polymorphic and sufficiently variable in 62 individuals (expected heterozygosity ranged from 0.707 to 0.880) to investigate population structure. All loci conformed to HWE except Dfr‐14, which showed heterozygote excess, and no two loci deviated from linkage equilibrium. The loci were tested for cross‐species amplification in four species of Dendroctonus (D. valens, D. terebrans, D. brevicomis, and D. ponderosae). Seven loci were polymorphic in at least one of the species tested.     

Isolation and characterization of polymorphic microsatellites in the tropical plant‐ant Cataulacus mckeyi (Formicidae: Myrmicinae)

Eleven microsatellite loci were isolated from the plant‐ant Cataulacus mckeyi (Hymenoptera: Myrmicinae) and their polymorphism was characterized. High levels of within‐population variation were observed at most loci, with number of alleles ranging from one to 16, and heterozygosity from 0 to 0.929 per population sample. Cross‐species amplification of these loci was also tested in four other species of the ant genus Cataulacus.     

PCR primers for polymorphic microsatellite loci in the facultatively polygynous plant‐ant Petalomyrmex phylax (Formicidae)

Fourteen microsatellite loci were isolated and their level of polymorphism characterized in two populations of the facultatively polygynous plant‐ant Petalomyrmex phylax (Formicinae). High levels of within‐population variation were observed at most loci, with number of alleles ranging from two to 15, and heterozygosity from 0.050 to 0.925. Cross‐species amplification of these loci was also tested in four plant‐ant species belonging to three other genera, Aphomomyrmex, Cladomyrma (both Formicinae) and Cataulacus (Myrmicinae).     

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.     

Isolation and characterization of 27 polymorphic microsatellite loci for the common snook,Centropomus undecimalis

Here we describe 32 di‐, tri‐ and tetranucleotide microsatellite loci isolated by PIMA, a polymerase chain reaction (PCR)‐based procedure, for the common snook (Centropomus undecimalis). Five loci were monomorphic, and the remaining loci averaged 6.7 alleles per locus in a sample of 45 common snook. For polymorphic loci, expected heterozygosities ranged from 0.02 to 0.91 (mean = 0.538). Significant departures from Hardy–Weinberg equilibrium expectations occurred in two loci. Exact tests for genotype disequilibrium gave evidence for linkage at one pair of loci. Many cross‐species primer assays yielded PCR fragments of the expected size for 11 species of Centropomus and two species of the confamilial genus Lates.     

Characterization of polymorphic microsatellites in the castration parasite plant‐ant Allomerus octoarticulatus cf. demerarae (Formicidae: Myrmicinae)

Fifteen microsatellite loci were isolated from the Peruvian tropical plant‐ant Allomerus octoarticulatus cf. demerarae (Hymenoptera: Myrmicinae) and their polymorphism was characterized. High levels of within‐population variation were observed at most loci, with number of alleles ranging from one to 21, and heterozygosity from 0 to 1 per population sample. Cross‐species amplification of these loci was also tested in one other species of the ant genus Allomerus (Allomerus decemarticulatus), displaying similar life history.