Novel tetranucleotide microsatellite DNA markers for the wood frog,Rana sylvatica

Fifteen tetranucleotide microsatellite loci were identified and characterized for wood frogs (Rana sylvatica) collected from three vernal pools in the southeastern US. These markers revealed a high degree of genetic diversity (nine to 34 alleles per locus), heterozygosity (30.6–92.3%) and allelic heterogeneity (69% of comparisons were statistically significant). Considerable differentiation among populations was observed as genetic distances (chord) ranged between 0.40 and 0.55 and all F_ST values (0.02–0.05) were statistically significant. Genotypic assignment tests correctly classified 103 of 113 individuals to their respective collection. These markers should prove useful for investigating fine‐scale population structure and metapopulation dynamics.     

Polymorphic microsatellite DNA loci identified in the common frog (Rana temporaria,Amphibia, Ranidae)

We developed seven microsatellite loci in the common frog, Rana temporaria. There were between 2 and 27 alleles per locus and the expected heterozygosities ranged from 0.28 to 0.96 in a sample of frogs collected in the French Alps. Adding these seven markers to the 15 previously available microsatellite loci for this species should facilitate studies of genetic structure of Rana temporaria populations at a fine geographical scale.     

Canopy Closure,Competition, and the Endangered Dusky Gopher Frog

Abstract: A major challenge facing wildlife biologists is understanding why some species go extinct while others persist in the same habitat. To address this question, we investigated whether tree canopy closure over ponds affects growth and survival of rare and common tadpoles within ponds and mediates competitive interactions among species. We conducted 2 experiments to test whether canopy closure and competition may have contributed to the decline of the endangered dusky gopher frog (Rana sevosa), but allowed the persistence of the southern leopard frog (R. sphenocephala). We explored the response of both species to canopy closure in single-species and mixed- (1:1) species treatments of identical total tadpole density. An experiment using aquatic enclosures in temporary ponds showed that canopy closure reduced tadpole growth approximately 20% for both species. Survival of dusky gopher frog tadpoles was higher in mixed-species enclosures than in single-species enclosures. In a complementary experiment using artificial ponds, dusky gopher frogs had lower survival to metamorphosis, reduced size at metamorphosis, and produced a lower total biomass of metamorphosed juveniles in shaded ponds. Southern leopard frogs exhibited reduced body size at metamorphosis only when shaded. These studies suggest that pond canopy closure, not larval competition, may be contributing to the decline of the dusky gopher frog. The different responses to canopy closure suggest a potential mechanism for the loss of dusky gopher frogs and the persistence of southern leopard frogs. Removal of trees from historically open-canopy ponds may help facilitate the recovery of dusky gopher frogs and benefit similar species.     

Isolation and characterization of polymorphic microsatellite loci in yellowtail catfish, Pangasius pangasius (Hamilton, 1822)

A total of nine polymorphic microsatellite loci were obtained from a genomic library of Pangasius pangasius (order Siluriformes, family Pangasiidae). Samples from rivers Bhagirathi (n = 22) and Mahanadi (n = 20) were genotyped for each of the nine microsatellite loci to determine genetic variation. The mean number of alleles per locus was 5.22 in Bhagirathi and 5.78 in Mahanadi; and expected heterozygosity ranged from 0.567 (Bhagirathi) to 0.578 (Bhagirathi). Significant deviation (P < 0.003) from Hardy–Weinberg expectations was evident at three loci, Ppa2 (Bhagirathi), Ppa14 (Mahanadi) and Ppa28 (Bhagirathi and Mahanadi). The identified microsatellite loci were found to be promising for population genetics studies of P. pangasius.     

Microsatellite DNA markers for the Chinese wood frog (Rana chensinensis) and tests for their cross-utility in 15 ranid frog species

We developed 22 microsatellite markers for the Chinese wood frog (Rana chensinensis) to study the impact of landscape features on its population structure. Thirty‐four individuals from one breeding site were examined and 14 loci were polymorphic. The number of alleles, expected heterozygosity and observed heterozygosity varied from two to 14, from 0.0833 to 0.9118, and from 0.1376 to 0.8667, respectively. Cross‐species amplification was tested for 15 ranid frog species. The Plateau brown frog, Rana kukunoris (n = 23), was successfully amplified at 18 loci, and 15 were polymorphic with number of alleles varying from two to 18. Ten other species were also amplified at a limited number of loci.     

Tetranucleotide microsatellite loci from the striped dolphin (Stenella coeruleoalba Meyen, 1833)

Five tetranucleotide microsatellite loci were isolated from the striped dolphin (Stenella coeruleoalba). Characterization of these loci revealed between three and 11 alleles at each locus and observed heterozygosity ranged from 0.192 to 0.775. All sets of primers amplified homologous loci when tested on four related species and showed polymorphisms in most locus–species combinations. A sixth microsatellite, which appeared to be monomorphic in the striped dolphin, was also shown to be polymorphic in three related species.     

Polymorphic tetranucleotide microsatellite loci in the Hwamei (Garrulax canorus canorus) (Timaliidae)

Eight polymorphic tetrarepeat (GATA)_n, microsatellite loci were isolated from a babbler, Hwamei (Garrulax canorus canorus). We evaluated the polymorphism of these microsatellite loci by genotyping 36–48 individuals from the Asian mainland. The number of alleles for each locus ranged from eight to 29. The heterozygosity was between 0.587 and 0.978. Except for one locus, genotype frequencies of these microsatellites did not significantly deviate from the Hardy–Weinberg expectation. These markers should be useful for monitoring potential hybridization between different Hwamei subspecies and provide new insights into the mating system and geographical differentiation of these birds.     

Nine new polymorphic microsatellite loci for the amplification of archived otolith DNA of Atlantic cod,Gadus morhua L.

Nine out of 22 microsatellite primers tested were successfully amplified on three samples of cod Gadus morhua L. (two contemporary and one archived otolith samples). All loci were polymorphic (5–23 alleles/locus). The average observed heterozygosity across loci and samples was 0.625, ranging from 0.294 to 0.895 at each locus. All loci were under Hardy–Weinberg equilibrium, except PGmo56 that showed significant excess of heterozygotes in all studied samples. The isolated loci were suitable for degraded DNA and therefore useful for conducting a long‐term temporal study with DNA obtained from archived otoliths of cod.     

Isolation and characterization of 145 polymorphic microsatellite loci for the common frog (Rana temporaria)

We describe primers and polymerase chain reaction conditions to amplify 145 di‐, tri‐ and tetranucleotide microsatellite loci from the common frog (Rana temporaria), a species commonly used as a model in ecological and evolutionary research. Primers were tested on 46 individuals from two Fennoscandian populations and yielded an average of six to nine alleles per locus (range = 1–30) depending on the population. Average observed heterozygosities in the two populations were 0.16 (range = 0–0.91) and 0.36 (range = 0–1).     

Development and cross-species testing of western bluebird (Sialia mexicana) microsatellite primers

Western and eastern bluebirds (Sialia mexicana and S. sialis) are socially monogamous passerines that engage in extra‐pair copulations. We obtained microsatellites from S. mexicana and optimized and characterized 15 microsatellite DNA loci in 60 individuals of this species. Primer pairs yielded an average of 13 alleles per locus in western bluebirds (range 3–35 alleles) with an average observed heterozygosity of 0.68 (range 0.27–0.88). All 15 loci also successfully amplified in S. sialis (n = 24), with an average of 11.5 alleles per locus (range 4–26) and an average observed heterozygosity of 0.59 (range 0.22–0.90).     

Development of microsatellite loci for population studies of the pink ling,Genypterus blacodes (Teleostei: Ophidiidae)

Fifteen microsatellite loci were developed for pink ling (Genypterus blacodes), a fish of significant commercial importance to Australasia. Nine loci were examined in samples from five regions of Australia’s South East Fishery. All nine were highly polymorphic; numbers of alleles per locus ranged from 11 to 52 in total samples of 270–306 individuals. The average observed heterozygosity per locus per sample (0.823) was a little lower than the average Hardy–Weinberg expected heterozygosity per sample (0.895), perhaps reflecting the possible presence of null alleles at two loci. There was no significant evidence of genetic stock heterogeneity.     

Characterization of microsatellite markers for the North African gundi,Ctenodactylus gundi (Rodentia: Ctenodactylidae)

Eleven highly polymorphic microsatellite loci were isolated from the North African gundi (Ctenodactylus gundi), a gregarious rock‐dwelling rodent found in northern Africa. These loci exhibited high genetic diversity (eight to 19 alleles per locus) and high levels of heterozygosity (62–98%). Collectively, these microsatellite markers are sufficiently variable to estimate levels of relatedness and population genetic structure for C. gundi.     

Cloning and characterization of novel microsatellite DNA markers for the grass rockfish,Sebastes rastrelliger,and cross‐species amplification in 10 related Sebastes spp

Here we report development and characterization of seven polymorphic loci derived from grass rockfish (Sebastes rastrelliger) genomic DNA phagemid libraries enriched for microsatellite motifs. Within grass rockfish, average allelic diversity was 11.3 alleles per locus and average heterozygosity was 0.73. The seven loci also were surveyed in 10 related species of Sebastes, where allelic diversity ranged from highly polymorphic to monomorphic. Deviations from Hardy–Weinberg were nonsignificant in all but one species/locus combination suggesting low occurrence of null alleles. Significant linkage disequilibrium was detected among three loci, but these events were limited to a single species in each case.     

Polymorphic tetranucleotide microsatellite markers in the Caribbean spiny lobster,Panulirus argus

Ten tetranucleotide microsatellite loci are described for the Caribbean spiny lobster Panulirus argus. Loci were polymorphic (4–15 alleles per locus) and exhibited high levels of expected (0.553–0.921) and observed heterozygosity (0.469–0.906) from samples caught off Belize and Puerto Rico coasts. No significant departure from Hardy–Weinberg equilibrium conditions were observed for any locus. All microsatellite loci should be useful for assessing population discrimination for this valuable marine animal currently subjected to excessive fishing efforts.     

Isolation and characterization of microsatellite DNA markers from the Lanyu scops owl (Otus elegans botelensis)

From a genomic library enriched for GATA/CTAT and GAAA/CTTT repeats, 12 polymorphic microsatellite markers were developed for the Lanyu scops owl (Otus elegans botelensis). Polymorphism of these loci was evaluated in a sample of 58 adult individuals of unknown relationship. The allele numbers of each locus were from five to 25 and the observed heterozygosity of each locus ranges from 0.707 to 0.914. When using these loci in parentage assignment, the probability of false parent exclusion is greater than 0.999. All loci conformed to Hardy–Weinberg expectations.     

Characterization of 24 microsatellite loci in delta smelt, Hypomesus transpacificus, and their cross-species amplification in two other smelt species of the Osmeridae family

We characterized 24 polymorphic tetranucleotide microsatellite loci for delta smelt (Hypomesus transpacificus) endemic to the San Francisco Bay Estuary, CA, USA. Screening of samples (n = 30) yielded two to 26 alleles per locus with observed levels of heterozygosity ranging from 0.17 to 1.0. Only one locus deviated from Hardy–Weinberg equilibrium, suggesting these individuals originate from a single panmictic population. Linkage disequilibrium was found in two pairs of loci after excluding the locus out of Hardy–Weinberg equilibrium. Twenty‐two primer pairs cross‐amplified in wakasagi smelt (Hypomesus nipponensis), and 15 primer pairs cross‐amplified in longfin smelt (Spirinchus thaleichthys).     

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.     

Evidence for Directional Selection at a Novel Major Histocompatibility Class I Marker in Wild Common Frogs (Rana temporaria) Exposed to a Viral Pathogen (Ranavirus)

Whilst the Major Histocompatibility Complex (MHC) is well characterized in the anuran Xenopus, this region has not previously been studied in another popular model species, the common frog (Rana temporaria). Nor, to date, have there been any studies of MHC in wild amphibian host-pathogen systems. We characterise an MHC class I locus in the common frog, and present primers to amplify both the whole region, and specifically the antigen binding region. As no more than two expressed haplotypes were found in over 400 clones from 66 individuals, it is likely that there is a single class I locus in this species. This finding is consistent with the single class I locus in Xenopus, but contrasts with the multiple loci identified in axolotls, providing evidence that the diversification of MHC class I into multiple loci likely occurred after the Caudata/Anura divergence (approximately 350 million years ago) but before the Ranidae/Pipidae divergence (approximately 230 mya). We use this locus to compare wild populations of common frogs that have been infected with a viral pathogen (Ranavirus) with those that have no history of infection. We demonstrate that certain MHC supertypes are associated with infection status (even after accounting for shared ancestry), and that the diseased populations have more similar supertype frequencies (lower F_ST) than the uninfected. These patterns were not seen in a suite of putatively neutral microsatellite loci. We interpret this pattern at the MHC locus to indicate that the disease has imposed selection for particular haplotypes, and hence that common frogs may be adapting to the presence of Ranavirus, which currently kills tens of thousands of amphibians in the UK each year.     

Microsatellite loci for behavioural studies of Eclectus parrot (Eclectus roratus: Aves)

Ten polymorphic microsatellite loci were isolated from the cooperatively breeding and sexually dichromatic Eclectus parrot (Eclectus roratus). Nine loci were in Hardy–Weinberg equilibrium and unlinked. One locus, Ero1, was presumed to be sex‐linked since females, the heterogametic sex, were all homozygous, whereas 72% of males were heterozygous. A DNA database search revealed that Ero8 is probably an independent isolation of the microsatellite locus AgGT83 of the parrot Amozona guildingii. With high heterozygosity (0.41–0.89) and number of alleles (two to 13), these loci should prove useful for investigating the mating system of these unusual Australasian parrots.     

Isolation and characterization of 12 tetranucleotide repeat microsatellite loci from the green‐backed tit (Parus monticolus)

From a partial genomic library enriched for GATA short tandem repeats, we developed 12 polymorphic microsatellite loci from the green‐backed tit (Parus monticolus). We characterized these loci by genotyping 30 adult individuals with unknown relationship. The number of alleles ranged from four to 17 per locus (mean = 9.3 alleles) and the observed heterozygosity for each locus ranged from 0.633 to 0.933 (mean = 0.789). All loci conformed to Hardy–Weinberg expectations. Four of 66 possible pairwise comparisons between loci showed significant gametic disequilibrium.