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.     

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.     

Isolation and characterization of novel tetranucleotide microsatellite DNA markers for the spotted salamander,Ambystoma maculatum

Fifteen tetranucleotide microsatellite loci were identified and characterized for spotted salamanders (Ambystoma maculatum) collected from three vernal pools in the south‐eastern USA. These markers revealed a high degree of genetic diversity (7–32 alleles per locus), heterozygosity (31.6–86.3%) and allelic heterogeneity (91% of comparisons were statistically significant). Considerable differentiation among populations was observed as genetic distances (chord) ranged between 0.50 and 0.65, and all F_ST values (0.08–0.14) were statistically significant. Moreover, genotypic assignment tests correctly classified all individuals to their respective collection. These markers should prove useful for investigating fine‐scale population structure and mating system.     

Postglacial range expansion from northern refugia by the wood frog, Rana sylvatica

Although the range dynamics of North American amphibians during the last glacial cycle are increasingly better understood, the recolonization history of the most northern regions and the impact of southern refugia on patterns of intraspecific genetic diversity and phenotypic variation in these regions are not well reconstructed. Here we present the phylogeographic history of a widespread and primarily northern frog, Rana sylvatica . We surveyed 551 individuals from 116 localities across the species' range for a 650-bp region of the NADH dehydrogenase subunit 2 and tRNA^TRP mitochondrial genes. Our phylogenetic analyses revealed two distinct clades corresponding to eastern and western populations, as well as a Maritime subclade within the eastern lineage. Patterns of genetic diversity support multiple refugia. However, high-latitude refugia in the Appalachian highlands and modern-day Wisconsin appear to have had the biggest impact on northern populations. Clustering analyses based on morphology further support a distinction between eastern and western wood frogs and suggest that postglacial migration has played an important role in generating broad-scale patterns of phenotypic variation in this species.     

Isolation of polymorphic tetranucleotide microsatellite markers for the ornate nursery frog Cophixalus ornatus

We have isolated 18 polymorphic microsatellite loci for Cophixalus ornatus from genomic libraries enriched for (AAAG)_n, (AACC)_n and (AAGG)_n repetitive elements. The number of alleles ranges from five to 22 per locus with the observed heterozygosity ranging from 0.10 to 0.92. These markers will be useful for the analysis of population structure in C. ornatus and testing alternative models of speciation.     

Isolation and characterization of 19 polymorphic microsatellite DNA markers in the Japanese brown frog (Rana japonica)

We report the isolation and characterization of 19 polymorphic microsatellite DNA markers in the Japanese brown frog (Rana japonica). These markers were tested in 24 individuals each collected from three distinct populations in Ichikai-machi, Tochigi Prefecture. The number of observed alleles per locus ranged from 3 to 24 across all populations, and the values of observed and expected heterozygosities ranged from 0.130 to 1 and from 0.125 to 0.941, respectively. These microsatellite loci will be useful for investigating the intraspecific genetic variation and population structure of this species.     

Microsatellite variation and fine-scale population structure in the wood frog (Rana sylvatica)

We investigated genetic population structure in wood frogs (Rana sylvatica) from a series of Prairie Pothole wetlands in the northern Great Plains. Amphibians are often thought to exist in demographic metapopulations, which require some movement between populations, yet genetic studies have revealed strong subdivision among populations, even at relatively fine scales (several km). Wood frogs are highly philopatric and studies of dispersal suggest that they may exhibit subdivision on a scale of approximately 1-2 km. We used microsatellites to examine population structure among 11 breeding assemblages separated by as little as 50 m up to approximately 5.5 km, plus one population separated from the others by 20 km. We found evidence for differentiation at the largest distances we examined and among a few neighbouring ponds, but most populations were strikingly similar in allele frequencies, suggesting high gene flow among all but the most distant populations. We hypothesize that the few significant differences among neighbouring populations at the finest scale may be a transient effect of extinction-recolonization founder events, driven by periodic drying of wetlands in this hydrologically dynamic landscape.     

Isolation of polymorphic tetranucleotide microsatellite markers for the green‐eyed tree frog (Litoria genimaculata)

We have isolated 16 polymorphic microsatellite markers for the green‐eyed tree frog, Litoria genimaculata, from genomic libraries enriched for (AAGG)_n and (AAAG)_n repetitive elements. The number of alleles ranges from four to 14 per locus with the observed heterozygosity ranging from 0.36 to 1.00. These markers will be useful for analysis of questions concerning population genetic structure and speciation.     

Characterization of γ-glutamyltranspeptidase in the liver of the frog: 3. Response to freezing and thawing in the freeze-tolerant wood frog Rana sylvatica

The freeze tolerant wood frog Rana sylvatica was studied to determine the impact of the freezing and thawing of this frog on the activity of γ-glutamyltranspeptidase in the liver. On exposure to ?2·5°C, for 1, 12 and 24 h, frogs were found to be cool, covered with ice crystals and frozen, respectively. Thawing for 24 h at 4°C recovered the frogs completely. A 45 per cent decrease in the liver weight: body weight ratio was notable after 1 h at ?2·5°C, suggestive of an early hepatic capacitance response. A glycemic response to freezing was observed: blood glucose levels exhibited a 55 per cent decrease after 1 h at ?2·5°C on cooling; a 10·5-fold increase after 12 h at ?2·5°C on the initiation of freezing; and a 22-fold increase after 24 h at ?2·5°C in the fully frozen state. Blood glucose levels remained elevated four-fold in the thawed state. Plasma insulin levels were increased twofold in the frozen state and 1·8-fold in the thawed state, while plasma ketone levels were increased 1·8-fold in the frozen state and 1·5-fold in the thawed state. Plasma total T₃ levels were decreased by 22 per cent in the frozen state and normalized on thawing. In homogenates and plasma membranes isolated from the livers of Rana sylvatica, the activity of γ-glutamyltranspeptidase was found to be elevated at all stages of the freeze–thaw process. After 1, 12 and 24 h at ?2·5°C, activities were increased 2·5-, 2·3-, 2·4-fold respectively in the homogenates and 2·5-, 2·2-, 2·4-fold respectively in the plasma membranes. After thawing, activities were still increased 1·9-fold in both homogenates and plasma membranes. In homogenates prepared from the kidneys of Rana sylvatica, the activity of γ-glutamyltranspeptidase was increased 1·4-fold after 1 h at ?2·5°C after which it returned to normal. The role of thyroid hormone in producing the increase in γ-glutamyltranspeptidase in the liver of Rana sylvatica in response to freezing is discussed as is the significance of the enzyme increase in terms of hepatic cytoprotection and freeze tolerance.     

Novel Jefferson salamander,Ambystoma jeffersonianum,microsatellite DNA markers detect population structure and hybrid complexes

Twenty polymorphic microsatellite DNA markers were isolated and characterized in Ambystoma jeffersonianum collected from three vernal pools in the mid‐Atlantic region of the U.S. These markers revealed a high degree of genetic diversity (7–23 alleles per locus), heterozygosity (46.7% to 100%), and allelic heterogeneity (96% of comparisons were statistically significant). Genetic distances were greatest in comparisons between collections, intermediate within collections, and least among sibling pairs. Six markers were trisomic in A. jeffersonianum‐A. laterale hybrids. These microsatellite DNA loci should allow delineation of genetic structure within and among populations of the diploid A. jeffersonianum and provide an effective method for identification of triploid hybrid individuals.     

Eight new tetranucleotide microsatellite loci for the agile frog (Rana dalmatina)

We describe eight new polymorphic tetranucleotide microsatellite loci isolated from the agile frog (Rana dalmatina). In 25 individuals from the Nature Reserve Lüneburger Heide (Lower Saxony, Germany), the number of alleles per locus ranged from four to nine and average observed heterozygosities from 69.1% to 80.7%. No evidence for linkage disequilibrium was found and none of the loci showed significant deviation from Hardy-Weinberg expectations. These microsatellite DNA markers are suitable tools for addressing population genetics issues in this endangered species.     

Nine polymorphic microsatellite loci for the northern leopard frog (Rana pipiens)

We describe the cloning and characterization of nine microsatellite loci from the northern leopard frog. Seven loci consist of tetranucleotide repeats, one locus consists of a dinucleotide repeat and one locus consists of a GT repeat juxtaposed with a GATA repeat. In a sample of 36 frogs from a natural population, polymorphism at these loci ranged from two to 13 alleles per locus with expected heterozygosities ranging from 0.5 to 0.91. These loci will be useful to researchers since this species is used for a broad range of studies.     

Isolation of polymorphic tri- and tetranucleotide microsatellite loci for the highly endangered Baw Baw frog (Philoria frosti)

We isolated 15 polymorphic microsatellite loci for the Baw Baw frog, Philoria frosti, from a genomic library enriched for (AAC)(n) and (AAAG)(n) repetitive elements. The number of alleles ranges from two to 14 per locus with the observed heterozygosity ranging from 0.04 to 0.96.     

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.     

Eight novel tetranucleotide and five cross-species dinucleotide microsatellite loci for the ornate chorus frog (Pseudacris ornata)

We describe the cloning and characterization of eight novel tetranucleotide microsatellite loci in the ornate chorus frog (Pseudacris ornata). We also screened 26 loci from GenBank that were isolated from other Pseudacris species and obtained consistent product from five of these dinucleotide loci. All loci are polymorphic. In our sample of 26 frogs from a natural population, polymorphism ranged from 1 to 22 alleles per locus with expected heterozygosities ranging from 0 to 0.958. These loci enable high-resolution studies of P. ornata. Moreover, cross-species amplification success suggests they will also be useful for other chorus frog species.     

Kin recognition by tadpoles and froglets of the wood frog Rana sylvatica

Summary We investigated kin recognition by the wood frog Rana sylvatica in blind laboratory experiments using spatial proximity as a recognition assay. Tadpoles were tested for the ability to discriminate between: 1) familiar full-sibs and unfamiliar non-kin, 2) unfamiliar paternal half-sibs and unfamiliar non-kin, and 3) familiar and unfamiliar full-sibs. Tadpoles discriminated full- and paternal half-sibs from unrelated conspecifics, but did not discriminate between familiar and unfamiliar full-sibs. Froglets from the same laboratory population were tested for the ability to discriminate between 1) familiar full-sibs and unfamiliar non-kin, and 2) unfamiliar paternal half-sibs and unfamiliar non-kin. Froglets preferentially associated with full- and half-sibs over unrelated conspecifics. Our results show that familiarity, i.e., prior association, is not necessary for kin recognition in tadpoles and froglets. The ability of tadpoles and froglets to recognize unfamiliar paternal half-sibs demonstrates that a common maternal factor is not necessary for kin recognition, and indicates that the recognition cue has a genetic component. Our results add to the increasing evidence that a variety of vertebrate and invertebrate animals have the ability to recognize unfamiliar kin by using genetically specified recognition cues.     

Post-freeze recovery of peripheral nerve function in the freeze-tolerant wood frog,Rana sylvatica

We investigated the restoration of peripheral nerve function and simple neurobehavioral reflexes in the freeze-tolerant wood frog (Rana sylvatica). Thirty-two specimens, allowed to freeze for 39 h and ultimately cooled to-2.2°C, were sampled at various time intervals up to 60 h after thawing at 5°C was initiated. The sciatic nerves of treated frogs were initially unresponsive to stimulation, but usually regained excitability within 5 h. Except for a slight reduction in nerve excitability characteristics of the compound action potentials of treated frogs were indistinguishable from those of control frogs. Recovery times for the hindlimb retraction and righting reflexes were 8 h and 14 h, respectively. Concentrations of the cryoprotectant glucose increased 8.2-fold in the sciatic nerve and 10.5-fold in the underlying semimembranosis muscle of treated frogs, and remained elevated for at least 60 h after thawing was initiated. These organs lost 47.2% and 15.9%, respectively, of their water during freezing, but were rehydrated within 2 h of the onset of thawing. The accumulation of glucose and the withdrawal of tissue water apparently are cryoprotective responses which enable this species to survive freezing.