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.     

Phylogeography of postglacial range expansion in Nigronia serricornis Say (Megaloptera: Corydalidae)

We have examined the effects of post-Wisconsinan glacial range expansion on the phylogeography of the saw-combed fishfly, Nigronia serricornis Say (Megaloptera: Corydalidae) because aquatic insects are under-represented in postglacial studies (and in phylogeography in general), and because the effects of ecological degradation on the population genetics of environmental indicator species like N. serricornis cannot be measured unless the underlying phylogeography is understood. Sequence data from a 630-base fragment of the mitochondrial cytochrome oxidase I (COI) gene were subjected to amova and nested clade analysis for 30 populations (n = 344) of N. serricornis. Both the amova and nested clade analysis revealed substantial population structure; 44.4% of the variance occured among populations. Three northward migrations are apparent: one from Tennessee into Illinois, Indiana, Wisconsin, Michigan and Ontario, a second that radiated eastward from Pennsylvania, and a third that moved along the coast from North Carolina into Connecticut, Massachusetts, Maine and then into New York. The latter two of these migrations were the result of contiguous range expansions, while the former expansion, out of Tennessee, appears to have been rapid with little gene flow from the source population. Additional clades included a group of haplotypes in central Kentucky that appear to have expanded along preglacial drainages, and clades in North Carolina and Georgia that have remained centrally located. Haplotype diversity decreased from south to north, a pattern that has been widely reported for animal and plant populations that expanded with the retreat of the Wisconsinan glaciation.     

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.     

Population genetic structure and diversity of the Apennine endemic stream frog, Rana italica--insights on the Pleistocene evolutionary history of the Italian peninsular biota

For most species in the Western Palaearctic region, southern Mediterranean peninsulas have been identified as major Quaternary refugia and hotspots of intraspecific diversity, and thus, as areas of particular relevance for the conservation of the evolutionary potential. We analysed the patterns of geographical variation among 26 populations of the Italian stream frog, using both nuclear (allozymes) and mitochondrial (partial cytochrome b sequences) markers. Phylogenetic, phylogeographical and population genetic analyses suggested that the species survived the last glacial–interglacial cycles in two distinct refugia, one restricted to the tip of the Calabrian peninsula, at the extreme south of the species' range, the other spanning from central Calabria to central Apennines and showing evidences for further population subdivision therein. Historical demographic tests suggested a significant population expansion from the latter, which most likely began around the last pleniglacial. This expansion would have led to the rapid colonization of the northern Apennines to the north, and to a secondary contact and population admixture with the population from the southern refugium in southern central Calabria. A comparison of the evolutionary history inferred for the Italian stream frog with the data emerging for other codistributed species suggests: (i) the generality of a multiple-refugia scenario for the Italian peninsula, (ii) the possible occurrence of at least one suture zone in southern Italy, and (iii) that for most species, this Pleistocene refugium is not only a hotspot, but also a melting pot of intraspecific genetic diversity. Finally, the conservation implications of these results are also briefly highlighted.     

Postfreeze reduction of locomotor endurance in the freeze-tolerant wood frog,Rana sylvatica

Considerable study has focused on the physiological adaptations for freeze tolerance in the wood frog, Rana sylvatica, a northern species that overwinters within the frost zone, but little attention has been paid to the associated costs to organismal performance. Here we report that freezing causes transient impairment of locomotor endurance and adverse changes in exercise physiology that persist for at least 96 h. Wood frogs frozen at -2 degrees C for 36 h exhibited normal behaviors and hydro-osmotic status and near-normal metabolite (glycogen, glucose, and lactate) levels within 24 h after thawing began. However, when exercised to exhaustion on a treadmill, these frogs showed a 40% reduction in endurance as compared to sham-treated (unfrozen) controls, a reduction that persisted for at least 96 h. Previously frozen frogs exhibited higher rates of lactate accumulation during exercise than controls, suggesting that prior freezing forces greater reliance on the glycolytic pathways of energy production to support exercise. Given that this species breeds in late winter, when subzero temperatures are common, freezing may result in reduced fitness by hampering their ability to reach the pond, avoid predators, and successfully obtain mates.     

The relationship among egg size,density and food level on larval development in the wood frog (Rana sylvatica)

Summary Although inter- and intraspecific variation in egg size among amphibians has been well documented, the relationship between egg size and fitness remains unclear. Recent attempts to correlate egg size intraspecifically with larval developmental patterns have been equivocal. In this study the development of larvae derived from large eggs and small eggs, from a single population in Maryland were compared under a range of food levels and larval population densities. Both food level and density had significant effects on the length of the larval period and size at metamorphosis. However, the response among larvae derived from different egg sizes was not additive. At low densities and high food levels, larvae from small eggs had longer larval periods and a larger size at metamorphosis than larvae derived from large eggs. In contrast, at high densities larvae from small eggs had longer developmental periods but were smaller at metamorphosis than larvae from large eggs. In addition, larvae from small eggs were more sensitive to density irrespective of food level. These results suggest that optimal egg size is correlated with environmental factors, which may explain the maintenance of both geographic and within population variation in egg size commonly observed in amphibians.     

Bosmina (Eubosmina) longicornis longicornis SCHOEDLER, 1866 (Crustacea,Cladocera: Bosminidae) from the Postglacial Sediments of the Neuendorfer See,East-Germany

Analyses were made of subfossil Eubosmina remains from the Lake Neuendorfer See, a lake in the middle part of the river Spree in the “Urstromtal” of Baruth situated in the eastern part of Germany. In addition to Bosmina (Bosmina) longirostris (O. F. M., 1785) Bosmina (Eub.) longicornis longicornis Schoedler, 1866 occured in the lake from the Boreal (sediment depth 15.85 m) to the recent surface layers of the core. Other eubosminids did not occur during the history of the Lake Neuendorfer See with the exception of few remains of B. (Eub.) longispina, which were detected in the lowermost layers of the core during the late Pleistocene. In the present-day plankton, Bosmina (Eub.) l. longicornis was the only eubosminid found in the Lake Neuendorfer See. During the long period between colonization during the Boreal to the present, no changes were detected in typical morphological features of B. (Eub.) l. longicornis. All remains of Eubosmina in the layers above 15.85 m belong undoubtedly to the same taxon. No signs of any morphological instability (other then ontogenetic and cyclomorphic formchanges) or “morphological transition” (Hofmann, 1977) to other taxa could be found. By using paleolimnological analyses, the named taxon has proven to be a “good” entity within the group of long-spined taxa of the subgenus Eubosmina.     

Concordant molecular and phenotypic data delineate new taxonomy and conservation priorities for the endangered mountain yellow-legged frog

The mountain yellow-legged frog Rana muscosa sensu lato , once abundant in the Sierra Nevada of California and Nevada, and the disjunct Transverse Ranges of southern California, has declined precipitously throughout its range, even though most of its habitat is protected. The species is now extinct in Nevada and reduced to tiny remnants in southern California, where as a distinct population segment, it is classified as Endangered. Introduced predators (trout), air pollution and an infectious disease (chytridiomycosis) threaten remaining populations. A Bayesian analysis of 1901 base pairs of mitochondrial DNA confirms the presence of two deeply divergent clades that come into near contact in the Sierra Nevada. Morphological studies of museum specimens and analysis of acoustic data show that the two major mtDNA clades are readily differentiated phenotypically. Accordingly, we recognize two species, Rana sierrae , in the northern and central Sierra Nevada, and R. muscosa , in the southern Sierra Nevada and southern California. Existing data indicate no range overlap. These results have important implications for the conservation of these two species as they illuminate a profound mismatch between the current delineation of the distinct population segments (southern California vs. Sierra Nevada) and actual species boundaries. For example, our study finds that remnant populations of R. muscosa exist in both the southern Sierra Nevada and the mountains of southern California, which may broaden options for management. In addition, despite the fact that only the southern California populations are listed as Endangered, surveys conducted since 1995 at 225 historic (1899–1994) localities from museum collections show that 93.3% ( n =146) of R. sierrae populations and 95.2% ( n =79) of R. muscosa populations are extinct. Evidence presented here underscores the need for revision of protected population status to include both species throughout their ranges.     

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).     

A test of the effect of supplemental UV-B radiation on the common frog, Rana temporaria L., during embryonic development

The release of certain man-made chemicals has led to recurrent, seasonal destruction of ozone in the upper atmosphere, allowing more solar radiation in the UV-B waveband to reach the Earth. Consequently, many amphibians may suffer increased exposure to UV-B at various stages in their lives. Embryonic stages of species which spawn in the spring, in shallow, open water, are at high risk of increased exposure. We exposed newly fertilized eggs of one such species, Rana temporaria L., to sunlight with and without supplemental UV-B. We used outdoor arrays of lamps to simulate the increase in UV-B which might result from previously documented ozone depletion. From immediately after fertilization to when hatchlings began feeding, ambient solar UV-B, weighted for DNA-damaging potential, was supplemented by ≈ 81% in 1995 and 113% in 1996. These levels of supplementation approximated the increase in solar UV-B expected to result from losses of 21% and 25%, respectively, of the total amount of ozone in the atmospheric column, relative to pre-ozone-depletion values. We found no evidence that these additions of UV-B radiation increased the incidence of mortality or overt developmental abnormality among embryos. We stress the need for appropriate dosimetry in studies of effects of UV-B on organisms.     

Membrane adaptation in phospholipids and cholesterol in the widely distributed,freeze-tolerant wood frog,Rana sylvatica

Maintaining proper membrane phase and fluidity is important for preserving membrane structure and function, and by altering membrane lipid composition many organisms can adapt to changing environmental conditions. We compared the phospholipid and cholesterol composition of liver and brain plasma membranes in the freeze-tolerant wood frog, Rana sylvatica, from southern Ohio and Interior Alaska during summer, fall, and winter. We also compared membranes from winter-acclimatized frogs from Ohio that were either acclimated to 0, 4, or 10 °C, or frozen to ?2.5 °C and sampled before or after thawing. Lipids were extracted from isolated membranes, separated by one-dimensional thin-layer chromatography, and analyzed via densitometry. Liver membranes underwent seasonal changes in phospholipid composition and lipid ratios, including a winter increase in phosphatidylethanolamine, which serves to increase fluidity. However, whereas Ohioan frogs decreased phosphatidylcholine and increased sphingomyelin, Alaskan frogs only decreased phosphatidylserine, indicating that these phenotypes use different adaptive strategies to meet the functional needs of their membranes. Liver membranes showed no seasonal variation in cholesterol abundance, though membranes from Alaskan frogs contained relatively less cholesterol, consistent with the need for greater fluidity in a colder environment. No lipid changed seasonally in brain membranes in either population. In the thermal acclimation experiment, cold exposure induced an increase in phosphatidylethanolamine in liver membranes and a decrease in cholesterol in brain membranes. No changes occurred during freezing and thawing in membranes from either organ. Wood frogs use tissue-specific membrane adaptation of phospholipids and cholesterol to respond to changing environmental factors, particularly temperature, though not with freezing.     

Sex reversal and primary sex ratios in the common frog (Rana temporaria)

Sex reversal has been suggested to have profound implications for the evolution of sex chromosomes and population dynamics in ectotherms. Occasional sex reversal of genetic males has been hypothesized to prevent the evolutionary decay of nonrecombining Y chromosomes caused by the accumulation of deleterious mutations. At the same time, sex reversals can have a negative effect on population growth rate. Here, we studied phenotypic and genotypic sex in the common frog (Rana temporaria) in a subarctic environment, where strongly female‐biased sex ratios have raised the possibility of frequent sex reversals. We developed two novel sex‐linked microsatellite markers for the species and used them with a third, existing marker and a Bayesian modelling approach to study the occurrence of sex reversal and to determine primary sex ratios in egg clutches. Our results show that a significant proportion (0.09, 95% credible interval: 0.04–0.18) of adults that were genetically female expressed the male phenotype, but there was no evidence of sex reversal of genetic males that is required for counteracting the degeneration of Y chromosome. The primary sex ratios were mostly equal, but three clutches consisted only of genetic females and three others had a significant female bias. Reproduction of the sex‐reversed genetic females appears to create all‐female clutches potentially skewing the population level adult sex‐ratio consistent with field observations. However, based on a simulation model, such a bias is expected to be small and transient and thus does not fully explain the observed female‐bias in the field.     

Glacial vicariance in the Pacific Northwest: evidence from a lodgepole pine mitochondrial DNA minisatellite for multiple genetically distinct and widely separated refugia

The Canadian side of the Pacific Northwest was almost entirely covered by ice during the last glacial maximum, which has induced vicariance and genetic population structure for several plant and animal taxa. Lodgepole pine ( Pinus contorta Dougl. ex. Loud.) has a wide latitudinal and longitudinal distribution in the Pacific Northwest. Our main objective was to identify relictual signatures of glacial vicariance in the population structure of the species and search for evidence of distinct glacial refugia in the Pacific Northwest. A maternally inherited mitochondrial DNA minisatellite-like marker was used to decipher haplotype diversity in 91 populations of lodgepole pine located across the natural range. Overall population differentiation was sizeable ( G _ST = 0.365 and R _ST = 0.568). Four relatively homogeneous groups of populations, possibly representative of as many genetically distinct glacial populations, were identified for the two main subspecies, ssp. latifolia and ssp. contorta . For ssp. contorta , one glacial lineage is suggested to have been located at high latitudes and possibly off the coast of mainland British Columbia (BC), while the other is considered to have been located south of the ice sheet along the Pacific coast. For ssp. latifolia , two genetically distinct glacial populations probably occurred south of the ice sheet: in the area bounded by the Cascades and Rocky Mountains ranges, and on the eastern side of the Rockies. A possible fifth refugium located in the Yukon may have also been present for ssp. latifolia . Zones of contact between these ancestral lineages were also apparent in interior and northern BC. These results indicate the role of the Queen Charlotte Islands and the Alexander Archipelago as a refugial zone for some Pacific Northwest species and the vicariant role played by the Cascades and the American Rocky Mountains during glaciation.     

Experimental and model analyses of the effects of competition on individual size variation in wood frog (Rana sylvatica) tadpoles

1. Size variation is a ubiquitous feature of animal populations and is predicted to strongly influence species abundance and dynamics; however, the factors that determine size variation are not well understood. 2. In a mesocosm experiment, we found that the relationship between mean and variation in wood frog (Rana sylvatica) tadpole size is qualitatively different at different levels of competition created by manipulating resource supply rates or tadpole density. At low competition, relative size variation (as measured by the coefficient of variation) decreased as a function of mean size, while at high competition, relative size variation increased. Therefore, increased competition magnified differences in individual performance as measured by growth rate. 3. A model was developed to estimate the contribution of size-dependent factors (i.e. based on size alone) and size-independent factors (i.e. resulting from persistent inherent phenotypic differences other than size that affect growth) on the empirical patterns. 4. Model analysis of the low competition treatment indicated that size-dependent factors alone can describe the relationship between mean size and size variation. To fit the data, the size scaling exponent that describes the dependence of growth rate on size was determined. The estimated value, 0-83, is in the range of that derived from physiological studies. 5. At high competition, the model analysis indicated that individual differences in foraging ability, either size-based or due to inherent phenotypic differences (size-independent factors), were much more pronounced than at low competition. The model was used to quantify the changes in size-dependent or size-independent factors that underlie the effect of competition on size-variation. In contrast to results at low competition, parameters derived from physiological studies could not be used to describe the observed relationships. 6. Our experimental and model results elucidate the role of size-dependent and size-independent factors in the development of size variation, and highlight and quantify the context dependence of individual (intrapopulation) differences in competitive abilities.