Conservation phylogeography: does historical diversity contribute to regional vulnerability in European tree frogs (Hyla arborea)?

Documenting and preserving the genetic diversity of populations, which conditions their long‐term survival, have become a major issue in conservation biology. The loss of diversity often documented in declining populations is usually assumed to result from human disturbances; however, historical biogeographic events, otherwise known to strongly impact diversity, are rarely considered in this context. We apply a multilocus phylogeographic study to investigate the late‐Quaternary history of a tree frog (Hyla arborea) with declining populations in the northern and western part of its distribution range. Mitochondrial and nuclear polymorphisms reveal high genetic diversity in the Balkan Peninsula, with a spatial structure moulded by the last glaciations. While two of the main refugial lineages remained limited to the Balkans (Adriatic coast, southern Balkans), a third one expanded to recolonize Northern and Western Europe, loosing much of its diversity in the process. Our findings show that mobile and a priori homogeneous taxa may also display substructure within glacial refugia (‘refugia within refugia’) and emphasize the importance of the Balkans as a major European biodiversity centre. Moreover, the distribution of diversity roughly coincides with regional conservation situations, consistent with the idea that historically impoverished genetic diversity may interact with anthropogenic disturbances, and increase the vulnerability of populations. Phylogeographic models seem important to fully appreciate the risks of local declines and inform conservation strategies.     

SPECIATION BY POLYPLOIDY IN TREEFROGS: MULTIPLE ORIGINS OF THE TETRAPLOID,HYLA VERSICOLOR

Speciation by polyploidy is rare in animals, yet, in vertebrates, there is a disproportionate concentration of polyploid species in anuran amphibians. Sequences from the cytochrome b gene of the mitochondrial DNA (mtDNA) were used to determine phylogenetic relationships among 37 populations of the diploid-tetraploid species pair of gray treefrogs, Hyla chrysoscelis and Hyla versicolor. The diploid species, H. chrysoscelis, consists of an eastern and a western lineage that have 2.3% sequence divergence between them. The tetraploid species, H. versicolor, had at least three separate, independent origins. Two of the tetraploid lineages are more closely related to one or the other of the diploid lineages (0.18%–1.4% sequence divergence) than they are to each other (1.9%–3.4% sequence divergence). The maternal ancestor of the third tetraploid lineage is unknown. The phylogenetic relationships between the two species and among lineages within each species support the hypothesis of multiple origins of the tetraploid lineages.     

A tree of tree frogs around the Black Sea

Speciation, the process by which one species evolves into two or more, is a major focus of ongoing debate, particularly regarding the geographic context in which it occurs. Geographic models of speciation tend to fall into discrete categories, typically referred to as allopatric, parapatric and sympatric speciation, according to whether two groups evolve reproductive isolation while geographically isolated, differentiated but connected by gene flow, or completely co‐occurring. Yet molecular studies indicate that full development of reproductive isolation can take very long compared with the timescale at which climatic oscillations occur, such that the geographic context of differentiating forms might change often during the long process to full species. Studies of genetic relationships across the ranges of organisms with low‐dispersal distances have the potential to reveal these complex histories. In a particularly elegant example in this issue, Dufresnes et al. ( 2016 ) use genetic variation and ecological niche modelling to show that a ring of populations of the eastern tree frog (Hyla orientalis) surrounding the Black Sea had a complex history of geographic differentiation. Alternating phases of geographic fragmentation and phases of gene flow between neighbouring populations have produced a pattern of gradual genetic change connecting the western, southern and eastern sides of the ring, with the northwestern and northeastern forms being most differentiated. In the north, a population in Crimea appears to have been produced through mixture of the two extreme forms. The overall genetic relationships are reminiscent of those found in ring species, which have been used as prime demonstrations of the process of speciation. The difference, however, is that the terminal forms appear to have mixed rather than be reproductively isolated, although more research is needed to infer whether there might be some reproductive isolation on the northern side of the ring.     

Buccal swabs allow efficient and reliable microsatellite genotyping in amphibians

Buccal swabs have recently been used as a minimally invasive sampling method in genetic studies of wild populations, including amphibian species. Yet it is not known to date what is the level of reliability for microsatellite genotypes obtained using such samples. Allelic dropout and false alleles may affect the genotyping derived from buccal samples. Here we quantified the success of microsatellite amplification and the rates of genotyping errors using buccal swabs in two amphibian species, the Alpine newt Triturus alpestris and the Green tree frog Hyla arborea, and we estimated two important parameters for downstream analyses, namely the number of repetitions required to achieve typing reliability and the probability of identity among genotypes. Amplification success was high, and only one locus tested required two to three repetitions to achieve reliable genotypes, showing that buccal swabbing is a very efficient approach allowing good quality DNA retrieval. This sampling method which allows avoiding the controversial toe-clipping will likely prove very useful in the context of amphibian conservation.     

Effects of food-deprivation on migratory restlessness and diurnal activity in the garden warbler Sylvia borin

Summary The effects of tadpole body size, tadpole sibship, and fish body size on predation of gray treefrog tadpoles, Hyla chrysoscelis, were studied in laboratory and artificial pond experiments. Tadpole body size had a significantly positive effect on the survival of tadpoles in all experiments. The relationship between tadpole biomass eaten and biomass available suggested that fish were not satiated when consuming the largest tadpoles. Large tadpoles were probably better able to evade predators. A difference in survival among full sib families of tadpoles was only present in one family, suggesting that genetic differences in predator avoidance behavior or palatability were probably secondarily important to body size per se. Fish body size had a significantly negative effect on the survival of tadpoles. Larger fish consumed a larger number and proportion of tadpoles as well as greater biomass. These results indicate that environmental factors affecting the growth rate of tadpoles cand dramatically alter their vulnerability to gape-limited predators.     

Mid-frequency suppression in the green treefrog (<Emphasis Type="Italic">Hyla cinerea</Emphasis>): mechanisms and implications for the evolution of acoustic communication

Advertisement calls of green treefrogs (Hyla cinerea) have two spectral peaks centered at about 1 kHz and 3 kHz. Addition of a component of intermediate frequency (1.8 kHz) to a synthetic call reduced its attractiveness to females relative to an alternative lacking this component. This mid-frequency suppression occurred over a 20-dB range of playback levels. Addition of other intermediate frequencies had weak effects on preferences at some playback levels, in some localities, and at lower-than-normal temperatures. These effects correlate well with the response properties of a population of low-frequency-tuned auditory neurons innervating the amphibian papilla. Males of a closely related species (H. gratiosa) produce calls with emphasized frequencies within the range of suppression in H. cinerea; however, suppression also occurred in localities well outside the area of geographical overlap with this species. Thus, previous speculation that mid-frequency suppression evolved to enhance species discrimination is probably incorrect. This phenomenon is more likely to reflect a general sensory bias in anurans and other vertebrates, tone-on-tone inhibition. Such negative biases, and other inhibitory mechanisms, almost certainly play an important role in the evolution of communication systems but have received far less attention than positive biases that enhance signal attractiveness.     

Parallel changes in mate-attracting calls and female preferences in autotriploid tree frogs

For polyploid species to persist, they must be reproductively isolated from their diploid parental species, which coexist at the same time and place at least initially. In a complex of biparentally reproducing tetraploid and diploid tree frogs in North America, selective phonotaxis--mediated by differences in the pulse-repetition (pulse rate) of their mate-attracting vocalizations--ensures assortative mating. We show that artificially produced autotriploid females of the diploid species (Hyla chrysoscelis) show a shift in pulse-rate preference in the direction of the pulse rate produced by males of the tetraploid species (Hyla versicolor). The estimated preference function is centred near the mean pulse rate of the calls of artificially produced male autotriploids. Such a parallel shift, which is caused by polyploidy per se and whose magnitude is expected to be greater in autotetraploids, may have facilitated sympatric speciation by promoting reproductive isolation of the initially formed polyploids from their diploid parental forms. This process also helps to explain why tetraploid lineages with different origins have similar advertisement calls and freely interbreed.     

Comparative skeletogenesis of the Oriental Tree Frog Hyla orientalis (Anura: Hylidae)

The ontogeny of skeletal development of the Oriental Tree Frog, Hyla orientalis (Bedriaga, 1890), is followed from tadpole to froglet and compared with four other hylid species i.e., Hypsiboas lanciformis, Hypsiboas pulchellus, Phyllomedusa vaillanti and Pseudis platensis. Our analysis and comparison is based on cleared and double-stained specimens. The parasphenoid is the first skeletal element that ossified in Hyla orientalis is followed by the exoocipital, prootic, axial and appendicular skeletal elements, frontoparietal, nasal, upper jaw, squamosal, pterygoid, lower jaw, vomer and quadratojugal. Ossification initiated at early Stage 33 in Hyla orientalis whereas in other hylids it begins after Stage 37. Major transformations of the larval jaw were visible after Stage 42. Cranial ossifications were completed after appendicular and axial ossifications of Hyla orientalis. All skeletal elements ossified before the end of the metamorphosis, a unique feature of Hyla orientalis in comparison to the other hylids studied here.     

A sex-specific marker reveals male heterogamety in European tree frogs

Most amphibians examined so far show undifferentiated sex chromosomes. The heterogametic sex's identity, usually revealed through indirect means, often varies among closely related species or even populations (as do sex-linkage groups), suggesting great evolutionary instability of the sex-determining genes. Here we take advantage of a sex-specific marker that amplifies in several related species of European tree frogs (Hyla arborea group) to disclose a homogeneous pattern of male heterogamety. Besides relevance for evolutionary studies of sex determination in amphibians, our results have potential for addressing practical issues in conservation biology because sex reversal by anthropogenic endocrine disruptors is considered one possible cause of amphibian decline.