Amaël Borzée

Cryptic diversity among Western Palearctic tree frogs: Postglacial range expansion, range limits, and secondary contacts of three European tree frog lineages (Hyla arborea group)

We characterize divergence times, intraspecific diversity and distributions for recently recognized lineages within the Hyla arborea species group, based on mitochondrial and nuclear sequences from 160 localities spanning its whole distribution. Lineages of H. arborea, H. orientalis, H. molleri have at least Pliocene age, supporting species level divergence. The genetically uniform Iberian H. molleri, although largely isolated by the Pyrenees, is parapatric to H. arborea, with evidence for successful hybridization in a small Aquitanian corridor (southwestern France), where the distribution also overlaps with H. meridionalis. The genetically uniform H. arborea, spread from Crete to Brittany, exhibits molecular signatures of a postglacial range expansion. It meets different mtDNA clades of H. orientalis in NE-Greece, along the Carpathians, and in Poland along the Vistula River (there including hybridization). The East-European H. orientalis is strongly structured genetically. Five geographic mitochondrial clades are recognized, with a molecular signature of postglacial range expansions for the clade that reached the most northern latitudes. Hybridization with H. savignyi is suggested in southwestern Turkey. Thus, cryptic diversity in these Pliocene Hyla lineages covers three extremes: a genetically poor, quasi-Iberian endemic (H. molleri), a more uniform species distributed from the Balkans to Western Europe (H. arborea), and a well-structured Asia Minor-Eastern European species (H. orientalis).

Sex-Chromosome Homomorphy in Palearctic Tree Frogs Results from Both Turnovers and X–Y Recombination

Contrasting with birds and mammals, poikilothermic vertebrates often have homomorphic sex chromosomes, possibly resulting from high rates of sex-chromosome turnovers and/or occasional X-Y recombination. Strong support for the latter mechanism was provided by four species of European tree frogs, which inherited from a common ancestor (∼ 5 Ma) the same pair of homomorphic sex chromosomes (linkage group 1, LG1), harboring the candidate sex-determining gene Dmrt1. Here, we test sex linkage of LG1 across six additional species of the Eurasian Hyla radiation with divergence times ranging from 6 to 40 Ma. LG1 turns out to be sex linked in six of nine resolved cases. Mapping the patterns of sex linkage to the Hyla phylogeny reveals several transitions in sex-determination systems within the last 10 My, including one switch in heterogamety. Phylogenetic trees of DNA sequences along LG1 are consistent with occasional X-Y recombination in all species where LG1 is sex linked. These patterns argue against one of the main potential causes for turnovers, namely the accumulation of deleterious mutations on nonrecombining chromosomes. Sibship analyses show that LG1 recombination is strongly reduced in males from most species investigated, including some in which it is autosomal. Intrinsically low male recombination might facilitate the evolution of male heterogamety, and the presence of important genes from the sex-determination cascade might predispose LG1 to become a sex chromosome.

Empirical evidence for large X-effects in animals with undifferentiated sex chromosomes.

Reproductive isolation is crucial for the process of speciation to progress. Sex chromosomes have been assigned a key role in driving reproductive isolation but empirical evidence from natural population processes has been restricted to organisms with degenerated sex chromosomes such as mammals and birds. Here we report restricted introgression at sex-linked compared to autosomal markers in a hybrid zone between two incipient species of European tree frog, Hyla arborea and H. orientalis, whose homologous X and Y sex chromosomes are undifferentiated. This large X-effect cannot result from the dominance or faster-X aspects of Haldane’s rule, which are specific to degenerated sex chromosomes, but rather supports a role for faster-heterogametic-sex or faster-male evolutionary processes. Our data suggest a prominent contribution of undifferentiated sex chromosomes to speciation.

Temporal and spatial differentiation in microhabitat use: Implications for reproductive isolation and ecological niche specification

Niche differentiation enables ecologically similar species to coexist by lessening competition over food and/or shelters and may be critical for reproductive isolation between closely related species in close proximity. Because no extra traits need to evolve, spatial and temporal differentiation may readily take place to complement other isolating mechanisms. Two closely related treefrog species occur together in Korea: the endangered Hyla suweonensis and the widespread Hyla japonica. Advertisement calls are differentiated, but it is unclear whether call difference is sufficient for reproductive isolation. We tracked individuals of both species to study fine-scale differentiation in microhabitat use in the diel cycle of the breeding season using a harmonic direction finder. tracking male movement patterns of both species revealed spatial and temporal differentiation in microhabitat use for calling and resting during the breeding season. Males of both H. suweonensis and H. japonica occurred in all 5 microhabitats identified in this study: rice paddy, ground, buried, grass and bush. Both treefrog species showed general similarities in calling from rice paddies and resting in grass and bush. However, H. suweonensis moved into rice paddies and produced advertisement calls 3 h earlier than H. japonica. These differences likely minimize contact between the species and provide an additional isolating mechanism. In addition, the activity of H. suweonensis may be contributing to the decline of this species, as resting in grass would increase dangers from predatory birds and habitat disturbance.

Asymmetric competition over calling sites in two closely related treefrog species.

Interspecific competition occurs when one species using a resource limits the use of that resource by another species. A dominance relationship between the species competing over a resource may result in asymmetric competition. Here, we tested the hypothesis that two sympatric treefrog species, the endangered Hyla suweonensis and the abundant H. japonica, compete with each other over calling sites. We observed the locations of calling individuals of the two treefrog species in rice paddies and tested whether removing one species affected the calling locations of the other species. Individuals of the two species were spatially isolated within rice paddies, with H. japonica at the edges and H. suweonensis in the interior. Male H. suweonensis moved towards the edges of rice paddies when male H. japonica were removed from the area, whereas male H. japonica hardly moved when male H. suweonensis were removed. The results of both studies are consistent with asymmetric interspecific competition, in which the calling locations of H. suweonensis are affected by the calling activity of H. japonica. In addition, H. japonica were found “sitting” on the substrate during call production, whereas H. suweonensis were “holding” onto vegetation. The difference in calling posture may represent an adaptive response to asymmetric interspecific competition.

Phylogeography reveals an ancient cryptic radiation in East-Asian tree frogs ( Hyla japonica group) and complex relationships between continental and island lineages

BackgroundIn contrast to the Western Palearctic and Nearctic biogeographic regions, the phylogeography of Eastern-Palearctic terrestrial vertebrates has received relatively little attention. In East Asia, tectonic events, along with Pleistocene climatic conditions, likely affected species distribution and diversity, especially through their impact on sea levels and the consequent opening and closing of land-bridges between Eurasia and the Japanese Archipelago. To better understand these effects, we sequenced mitochondrial and nuclear markers to determine phylogeographic patterns in East-Asian tree frogs, with a particular focus on the widespread H. japonica.ResultsWe document several cryptic lineages within the currently recognized H. japonica populations, including two main clades of Late Miocene divergence (~5 Mya). One occurs on the northeastern Japanese Archipelago (Honshu and Hokkaido) and the Russian Far-East islands (Kunashir and Sakhalin), and the second one inhabits the remaining range, comprising southwestern Japan, the Korean Peninsula, Transiberian China, Russia and Mongolia. Each clade further features strong allopatric Plio-Pleistocene subdivisions (~2-3 Mya), especially among continental and southwestern Japanese tree frog populations. Combined with paleo-climate-based distribution models, the molecular data allowed the identification of Pleistocene glacial refugia and continental routes of postglacial recolonization. Phylogenetic reconstructions further supported genetic homogeneity between the Korean H. suweonensis and Chinese H. immaculata, suggesting the former to be a relic population of the latter that arose when the Yellow Sea formed, at the end of the last glaciation.ConclusionsPatterns of divergence and diversity were likely triggered by Miocene tectonic activities and Quaternary climatic fluctuations (including glaciations), causing the formation and disappearance of land-bridges between the Japanese islands and the continent. Overall, this resulted in a ring-like diversification of H. japonica around the Sea of Japan. Our findings urge for important taxonomic revisions in East-Asian tree frogs. First, they support the synonymy of H. suweonensis (Kuramoto, 1980) and H. immaculata (Boettger, 1888). Second, the nominal H. japonica (Günther, 1859) represents at least two species: an eastern (new taxon A) on the northern Japanese and Russian Far East islands, and a southwestern species (n. t. B) on southern Japanese islands and possibly also forming continental populations. Third, these continental tree frogs may also represent an additional entity, previously described as H. stepheni Boulenger, 1888 (senior synonym of H. ussuriensis Nikolskii, 1918). A complete revision of this group requires further taxonomic and nomenclatural analyses, especially since it remains unclear to which taxon the species-epitheton japonica corresponds to.

Impact of land reclamation and agricultural water regime on the distribution and conservation status of the endangered Dryophytes suweonensis

Knowledge about the distribution and habitat preferences of a species is critical for its conservation. The Suweon Treefrog (Dryophytes suweonensis) is an endangered species endemic to the Republic of Korea. We conducted surveys from 2014 to 2016 at 890 potentially suitable sites across the entire range of the species in South Korea. We then assessed whether D. suweonensis was found in the current and ancestral predicted ranges, reclaimed and protected areas, and how the presence of agricultural floodwater affected its occurrence. Our results describe a 120 km increase in the southernmost known distribution of the species, and the absence of the species at lower latitudes. We then demonstrate a putative constriction on the species ancestral range due to urban encroachment, and provide evidence for a significant increase in its coastal range due to the colonisation of reclaimed land by the species. In addition, we demonstrate that D. suweonensis is present in rice fields that are flooded with water originating from rivers as opposed to being present in rice fields that are irrigated from underground water. Finally, the non-overlap of protected areas and the occurrence of the species shows that only the edge of a single site where D. suweonensis occurs is legally protected. Based on our results and the literature, we suggest the design of a site fitting all the ecological requirements of the species, and suggest the use of such sites to prevent further erosion in the range of D. suweonensis.

Introduced bullfrogs are associated with increased Batrachochytrium dendrobatidis prevalence and reduced occurrence of Korean treefrogs

Bullfrogs, Lithobates catesbeianus, have been described as major vectors of the amphibian chytrid fungus, Batrachochytrium dendrobatidis (Bd). Bd is widespread throughout the range of amphibians yet varies considerably within and among populations in prevalence and host impact. In our study, the presence of L. catesbeianus is correlated with a 2.5 increase in Bd prevalence in treefrogs, and the endangered Dryophytes suweonensis displays a significantly higher Bd prevalence than the more abundant D. japonicus for the 37 sites surveyed. In addition, the occurrence of L. catesbeianus was significantly correlated with a decrease in presence of D. suweonensis at sites. We could not determine if it is the presence of bullfrogs as competitors or predators that is limiting the distribution of D. suweonensis or whether this is caused by bullfrogs acting as a reservoir for Bd. However, L. catesbeianus can now be added to the list of factors responsible for the decline of D. suweonensis populations.

Impact of Water Quality on the Occurrence of Two Endangered Korean Anurans: Dryophytes suweonensis and Pelophylax chosenicus

Abstract Water contaminants play an important role in threatening amphibian species already in the midst of the sixth extinction. Species relying exclusively on agricultural habitats for breeding are under multiple, and sometimes synergistic, threats because of the high levels of contamination from chemicals used as plant fertilizers and pest control. In the Republic of Korea, two anuran species listed as endangered occur primarily in rice paddies: Suweon Treefrogs (Dryophytes suweonensis) and Gold-spotted Pond Frogs (Pelophylax chosenicus). The purpose of this study was to describe the relationship between the occurrence of these two species and water contaminants in rice paddies. Water quality and contaminants were assessed through multisite triplicate sampling. The rice paddies where the two anuran species occurred were characterized by higher concentrations of iron and lower concentrations of phosphate than rice paddies without the two species. Furthermore, low conductivity in the water was correlated to the occurrence of P. chosenicus, and low ammonium nitrate was a reliable predictor for the occurrence of D. suweonensis. These contaminants principally result from the application of chemical treatments on agricultural fields. The decline of these two endangered anuran species might be related to current agricultural practices.

Variations in boldness, behavioural and physiological traits of an endangered and a common hylid species from Korea

Animal populations with narrow distribution ranges due to recent constrictions are predicted to be physiologically and evolutionarily less fit than their counterparts with wider distributions. In anurans, muscle mass and body size are generally correlated with locomotor and physiological performances. These, in turn, may be representatives of species-specific strategies such as predator avoidance, exploration, habitat use, and dispersal. We conducted behavioural experiments and field observations to assess the differences in physiological capacity, exploratory behaviour, and response delay between the endangered Dryophytes suweonensis and the widespread D. japonicus. The complementary genetic analyses revealed a lower genetic diversity in D. suweonensis than in D. japonicus. Despite its smaller size, D. suweonensis displayed a significantly larger mass for locomotor muscles, but a longer delay for responses to stimulus and a lower endurance than D. japonicus. The combined lower cognitive delay for behavioural responses of D. suweonensis and the better endurance of D. japonicus make the later species more fit to its environment, allowing for faster escape and wider dispersal capabilities. Our results therefore highlight the correlation between the endangered D. suweonensis with a narrow ranges and depressed boldness, in comparison to the widespread and closely related D. japonicus. These results are clade specific, but open a potential new line of research into species decline.