Ines Van Bocxlaer

Late Cretaceous Vicariance in Gondwanan Amphibians

Overseas dispersals are often invoked when Southern Hemisphere terrestrial and freshwater organism phylogenies do not fit the sequence or timing of Gondwana fragmentation. We used dispersal-vicariance analyses and molecular timetrees to show that two species-rich frog groups, Microhylidae and Natatanura, display congruent patterns of spatial and temporal diversification among Gondwanan plates in the Late Cretaceous, long after the presumed major tectonic break-up events. Because amphibians are notoriously salt-intolerant, these analogies are best explained by simultaneous vicariance, rather than by oceanic dispersal. Hence our results imply Late Cretaceous connections between most adjacent Gondwanan landmasses, an essential concept for biogeographic and palaeomap reconstructions.

Discovery of a new family of amphibians from northeast India with ancient links to Africa

The limbless, primarily soil-dwelling and tropical caecilian amphibians (Gymnophiona) comprise the least known order of tetrapods. On the basis of unprecedented extensive fieldwork, we report the discovery of a previously overlooked, ancient lineage and radiation of caecilians from threatened habitats in the underexplored states of northeast India. Molecular phylogenetic analyses of mitogenomic and nuclear DNA sequences, and comparative cranial anatomy indicate an unexpected sister-group relationship with the exclusively African family Herpelidae. Relaxed molecular clock analyses indicate that these lineages diverged in the Early Cretaceous, about 140 Ma. The discovery adds a major branch to the amphibian tree of life and sheds light on both the evolution and biogeography of caecilians and the biotic history of northeast India—an area generally interpreted as a gateway between biodiversity hotspots rather than a distinct biogeographic unit with its own ancient endemics. Because of its distinctive morphology, inferred age and phylogenetic relationships, we recognize the newly discovered caecilian radiation as a new family of modern amphibians.

Low genetic diversity in tepui summit vertebrates

Summary The Pantepui region of South America, located in southern Venezuela, northern Brazil, and western Guyana, is characterized by table mountains (tepuis) made of Proterozoic (> 1.5 billion years old) sandstone — the highest reaching nearly 3 km — that are isolated from their surroundings by up to 1000 m high vertical cliffs (Figure 1A). Tepuis are among the most inaccessible places on earth (Supplemental information), and the majority of their summits have been visited less than the moon. Due to its age and topography [1,2], this region has been assumed to be an ideal nursery of speciation and a potential inland counterpart to oceanic islands [3,4]. High endemism has been reported for the flora (25% in vascular plants) and fauna (68.5% in amphibians and reptiles) of single tepuis [5,6], and an ancient origin has been postulated for some of these organisms. But, it has also been suggested that a few taxa living in habitats extending from lowlands to summits (e.g., savannah) invaded some of the more accessible tepuis only recently [6–8]. Taken at face value, the overall timing and extent of biotic interchange between tepui summits has remained unstudied. Here, we show that recent faunal interchange among currently isolated tepui summits has been extensive, and affected even taxa living in some of the most tepui-specific habitats and on the most inaccessible summits.

Love Is Blind: Indiscriminate Female Mating Responses to Male Courtship Pheromones in Newts (Salamandridae)

Internal fertilization without copulation or prolonged physical contact is a rare reproductive mode among vertebrates. In many newts (Salamandridae), the male deposits a spermatophore on the substrate in the water, which the female subsequently takes up with her cloaca. Because such an insemination requires intense coordination of both sexes, male newts have evolved a courtship display, essentially consisting of sending pheromones under water by tail-fanning towards their potential partner. Behavioral experiments until now mostly focused on an attractant function, i.e. showing that olfactory cues are able to bring both sexes together. However, since males start their display only after an initial contact phase, courtship pheromones are expected to have an alternative function. Here we developed a series of intraspecific and interspecific two-female experiments with alpine newt (Ichthyosaura alpestris) and palmate newt (Lissotriton helveticus) females, comparing behavior in male courtship water and control water. We show that male olfactory cues emitted during tail-fanning are pheromones that can induce all typical features of natural female mating behavior. Interestingly, females exposed to male pheromones of their own species show indiscriminate mating responses to conspecific and heterospecific females, indicating that visual cues are subordinate to olfactory cues during courtship.

Origin and Diversification of a Salamander Sex Pheromone System

Sex pheromones form an important facet of reproductive strategies in many organisms throughout the Animal Kingdom. One of the oldest known sex pheromones in vertebrates are proteins of the Sodefrin Precursor-like Factor (SPF) system, which already had a courtship function in early salamanders. The subsequent evolution of salamanders is characterized by a diversification in courtship and reproduction, but little is known on how the SPF pheromone system diversified in relation to changing courtship strategies. Here, we combined transcriptomic, genomic, and phylogenetic analyses to investigate the evolution of the SPF pheromone system in nine salamandrid species with distinct courtship displays. First, we show that SPF originated from vertebrate three-finger proteins and diversified through multiple gene duplications in salamanders, while remaining a single copy in frogs. Next, we demonstrate that tail-fanning newts have retained a high phylogenetic diversity of SPFs, whereas loss of tail-fanning has been associated with a reduced importance or loss of SPF expression in the cloacal region. Finally, we show that the attractant decapeptide sodefrin is cleaved from larger SPF precursors that originated by a 62 bp insertion and consequent frameshift in an ancestral Cynops lineage. This led to the birth of a new decapeptide that rapidly evolved a pheromone function independently from uncleaved proteins.

Mountain-associated clade endemism in an ancient frog family (Nyctibatrachidae) on the Indian subcontinent

Night frogs (Nyctibatrachidae) form a family endemic to the Western Ghats, a hill chain along the west coast of southern India. Extant members of this family are descendants of a lineage that originated on the subcontinent during its longtime isolation in the Late Cretaceous. Because the evolutionary history of Nyctibatrachidae has always been tightly connected to the subcontinent, these tropically-adapted frogs are an ideal group for studying how patterns of endemism originated and evolved during the Cenozoic in the Western Ghats. We used a combined set of mitochondrial and nuclear DNA fragments to investigate the phylogenetic relationships of 120 ingroup specimens of all known species of Nyctibatrachidae. Our analyses indicate that, although this family had an early origin on the Indian subcontinent, the early diversification of extant nyctibatrachids happened only in the Eocene. Biogeographic analyses show that dispersal across the Palghat gap and Shencottah gap was limited, which led to clade endemism within mountain ranges of the Western Ghats. It is likely that multiple biota have been affected simultaneously by these prominent geographical barriers. Our study therefore further highlights the importance of considering the Western Ghats-Sri Lanka biodiversity hotspot as an assemblage of distinct mountain regions, each containing endemism and deserving attention in future conservation planning.

Frog nuptial pads secrete mating season-specific proteins related to salamander pheromones

SUMMARY Males of many frog species develop spiny nuptial pads with underlying glands on their thumbs during the mating period. We used 3D visualization on the European common frog Rana temporaria to show that the morphology of these glands allows the channelling of secreted molecules to the pads surface during amplexus. Combined transcriptome and proteome analyses show that proteins of the Ly-6/uPAR family, here termed amplexins, are highly expressed in the nuptial glands during the mating season, but are totally absent outside that period. The function of amplexins remains unknown, but it is interesting to note that they share structural similarities with plethodontid modulating factors, proteins that influence courtship duration in salamanders.

Side-by-side secretion of Late Palaeozoic diverged courtship pheromones in an aquatic salamander

Males of the advanced salamanders (Salamandroidea) attain internal fertilization without a copulatory organ by depositing a spermatophore on the substrate in the environment, which females subsequently take up with their cloaca. The aquatically reproducing modern Eurasian newts (Salamandridae) have taken this to extremes, because most species do not display close physical contact during courtship, but instead largely rely on females following the male track at spermatophore deposition. Although pheromones have been widely assumed to represent an important aspect of male courtship, molecules able to induce the female following behaviour that is the prelude for successful insemination have not yet been identified. Here, we show that uncleaved sodefrin precursor-like factor (SPF) protein pheromones are sufficient to elicit such behaviour in female palmate newts (Lissotriton helveticus). Combined transcriptomic and proteomic evidence shows that males simultaneously tail-fan multiple ca 20 kDa glycosylated SPF proteins during courtship. Notably, molecular dating estimates show that the diversification of these proteins already started in the late Palaeozoic, about 300 million years ago. Our study thus not only extends the use of uncleaved SPF proteins outside terrestrially reproducing plethodontid salamanders, but also reveals one of the oldest vertebrate pheromone systems.

Recurrent functional divergence of early tetrapod keratins in amphibian toe pads and mammalian hair

Amphibians have invaded arboreal habitats multiple times independently during their evolution. Adaptation to these habitats was nearly always accompanied by the presence or appearance of toe pads, flattened enlargements on tips of fingers and toes that provide adhesive power in these environments. The strength and elasticity of the toe pad relies on polygonal arrayed cells ending in nanoscale projections, which are densely packed with cytoskeletal proteins. Here, we characterized and determined the evolutionary origin of these proteins in the toe pad of the tree frog Hyla cinerea. We created a subtracted cDNA library enriching genes that are expressed in the toe pad, but nowhere else in the toe. Our analyses revealed five alpha keratins as main structural proteins of the amphibian toe pad. Phylogenetic analyses show that these proteins belong to different keratin lineages that originated in an early tetrapod ancestor and in mammals evolved to become the major keratin types of hair. The ancestral keratins were probably already expressed in areas that required skin reinforcement in early tetrapods, and subsequently diverged to support fundamentally different adaptive structures in amphibians and mammals.

Frankixalus, a New Rhacophorid Genus of Tree Hole Breeding Frogs with Oophagous Tadpoles.

Despite renewed interest in the biogeography and evolutionary history of Old World tree frogs (Rhacophoridae), this family still includes enigmatic frogs with ambiguous phylogenetic placement. During fieldwork in four northeastern states of India, we discovered several populations of tree hole breeding frogs with oophagous tadpoles. We used molecular data, consisting of two nuclear and three mitochondrial gene fragments for all known rhacophorid genera, to investigate the phylogenetic position of these new frogs. Our analyses identify a previously overlooked, yet distinct evolutionary lineage of frogs that warrants recognition as a new genus and is here described as Frankixalus gen. nov. This genus, which contains the enigmatic ‘Polypedates’ jerdonii described by Günther in 1876, forms the sister group of a clade containing Kurixalus, Pseudophilautus, Raorchestes, Mercurana and Beddomixalus. The distinctiveness of this evolutionary lineage is also corroborated by the external morphology of adults and tadpoles, adult osteology, breeding ecology, and life history features.