Christelle Tougard

Biogeography and migration routes of large mammal faunas in South–East Asia during the Late Middle Pleistocene: focus on the fossil and extant faunas from Thailand

Abstract Thailand has long held a key position in South–East Asia because of its location at the boundary of the Indochinese and Sundaic provinces, the major biogeographical regions of South–East Asia. These provinces are distinct climatically, floristically and faunistically. The present-day limit between them is located at the Kra Isthmus, in peninsular Thailand. Previous studies of the Javanese large mammal fossil faunas and the recent study of fossil large mammal faunas from Thailand strengthen the hypothesis of a “continental” migration route (in contrast with the “insular” hypothesis via Taiwan and the Philippines) during the Late Middle/Late Pleistocene period. Thailand was even part of this migration route. During the glacial periods, the faunal exchanges were favored by the emersion of a huge continental shelf called Sundaland (South–East Asian continental area connected to Borneo and Indonesia islands by land bridges), when the sea level was low. No geological, biogeographical or paleobiogeographical evidence supports the hypothesis of a migration route via Taiwan and the Philippines. Analysis of the extant and Late Middle Pleistocene large mammal faunas (Carnivora, Primates, Proboscidea and Ungulata) points out the antiquity of the Indochinese and Sundaic provinces. This idea is also supported by the fact that mainland faunas already displayed modern character, whereas the Javanese faunas were mainly composed of endemic forms. However, the occurrence of the extinct species, Elephas namadicus found in a Malaysian site (Tambun) indicates that the limit between the two provinces, if not close to the present place, was located more southward than today, possibly in peninsular Malaysia. It is also confirmed by the presence of northern species, such as Ailuropoda melanoleuca baconi and Crocuta crocuta ultima , in Late Middle Pleistocene sites of Thailand. In fact, because of climatic cooling that occurred in the northern hemisphere during the Pleistocene, the northern faunas had to move southward. To our knowledge, the hypothesis of the antiquity of the South–East Asian provinces with a boundary south of the present-day one is mentioned here for the first time. Previous biogeographical studies were based mainly on insular data from the Philippine Archipelago (geological and mainly present-day zoogeographical data) but also from Indonesia and Borneo (paleontological data). However, the recent discovery of Thai large mammal faunas from Late Middle Pleistocene allows to bridge the gap in the fossil record between the northern faunas of South–East Asia and the southern ones. These Thai faunas are characterized mainly by extant forms but some are today absent from the Thai territory ( Ailuropoda melanoleuca , Crocuta crocuta , Pongo pygmaeus , Sus cf. barbatus , Rhinoceros cf. unicornis , Cervus eldii ).

Abnormal fossil upper molar of Pongo from Thailand : Quaternary climatic changes in southeast Asia as a possible cause

We interpret an unusual left M3attributed to Pongo from the Late Middle Pleistocene of Thailand to be an abnormal tooth rather than a supernumerary molar. Its peculiar morphology cannot be related to classical known causes that affect the dental germ: Gemination, schizodontia, synodontia. The ontogenic mechanism that might have led to the morphology of the tooth perhaps reflects stress related to a phase of marked climatic changes during the Late Middle Pleistocene in Southeast Asia.

A from-benchtop-to-desktop workflow for validating HTS data and for taxonomic identification in diet metabarcoding studies

The main objective of this work was to develop and validate a robust and reliable “from‐benchtop‐to‐desktop” metabarcoding workflow to investigate the diet of invertebrate‐eaters. We applied our workflow to faecal DNA samples of an invertebrate‐eating fish species. A fragment of the cytochrome c oxidase I (COI) gene was amplified by combining two minibarcoding primer sets to maximize the taxonomic coverage. Amplicons were sequenced by an Illumina MiSeq platform. We developed a filtering approach based on a series of nonarbitrary thresholds established from control samples and from molecular replicates to address the elimination of cross‐contamination, PCR/sequencing errors and mistagging artefacts. This resulted in a conservative and informative metabarcoding data set. We developed a taxonomic assignment procedure that combines different approaches and that allowed the identification of ~75% of invertebrate COI variants to the species level. Moreover, based on the diversity of the variants, we introduced a semiquantitative statistic in our diet study, the minimum number of individuals, which is based on the number of distinct variants in each sample. The metabarcoding approach described in this article may guide future diet studies that aim to produce robust data sets associated with a fine and accurate identification of prey items.

Phylogeography of the heavily poached African common pangolin (Pholidota, Manis tricuspis) reveals six cryptic lineages as traceable signatures of Pleistocene diversification

Knowledge on faunal diversification in African rainforests remains scarce. We used phylogeography to assess (i) the role of Pleistocene climatic oscillations in the diversification of the African common pangolin (Manis tricuspis) and (ii) the utility of our multilocus approach for taxonomic delineation and trade tracing of this heavily poached species. We sequenced 101 individuals for two mitochondrial DNA (mtDNA), two nuclear DNA and one Y‐borne gene fragments (totalizing 2602 bp). We used a time‐calibrated, Bayesian inference phylogenetic framework and conducted character‐based, genetic and phylogenetic delineation of species hypotheses within African common pangolins. We identified six geographic lineages partitioned into western Africa, Ghana, the Dahomey Gap, western central Africa, Gabon and central Africa, all diverging during the Middle to Late Pleistocene. MtDNA (cytochrome b + control region) was the sole locus to provide diagnostic characters for each of the six lineages. Tree‐based Bayesian delimitation methods using single‐ and multilocus approaches gave high support for ‘species’ level recognition of the six African common pangolin lineages. Although the diversification of African common pangolins occurred during Pleistocene cyclical glaciations, causative correlation with traditional rainforest refugia and riverine barriers in Africa was not straightforward. We conclude on the existence of six cryptic lineages within African common pangolins, which might be of major relevance for future conservation strategies. The high discriminative power of the mtDNA markers used in this study should allow an efficient molecular tracing of the regional origin of African common pangolin seizures.

Isolation of ten microsatellite markers using a pyrosequencing procedure and cross-priming in the Salaria genus

Ten new microsatellite markers were isolated from the freshwater blenny, Salaria fluviatilis. These loci were characterised for three distinct populations (Var River and Lake Bourget in France, and Lake Kinneret in Israel). S. fluviatilis populations in the North-West of the Mediterranean Sea are all polymorphic and under Hardy–Weinberg equilibrium. These microsatellite markers are suggested for studying the genetic structure in this area. Seven of the ten microsatellite markers are applicable for all the freshwater blenny populations of the entire Mediterranean basin. Cross-species characterisation was also performed on the peacock blenny, Salaria pavo. Seven of the ten microsatellite loci exhibited successful PCR amplification on this latter species. Polymorphism is observed on four of these seven loci.