Philippe Geniez

Next-generation monitoring of aquatic biodiversity using environmental DNA metabarcoding.

Global biodiversity in freshwater and the oceans is declining at high rates. Reliable tools for assessing and monitoring aquatic biodiversity, especially for rare and secretive species, are important for efficient and timely management. Recent advances in DNA sequencing have provided a new tool for species detection from DNA present in the environment. In this study, we tested whether an environmental DNA (eDNA) metabarcoding approach, using water samples, can be used for addressing significant questions in ecology and conservation. Two key aquatic vertebrate groups were targeted: amphibians and bony fish. The reliability of this method was cautiously validated in silico, in vitro and in situ. When compared with traditional surveys or historical data, eDNA metabarcoding showed a much better detection probability overall. For amphibians, the detection probability with eDNA metabarcoding was 0.97 (CI = 0.90–0.99) vs. 0.58 (CI = 0.50–0.63) for traditional surveys. For fish, in 89% of the studied sites, the number of taxa detected using the eDNA metabarcoding approach was higher or identical to the number detected using traditional methods. We argue that the proposed DNA‐based approach has the potential to become the next‐generation tool for ecological studies and standardized biodiversity monitoring in a wide range of aquatic ecosystems.

Relationships and evolution of the North African geckos, Geckonia and Tarentola (Reptilia: Gekkonidae), based on mitochondrial and nuclear DNA sequences.

Mitochondrial (cytochrome b and 12S rRNA) and nuclear (c-mos) genes, analyzed by a variety of methods, indicate that the distinctive northwest African gecko Geckonia chazaliae is a member of the Tarentola clade, being most closely related to the species of the western Canary and Cape Verde islands. Relationships in Tarentola as a whole are as follows: (T. americana ((T. mauritanica, T. angustimentalis) ((T. deserti, T. boehmei) ((T. b. boettgeri-South (T. b. boettgeri-North (T. b. bischoffi, T. b. hierrensis))) ((T. annularis, T. ephippiata) (Geckonia, T. delalandii, T. gomerensis, Cape Verde species)))))); nearly all nodes have high bootstrap support. Results confirm that T. americana of Cuba and the Bahamas separated at the most basal dichotomy of the phylogeny and give no positive support for the monophyly of the subgenera Tarentola s. str. and Makariogecko. The latter includes Geckonia and the subgenus Sahelogecko. Continental Tarentola appear to have invaded the Sahara desert from its northern edge. They have also colonized groups of Atlantic islands five times: a single invasion of the West Indies and three of the Canary islands, one of which then went on to invade the Cape Verde archipelago. The phylogeny corroborates anatomical evidence that the ground-dwelling Geckonia had a climbing ancestry, something that is paralleled in some southern African terrestrial gekkonids related to Pachydactylus. Distinctive derived features of Geckonia occur in other gekkonids that are ground dwelling in arid habitats and may be functionally related to this environment. The evolution of such features indicates that, although Tarentola is generally very uniform and may have been so for over 10 million years, this is not due to any overwhelming phylogenetic constraint. G. chazaliae should be included in Tarentola, as Tarentola chazaliae.

Conquering the Sahara and Arabian deserts: systematics and biogeography of Stenodactylus geckos (Reptilia: Gekkonidae)

BackgroundThe evolutionary history of the biota of North Africa and Arabia is inextricably tied to the complex geological and climatic evolution that gave rise to the prevalent deserts of these areas. Reptiles constitute an exemplary group in the study of the arid environments with numerous well-adapted members, while recent studies using reptiles as models have unveiled interesting biogeographical and diversification patterns. In this study, we include 207 specimens belonging to all 12 recognized species of the genus Stenodactylus. Molecular phylogenies inferred using two mitochondrial (12S rRNA and 16S rRNA) and two nuclear (c-mos and RAG-2) markers are employed to obtain a robust time-calibrated phylogeny, as the base to investigate the inter- and intraspecific relationships and to elucidate the biogeographical history of Stenodactylus, a genus with a large distribution range including the arid and hyper-arid areas of North Africa and Arabia.ResultsThe phylogenetic analyses of molecular data reveal the existence of three major clades within the genus Stenodactylus, which is supported by previous studies based on morphology. Estimated divergence times between clades and sub-clades are shown to correlate with major geological events of the region, the most important of which is the opening of the Red Sea, while climatic instability in the Miocene is hypothesized to have triggered diversification. High genetic variability is observed in some species, suggesting the existence of some undescribed species. The S. petrii - S. stenurus species complex is in need of a thorough taxonomic revision. New data is presented on the distribution of the sister species S. sthenodactylus and S. mauritanicus.ConclusionsThe phylogenetic hypothesis for the genus Stenodactylus presented in this work permits the reconstruction of the biogeographical history of these common desert dwellers and confirms the importance of the opening of the Red Sea and the climatic oscillations of the Miocene as major factors in the diversification of the biota of North Africa and Arabia. Moreover, this study traces the evolution of this widely distributed and highly specialized group, investigates the patterns of its high intraspecific diversity and elucidates its systematics.

Morphology and nuclear markers reveal extensive mitochondrial introgressions in the Iberian Wall Lizard species complex

Mitochondrial markers are still often used alone to identify evolutionary units, despite widespread evidence for processes such as incomplete lineage sorting or introgressive hybridization that may blur past population history. The combination of mitochondrial DNA data with other sources of information (morphology, nuclear genes) is a powerful tool to reveal when and why mitochondrial markers are potentially misleading. In this study, we evaluate the performance of mtDNA markers to unravel the evolutionary history of Spanish lizards from the Podarcis hispanicus species complex. We first uncover several cases of discordance between morphological and mitochondrial data in delimitation of taxa. To assess the origin of these discordances, we analysed the same populations using several independent nuclear loci. Both morphological and nuclear markers identified the same three evolutionary units in the region, while mitochondrial data revealed four deeply divergent lineages. We suggest here that the most likely scenario to explain this discordance is ancient mitochondrial introgression originating from a fourth evolutionary unit presently absent from the study area. Notably, this resulted in a complete replacement of the original lineage in a large part of the distribution of one of the taxa investigated. We discuss the potential evolutionary scenarios leading to this complete mitochondrial replacement and suggest why the previous studies have failed to recover the correct history of this species complex.

Molecular phylogenetics and historical biogeography of the west-palearctic common toads (Bufo bufo species complex).

In most pan-Eurasiatic species complexes, two phenomena have been traditionally considered key processes of their cladogenesis and biogeography. First, it is hypothesized that the origin and development of the Central Asian Deserts generated a biogeographic barrier that fragmented past continuous distributions in Eastern and Western domains. Second, Pleistocene glaciations have been proposed as the main process driving the regional diversification within each of these domains. The European common toad and its closest relatives provide an interesting opportunity to examine the relative contributions of these paleogeographic and paleoclimatic events to the phylogeny and biogeography of a widespread Eurasiatic group. We investigate this issue by applying a multiproxy approach combining information from molecular phylogenies, a multiple correspondence analysis of allozyme data and species distribution models. Our study includes 304 specimens from 164 populations, covering most of the distributional range of the Bufo bufo species complex in the Western Palearctic. The phylogenies (ML and Bayesian analyses) were based on a total of 1988 bp of mitochondrial DNA encompassing three genes (tRNAval, 16S and ND1). A dataset with 173 species of the family Bufonidae was assembled to estimate the separation of the two pan-Eurasiatic species complexes of Bufo and to date the main biogeographic events within the Bufo bufo species complex. The allozyme study included sixteen protein systems, corresponding to 21 presumptive loci. Finally, the distribution models were based on maximum entropy. Our distribution models show that Eastern and Western species complexes are greatly isolated by the Central Asian Deserts, and our dating estimates place this divergence during the Middle Miocene, a moment in which different sources of evidence document a major upturn of the aridification rate of Central Asia. This climate-driven process likely separated the Eastern and Western species. At the level of the Western Palearctic, our dating estimates place most of the deepest phylogenetic structure before the Pleistocene, indicating that Pleistocene glaciations did not have a major role in splitting the major lineages. At a shallow level, the glacial dynamics contributed unevenly to the genetic structuring of populations, with a strong influence in the European-Caucasian populations, and a more relaxed effect in the Iberian populations.

Phylogeny of North African Agama lizards (Reptilia: Agamidae) and the role of the Sahara desert in vertebrate speciation

The origin of Saharan biodiversity is poorly understood, in part because the geological and paleoclimatic events that presumably shaped species diversity are still controversial, but also because few studies have explored causal explanations for the origin of Saharan diversity using a phylogenetic framework. Here, we use mtDNA (16S and ND4 genes) and nDNA (MC1R and CMOS genes) to infer the relationships and biogeographic history of North African agamas (genus Agama). Agamas are conspicuous, diverse and abundant African lizards that also occur in the Saharan xeric and mesic environments. Our results revealed the presence of three Agama lineages in North Africa: one Afrotropical, one Sahelo-Saharan, and one broadly distributed in North Africa and mainly Saharan. Southern Mauritania contains the highest known diversity, with all three lineages present. Results suggest that agamas colonized the Sahara twice, but only one lineage was able to radiate and diversify there. Species in the Saharan lineage are mostly allopatric, and their splitting, genetic diversity and distribution are greatly explained by mountain ranges. One species in this lineage has colonized the Mediterranean climatic zone (A. impalearis), and another one the Sahel savannah (A. boueti). The other lineage to colonize the Sahara corresponds to A. boulengeri, an eminently Sahelian species that also inhabits Saharan mountain ranges in Mauritania and Mali. Phylogenetic analyses indicate that allopatric montane populations within some currently recognized species are also genetically divergent. Our study therefore concludes that vicariant speciation is a leading motor of species diversification in the area: Inside the Sahara, associated to mountain-ranges isolated by dune seas and bare plains; outside, associated to less harsh climates to the North and South. Paleoclimatic oscillations are suggested as causal explanations of the vicariant distribution and origin of species. Agamas are thought to have colonized northern Africa during wet periods, with subsequent dry periods fragmenting species distribution and leading to allopatric populations associated to milder and wetter climates in the Mediterranean, Sahel, and in Saharan mountains, in an island-model fashion. Finally, our results support the synonymization of A. castroviejoi with A. boueti, the reciprocal monophyly of all other North African agamas, and suggest one candidate species within A. boulengeri.

Radically different phylogeographies and patterns of genetic variation in two European brown frogs, genus Rana

We reconstruct range-wide phylogeographies of two widespread and largely co-occurring Western Palearctic frogs, Rana temporaria and R. dalmatina. Based on tissue or saliva samples of over 1000 individuals, we compare a variety of genetic marker systems, including mitochondrial DNA, single-copy protein-coding nuclear genes, microsatellite loci, and single nucleotide polymorphisms (SNPs) of transcriptomes of both species. The two focal species differ radically in their phylogeographic structure, with R. temporaria being strongly variable among and within populations, and R. dalmatina homogeneous across Europe with a single strongly differentiated population in southern Italy. These differences were observed across the various markers studied, including microsatellites and SNP density, but especially in protein-coding nuclear genes where R. dalmatina had extremely low heterozygosity values across its range, including potential refugial areas. On the contrary, R. temporaria had comparably high range-wide values, including many areas of probable postglacial colonization. A phylogeny of R. temporaria based on various concatenated mtDNA genes revealed that two haplotype clades endemic to Iberia form a paraphyletic group at the base of the cladogram, and all other haplotypes form a monophyletic group, in agreement with an Iberian origin of the species. Demographic analysis suggests that R. temporaria and R. dalmatina have genealogies of roughly the same time to coalescence (TMRCA ~3.5 mya for both species), but R. temporaria might have been characterized by larger ancestral and current effective population sizes than R. dalmatina. The high genetic variation in R. temporaria can therefore be explained by its early range expansion out of Iberia, with subsequent cycles of differentiation in cryptic glacial refugial areas followed by admixture, while the range expansion of R. dalmatina into central Europe is a probably more recent event.

Species on the rocks: Systematics and biogeography of the rock-dwelling Ptyodactylus geckos (Squamata: Phyllodactylidae) in North Africa and Arabia

The understanding of the diversity of species in the Palearctic and the processes that have generated it is still weak for large parts of the arid areas of North Africa and Arabia. Reptiles are among their most remarkable representatives, with numerous groups well adapted to the diverse environments. The Ptyodactylus geckos are a strictly rock-dwelling genus with homogeneous morphology distributed across mountain formations and rocky plateaus from the western African ranges in Mauritania and the Maghreb to the eastern tip of the Arabian Peninsula, with an isolated species in southern Pakistan. Here, we use a broad sampling of 378 specimens, two mitochondrial (12S and cytb) and four nuclear (c-mos, MC1R, ACM4, RAG2) markers in order to obtain the first time-calibrated molecular phylogeny of the genus and place its diversification in a temporal framework. The results reveal high levels of intraspecific variability, indicative of undescribed diversity, and they do not support the monophyly of one species (P. ragazzii). Ptyodactylus species are allopatric across most of their range, which may relate to their high preference for the same type of structural habitat. The onset of their diversification is estimated to have occurred in the Late Oligocene, while that of several deep clades in the phylogeny took place during the Late Miocene, a period when an increase in aridification in North Africa and Arabia initiated.

Area prioritization and performance evaluation of the conservation area network for the Moroccan herpetofauna: a preliminary assessment

The integration of spatial area prioritization algorithms and species distribution modelling has shown great promise in conservation planning in recent years. However, despite the fact that reptiles and amphibians have the highest threat status of all terrestrial vertebrates, these species are often under-represented in conservation planning. The Kingdom of Morocco possesses the richest and most varied herpetofauna in the Maghreb and the western Mediterranean, and is characterized by high species richness, endemism and number of European relict species. Despite the fact that Moroccan reptiles and amphibians have been the subject of numerous studies by a large number of international herpetologists since the beginning of the 20th century, few or none of these concerned their conservation. This study had three main objectives: (1) to identify those areas that harbour the highest species richness; (2) to evaluate the existing and proposed future ‘important biological and ecological sites’ (SIBES) conservation area network (CAN) with respect to their ability to protect the herpetofauna adequately; and (3) to identify priority areas into which the existing protected areas can be augmented. We used maximum-entropy species distribution modelling to run distribution models for 11 amphibian and 86 reptile species (27.6% endemics and 12.4% threatened) for which we had 2,170 single geographic records. A total of 97 models were used to create a richness map of the Moroccan herpetofauna and thereby detect both areas of high species richness and the distribution patterns of individual species. This map was subsequently used as a basis for performance evaluation of the CAN and area prioritization using the ConsNet conservation planning software initialized by “Rarity” first, while using representation targets of 5% and 10%. Additionally, the proposed future Moroccan CAN (SIBES) was evaluated in terms of its overlay and proximity with ConsNet solutions using visual interpretation and distance measurements in a GIS. Our results show that Moroccan herpetofauna is poorly protected under the existing and future CAN. Prioritization of areas shows that a major increase in conservation area is required to guarantee the persistence of individual herpetofauna species even with a global minimum representation target of only 10%. An increase of the existing CAN is especially needed along parts of the Atlantic coast, in the north-western Mediterranean region, on the north-eastern Moroccan coast, as well as in several areas in the Sahara, notably vast proportions of the Vallée du Haut and Bas Drâa.

Evolution around the Red Sea: Systematics and biogeography of the agamid genus Pseudotrapelus (Squamata: Agamidae) from North Africa and Arabia.

Since the Oligocene, regions adjacent to the Red Sea have experienced major environmental changes, from tectonic movements and continuous geological activity to shifting climatic conditions. The effect of these events on the distribution and diversity of the regional biota is still poorly understood. Agamid members of the genus Pseudotrapelus are diurnal, arid-adapted lizards distributed around the Red Sea from north-eastern Africa, across the mountains and rocky plateaus of the Sinai and Arabian Peninsulas northwards to Syria. Despite recent taxonomic work and the interest in the group as a model for studying biogeographic and diversity patterns of the arid areas of North Africa and Arabia, its taxonomy is poorly understood and a comprehensive phylogeny is still lacking. In this study, we analyzed 92 Pseudotrapelus specimens from across the entire distribution range of the genus. We included all known species and subspecies, and sequenced them for mitochondrial (16S, ND4 and tRNAs) and nuclear (MC1R, c-mos) markers. This enabled us to obtain the first time-calibrated molecular phylogeny of the genus, using gene trees, species trees and coalescent-based methods for species delimitation. Our results revealed Pseudotrapelus as a monophyletic genus comprised of two major clades and six independently evolving lineages. These lineages correspond to the five currently recognized species and a sixth lineage relating to the synonymized P. neumanni. The subspecific validity of P. sinaitus werneri needs further assessment as it does not form a distinct cluster relative to P. s. sinaitus. The onset of Pseudotrapelus diversification is estimated to have occurred in Arabia during the late Miocene. Radiation has likely resulted from vicariance and dispersal events due to the continued geological instability, sea level fluctuations and climatic changes within the region.