Optimizing detectability of the endangered fan mussel using eDNA and ddPCR.

Spatial and temporal monitoring of species threatened with extinction is of critical importance for conservation and ecosystem management. In the Mediterranean coast, the fan mussel (Pinna nobilis) is listed as critically endangered after suffering from a mass mortality event since 2016, leading to 100% mortality in most marine populations. Conventional monitoring for this macroinvertebrate is done using scuba, which is challenging in dense meadows or with low visibility. Here we developed an environmental DNA assay targeting the fan mussel and assessed the influence of several environmental parameters on the species detectability in situ. We developed and tested an eDNA molecular marker and collected 48 water samples in two sites at the Thau lagoon (France) with distinct fan mussel density, depths and during two seasons (summer and autumn). Our marker can amplify fan mussel DNA but lacks specificity since it also amplifies a conspecific species (Pinna rudis). We successfully amplified fan mussel DNA from in situ samples with 46 positive samples (out of 48) using ddPCR, although the DNA concentrations measured were low over almost all samples. Deeper sampling depth slightly increased DNA concentrations, but no seasonal effect was found. We highlight a putative spawning event on a single summer day with much higher DNA concentration compared to all other samples. We present an eDNA molecular assay able to detect the endangered fan mussel and provide guidelines to optimize the sampling protocol to maximize detectability. Effective and non-invasive monitoring tools for endangered species are promising to monitor remaining populations and have the potential of ecological restoration or habitat recolonization following a mass mortality event.

Diversification and evolutionary history of the African laminated-toothed rats (Rodentia, Otomyini).

The African continent was subjected to periodic climatic shifts during the Pliocene and Pleistocene. These habitat changes greatly affected the evolutionary processes and tempo of diversification in numerous, widely distributed mammals. The Otomyini (Family Muridae) comprises three African rodent genera, Parotomys, Otomys and Myotomys, characterized by unique laminated-shaped molars. Species within this tribe generally prefer open-habitat and show low dispersal capabilities, with previous studies suggesting that their diversification was closely associated with climatic oscillations over the last four million years. Our phylogenetic reconstructions, based on three mitochondrial (mtDNA) genes (Cytb, COI and 12S) and four nuclear introns (EF, SPTBN, MGF and THY), identified eight major genetic clades that are distributed across southern, eastern and western Africa. Our data permit the re-examination of the taxonomic status of the three genera as well as the previously proposed mesic-arid dichotomy of the 10 South African species. Moreover, multiple mtDNA species delimitation methods incorporating 168 specimens estimated the number of Otomyini species to be substantially higher than the âˆ¼ 30 recognized, suggesting that the current taxonomy will necessitate an integrative approach to delimit extant species diversity within the Otomyini. The data suggests that the origin of the tribe can be dated back to âˆ¼ 5.7 million years ago (Ma) in southern Africa. The distribution and phylogenetic associations among the eight major otomyine evolutionary lineages can best be explained by several waves of northward colonization from southern Africa, complemented by independent reversed dispersals from eastern back to southern Africa at different time periods. There is strong support for the hypothesis that the radiation, dispersion, and diversification of the otomyine rodents is closely linked to recent Plio-Pleistocene climatic oscillations.

Environmental DNA metabarcoding reveals and unpacks a biodiversity conservation paradox in Mediterranean marine reserves.

Although we are currently experiencing worldwide biodiversity loss, local species richness does not always decline under anthropogenic pressure. This conservation paradox may also apply in protected areas but has not yet received conclusive evidence in marine ecosystems. Here, we survey fish assemblages in six Mediterranean no-take reserves and their adjacent fishing grounds using environmental DNA (eDNA) while controlling for environmental conditions. We detect less fish species in marine reserves than in nearby fished areas. The paradoxical gradient in species richness is accompanied by a marked change in fish species composition under different managements. This dissimilarity is mainly driven by species that are often overlooked by classical visual surveys but detected with eDNA: cryptobenthic, pelagic, and rare fishes. These results do not negate the importance of reserves in protecting biodiversity but shed new light on how under-represented species groups can positively react to fishing pressure and how conservation efforts can shape regional biodiversity patterns.

New genomic resources for three exploited Mediterranean fishes.

Extensive fishing has led to fish stock declines throughout the last decades. While clear stock identification is required for designing management schemes, stock delineation is problematic due to generally low levels of genetic structure in marine species. The development of genomic resources can help to solve this issue. Here, we present the first mitochondrial and nuclear draft genome assemblies of three economically important Mediterranean fishes, the white seabream, the striped red mullet, and the comber. The assemblies are between 613 and 785 Mbp long and contain between 27,222 and 32,375 predicted genes. They were used as references to map Restriction-site Associated DNA markers, which were developed with a single-digest approach. This approach provided between 15,710 and 21,101 Single Nucleotide Polymorphism markers per species. These genomic resources will allow uncovering subtle genetic structure, identifying stocks, assigning catches to populations and assessing connectivity. Furthermore, the annotated genomes will help to characterize adaptive divergence.

eDNA Increases the Detectability of Ranavirus Infection in an Alpine Amphibian Population.

The early detection and identification of pathogenic microorganisms is essential in order to deploy appropriate mitigation measures. Viruses in the Iridoviridae family, such as those in the Ranavirus genus, can infect amphibian species without resulting in mortality or clinical signs, and they can also infect other hosts than amphibian species. Diagnostic techniques allowing the detection of the pathogen outside the period of host die-off would thus be of particular use. In this study, we tested a method using environmental DNA (eDNA) on a population of common frogs (Rana temporaria) known to be affected by a Ranavirus in the southern Alps in France. In six sampling sessions between June and September (the species' activity period), we collected tissue samples from dead and live frogs (adults and tadpoles), as well as insects (aquatic and terrestrial), sediment, and water. At the beginning of the breeding season in June, one adult was found dead; at the end of July, a mass mortality of tadpoles was observed. The viral DNA was detected in both adults and tadpoles (dead or alive) and in water samples, but it was not detected in insects or sediment. In live frog specimens, the virus was detected from June to September and in water samples from August to September. Dead tadpoles that tested positive for Ranavirus were observed only on one date (at the end of July). Our results indicate that eDNA can be an effective alternative to tissue/specimen sampling and can detect Ranavirus presence outside die-offs. Another advantage is that the collection of water samples can be performed by most field technicians. This study confirms that the use of eDNA can increase the performance and accuracy of wildlife health status monitoring and thus contribute to more effective surveillance programs.

Comparative phylogeography of amphibians and reptiles in Algeria suggests common causes for the east-west phylogeographic breaks in the Maghreb.

A series of phylogeographic studies in the Maghreb identified a repeated pattern of deep genetic divergence between an eastern (Tunisia) and western (Morocco) lineage for several taxa but lack of sampling in Algeria made it difficult to know if the range limits between the eastern and western lineages were shared among taxa or not. To address this question, we designed a comparative phylogeographic study using 8 reptile and 3 amphibian species with wide distribution in the Maghreb as models. We selected species where previous studies had identified an East-West phylogeographic divide and collected sampled in Algeria to 1) examine whether the simple East-West divergence pattern still holds after filling the sampling gap in Algeria or if more complex diversity patterns emerge; 2) if the E-W pattern still holds, test whether the limits between the E and W clades are shared between species, suggesting that common historical process caused the E-W divergences; 3) if E-W limits are shared between species, use information on the age of the divergence to identify possible geological or climatic events that could have triggered these E-W differentiations. We found that the E-W pattern was generally maintained after additional sampling in Algeria and identified two common disjunction areas, one around the Algeria-Morocco border, the other one in Kabylia (central Algeria), suggesting that common historical mechanisms caused the E-W divergences in the Maghreb. Our estimates for the times to most common recent ancestors to the E and W clades span a wide range between the Messinian salinity crisis and the Plio-Pleistocene limit (except for one older split), suggesting different origins for the initial divergences and subsequent preservation of the E and W lineages in common climatic refugia in the west and the east of the Maghreb.

Conservation Below the Species Level: Suitable Evolutionarily Significant Units among Mountain Vipers (the Montivipera raddei complex) in Iran.

Northern and western mountains of Iran are among the most important biodiversity and endemism hot spots for reptiles in the Middle East. Among herpetofauna, the montivipers represent an emblematic and fragmented endemic group for which estimating their level of genetic differentiation and defining conservation priorities is urgently needed. Here, we present the most comprehensive phylogenetic study on the Montivipera raddei species group comprising all 5 known taxa, among which 3 are endemic to Iran. Based on 2 mitochondrial genes, phylogenetic and phylogeographic analyses revealed 3 major lineages each presenting very contrasting distribution areas. The Iranian montivipers are highly structured in clades showing low genetic diversity and corresponding to high altitude summits. Molecular dating revealed the role of Quaternary paleo-climatic oscillations and altitudinal movements of montivipers in shaping genetic diversity and differentiation of these sky-island taxa. In addition, the best scenario of historical biogeography allowed identifying 3 possible refugial areas in Iran most likely arising by vicariance. Based on our mitochondrial results and pending additional data, we recognize 3 candidate species among the M. raddei complex: M. raddei, Montivipera latifii, and Montivipera kuhrangica that are coherent with their geographical distribution. We propose that the most appropriate evolutionary significant units for conservation of the montivipers are represented by 13 units among which 6 are recognized as high priority. Finally, we suggest some recommendations to the IUCN as well as to the Iranian conservation policies with respect to conservation prioritization.

Being cosmopolitan: evolutionary history and phylogeography of a specialized raptor, the Osprey Pandion haliaetus.

BACKGROUND: The Osprey (Pandion haliaetus) is one of only six bird species with an almost world-wide distribution. We aimed at clarifying its phylogeographic structure and elucidating its taxonomic status (as it is currently separated into four subspecies). We tested six biogeographical scenarios to explain how the species' distribution and differentiation took place in the past and how such a specialized raptor was able to colonize most of the globe. RESULTS: Using two mitochondrial genes (cyt b and ND2), the Osprey appeared structured into four genetic groups representing quasi non-overlapping geographical regions. The group Indo-Australasia corresponds to the cristatus ssp, as well as the group Europe-Africa to the haliaetus ssp. In the Americas, we found a single lineage for both carolinensis and ridgwayi ssp, whereas in north-east Asia (Siberia and Japan), we discovered a fourth new lineage. The four lineages are well differentiated, contrasting with the low genetic variability observed within each clade. Historical demographic reconstructions suggested that three of the four lineages experienced stable trends or slight demographic increases. Molecular dating estimates the initial split between lineages at about 1.16 Ma ago, in the Early Pleistocene. CONCLUSIONS: Our biogeographical inference suggests a pattern of colonization from the American continent towards the Old World. Populations of the Palearctic would represent the last outcomes of this colonization. At a global scale the Osprey complex may be composed of four different Evolutionary Significant Units, which should be treated as specific management units. Our study brought essential genetic clarifications, which have implications for conservation strategies in identifying distinct lineages across which birds should not be artificially moved through exchange/reintroduction schemes.

Congruent signals of population history but radically different patterns of genetic diversity between mitochondrial and nuclear markers in a mountain lizard.

Historical factors, current population size, population connectivity and selective processes at linked loci contribute to shaping contemporary patterns of neutral genetic diversity. It is now widely acknowledged that nuclear and mitochondrial markers react differently to current demography as well as to past history, so the use of both types of markers is often advocated to gain insight on both historical and contemporary processes. We used 12 microsatellite loci genotyped in 13 populations of a mountain lizard (Iberolacerta bonnali) to test whether the historical scenario favoured by a previous mitochondrial study was also supported by nuclear markers and thereby evaluated the consequences of postglacial range movements on nuclear diversity. Congruent signals of recent history were revealed by nuclear and mitochondrial markers using an Approximate Bayesian computation approach, but contemporary patterns of mtDNA and nuclear DNA diversity were radically different. Although dispersal in this species is probably highly restricted at all spatial scales, colonization abilities have been historically good, suggesting capability for reestablishment of locally extinct populations except in fully disconnected habitats.

High genetic differentiation among French populations of the Orsini's viper (Vipera ursinii ursinii) based on mitochondrial and microsatellite data: implications for conservation management.

The Orsini's viper (Vipera ursinii) is one of the most threatened snakes in Europe due to its highly fragmented distribution and specific open environment (steppic habitat) requirement. French populations are isolated on top of mountain massifs of the southern Prealps/Alps. Mitochondrial sequences (cytochrome b) and 6 microsatellite loci have been used to estimate the levels of genetic diversity and isolation within and among 11 French fragmented populations (a total of 157 individuals). Eleven cytochrome b haplotypes with a limited divergence were observed (mean divergence between haplotypes: 0.31%). However, we detected considerable genetic differentiation among populations (global F(ST) = 0.76 and 0.26 for mitochondrial and nuclear DNA, respectively). Results indicate that 3 populations possibly went through a bottleneck and 1 population showed low genetic diversity compared with the others. Although a significant isolation by distance was detected for both markers, strong differentiation was also observed between geographically close populations, probably due to the ragged landscape that constitutes a serious barrier to gene flow owing to the limited dispersal capability of the viper. Despite some discrepancies between the 2 markers, 8 Management Units have been identified and should be considered for future management projects.

Suprafamilial relationships among Rodentia and the phylogenetic effect of removing fast-evolving nucleotides in mitochondrial, exon and intron fragments.

BACKGROUND: The number of rodent clades identified above the family level is contentious, and to date, no consensus has been reached on the basal evolutionary relationships among all rodent families. Rodent suprafamilial phylogenetic relationships are investigated in the present study using approximately 7600 nucleotide characters derived from two mitochondrial genes (Cytochrome b and 12S rRNA), two nuclear exons (IRBP and vWF) and four nuclear introns (MGF, PRKC, SPTBN, THY). Because increasing the number of nucleotides does not necessarily increase phylogenetic signal (especially if the data is saturated), we assess the potential impact of saturation for each dataset by removing the fastest-evolving positions that have been recognized as sources of inconsistencies in phylogenetics. RESULTS: Taxonomic sampling included multiple representatives of all five rodent suborders described. Fast-evolving positions for each dataset were identified individually using a discrete gamma rate category and sites belonging to the most rapidly evolving eighth gamma category were removed. Phylogenetic tree reconstructions were performed on individual and combined datasets using Parsimony, Bayesian, and partitioned Maximum Likelihood criteria. Removal of fast-evolving positions enhanced the phylogenetic signal to noise ratio but the improvement in resolution was not consistent across different data types. The results suggested that elimination of fastest sites only improved the support for nodes moderately affected by homoplasy (the deepest nodes for introns and more recent nodes for exons and mitochondrial genes). CONCLUSION: The present study based on eight DNA fragments supports a fully resolved higher level rodent phylogeny with moderate to significant nodal support. Two inter-suprafamilial associations emerged. The first comprised a monophyletic assemblage containing the Anomaluromorpha (Anomaluridae + Pedetidae) + Myomorpha (Muridae + Dipodidae) as sister clade to the Castorimorpha (Castoridae + Geomyoidea). The second suprafamilial clustering identified a novel association between the Sciuromorpha (Gliridae + (Sciuridae + Aplodontidae)) and the Hystricomorpha (Ctenodactylidae + Hystricognathi) which together represents the earliest dichotomy among Rodentia. Molecular time estimates using a relaxed Bayesian molecular clock dates the appearance of the five suborders nearly contemporaniously at the KT boundary and this is congruent with suggestions of an early explosion of rodent diversity. Based on these newly proposed phylogenetic relationships, the evolution of the zygomasseteric pattern that has been used for a long time in rodent systematics is evaluated.