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The bushmeat trade in tropical Africa represents illegal, unsustainable off-takes of millions of tons of wild game - mostly mammals - per year. We sequenced four mitochondrial gene fragments (cyt b, COI, 12S, 16S) in >300 bushmeat items representing nine mammalian orders and 59 morphological species from five western and central African countries (Guinea, Ghana, Nigeria, Cameroon and Equatorial Guinea). Our objectives were to assess the efficiency of cross-species PCR amplification and to evaluate the usefulness of our multilocus approach for reliable bushmeat species identification. We provide a straightforward amplification protocol using a single 'universal' primer pair per gene that generally yielded >90% PCR success rates across orders and was robust to different types of meat preprocessing and DNA extraction protocols. For taxonomic identification, we set up a decision pipeline combining similarity- and tree-based approaches with an assessment of taxonomic expertise and coverage of the GENBANK database. Our multilocus approach permitted us to: (i) adjust for existing taxonomic gaps in GENBANK databases, (ii) assign to the species level 67% of the morphological species hypotheses and (iii) successfully identify samples with uncertain taxonomic attribution (preprocessed carcasses and cryptic lineages). High levels of genetic polymorphism across genes and taxa, together with the excellent resolution observed among species-level clusters (neighbour-joining trees and Klee diagrams) advocate the usefulness of our markers for bushmeat DNA typing. We formalize our DNA typing decision pipeline through an expert-curated query database - DNA BUSHMEAT - that shall permit the automated identification of African forest bushmeat items., (© 2014 John Wiley & Sons Ltd.)

Specimens stored in museum collections represent a crucial source of morphological and genetic information, notably for taxonomically problematic groups and extinct taxa. Although fluid-preserved specimens of groups such as teleosts may constitute an almost infinite source of DNA, few ancient DNA protocols have been applied to such material. In this study, we describe a non-invasive Guanidine-based (GuSCN) ancient DNA extraction protocol adapted to fluid-preserved specimens that we use to re-assess the systematics of the genus Orestias (Cyprinodontidae: Teleostei). The latter regroups pupfishes endemic to the inter-Andean basin that have been considered as a 'species flock', and for which the morphology-based taxonomic delimitations have been hotly debated. We extracted DNA from the type specimens of Orestias kept at the Muséum National d'Histoire Naturelle of Paris, France, including the extinct species O. cuvieri. We then built the first molecular (control region [CR] and rhodopsin [RH]) phylogeny including historical and recently collected representatives of all the Orestias complexes as recognized by Parenti (1984a): agassizii, cuvieri, gilsoni and mulleri. Our ancient DNA extraction protocol was validated after PCR amplification through an approach based on fragment-by-fragment chimera detection. After optimization, we were able to amplify < 200 bp fragments from both mitochondrial and nuclear DNA (CR and RH, respectively) from probably formalin-fixed type specimens bathed entirely in the extraction fluid. Most of the individuals exhibited few modifications of their external structures after GuSCN bath. Our approach combining type material and 'fresh' specimens allowed us to taxonomically delineate four clades recovered from the well-resolved CR tree into four redefined complexes: agassizii (sensu stricto, i.e. excluding luteus-like species), luteus, cuvieri and gilsoni. The mulleri complex is polyphyletic. Our phylogenetic analyses based on both mitochondrial and nuclear DNA revealed a main, deep dichotomy within the genus Orestias, separating the agassizii complex from a clade grouped under shallow dichotomies as (luteus, (cuvieri, gilsoni)). This 'deep and shallow' diversification pattern could fit within a scenario of ancient divergence between the agassizii complex and the rest of Orestias, followed by a recent diversification or adaptive radiation within each complex during the Pleistocene, in- and outside the Lake Titicaca. We could not recover the reciprocal monophyly of any of the 15 species or morphotypes that were considered in our analyses, possibly due to incomplete lineage sorting and/or hybridization events. As a consequence, our results starkly question the delineation of a series of diagnostic characters listed in the literature for Orestias. Although not included in our phylogenetic analysis, the syntype of O. jussiei could not be assigned to the agassizii complex as newly defined. The CR sequence of the extinct O. cuvieri was recovered within the cuvieri clade (same haplotype as one representative of O. pentlandii), so the mtDNA of the former species might still be represented in the wild.

The recent discovery of a lineage of gray wolf in North-East Africa suggests the presence of a cryptic canid on the continent, the African wolf Canis lupus lupaster. We analyzed the mtDNA diversity (cytochrome b and control region) of a series of African Canis including wolf-like animals from North and West Africa. Our objectives were to assess the actual range of C. l. lupaster, to further estimate the genetic characteristics and demographic history of its lineage, and to question its taxonomic delineation from the golden jackal C. aureus, with which it has been considered synonymous. We confirmed the existence of four distinct lineages within the gray wolf, including C. lupus/familiaris (Holarctic wolves and dogs), C. l. pallipes, C. l. chanco and C. l. lupaster. Taxonomic assignment procedures identified wolf-like individuals from Algeria, Mali and Senegal, as belonging to C. l. lupaster, expanding its known distribution c. 6,000 km to the west. We estimated that the African wolf lineage (i) had the highest level of genetic diversity within C. lupus, (ii) coalesced during the Late Pleistocene, contemporaneously with Holarctic wolves and dogs, and (iii) had an effective population size of c. 80,000 females. Our results suggest that the African wolf is a relatively ancient gray wolf lineage with a fairly large, past effective population size, as also suggested by the Pleistocene fossil record. Unique field observations in Senegal allowed us to provide a morphological and behavioral diagnosis of the African wolf that clearly distinguished it from the sympatric golden jackal. However, the detection of C. l. lupaster mtDNA haplotypes in C. aureus from Senegal brings the delineation between the African wolf and the golden jackal into question. In terms of conservation, it appears urgent to further characterize the status of the African wolf with regard to the African golden jackal.

Biofilms are complex communities playing an important role in aquatic ecosystems. Automated ribosomal intergenic spacer analysis (ARISA) has been used successfully to explore biofilm bacterial diversity. However, a gap remains to be filled as regards its application to biofilm eukaryotic populations. The aim of this study is to use ARISA to detect eukaryotic population shifts in biofilm. We designed a new set of primers to focus specifically on the ITS1-5.8S-ITS2 region of diatoms and tested it on natural biofilms. Additionally, we tested universal primers, used previously to perform ARISA on fungal communities. Cloning and sequencing showed that the universal primer set amplified various eukaryotes, whereas the new set was diatom specific. The new set amplified a wider variety of diatoms. Therefore, the universal set is appropriate to study the general eukaryotic population shifts in biofilms, whereas the new set is more appropriate to study diatoms specifically. We used both primer sets, along with a bacterial set, to study the population shifts in natural river biofilms. Principal component analysis of the ARISA fingerprints revealed seasonal shifts that did not coincide for bacterial and eukaryotic communities. Therefore, the use of both eukaryotic and bacterial primers provides a useful insight to assess microbial succession in biofilms., (© 2010 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

We used the supertree approach of matrix representation with parsimony to reconstruct to date the most exhaustive (genus-level) phylogeny of Cyprinidae. The supertree of Cyprinidae, representing 397 taxa (237 nominal genera) and 990 pseudocharacters, was well resolved (96%) through extended consensus majority rule, although 36 nodes (9.4%) were unsupported. The proportion of shared taxa among source trees was very low after calculation of the taxonomic coverage index (TCI = 0.059), which is proposed here as a more accurate alternative to the usual ratios calculated from the number of pseudo-characters or source trees per taxon. We define a new index for the calculation of partitioned qualitative clade support, the partitioned rQS ((p)rQS), which offers a straightforward visualization of the relative supports of source tree partitions at supertree nodes.The use of (p)rQS showed that the molecular source tree partition contributed to most node supports within the supertree of Cyprinidae (73%, contra 21% for the morphological partition) and evidenced a fair proportion of conflict at nodes between the two partitions (21%), notably reflecting (i) the greater number and resolution of molecular source trees, and (ii) potential morphological convergences. Most of the higher-level relationships within Cyprinidae were supported by both morphological and molecular source tree partitions. Our supertree showed a well-supported dichotomy between a clade consisting of a 'barbine' + 'rasborine' lineage, sister group to (Barbinae [paraphyletic], (Cyprininae, Labeoninae)), and a clade consisting of other rasborines (large polytomy) and the two monophyletic groups ((Tincinae, Tanichthys), (Ecocarpia, (Acheilognathinae, (Gobioninae, Leuciscinae)))) and (Squaliobarbinae, (Xenocyprinae, Cultrinae)). Through the non-monophyly of almost all the traditional subfamilies of Cyprinidae and 34 genera, our supertree exemplified the taxonomic chaos that reigns in the classification of the family. It also highlighted that further efforts should aim at increasing taxonomic sampling and generating alternative phylogenetic signals, notably for the still poorly apprehended Tincinae, Squaliobarbinae, Acheilognathinae, Gobioninae, and Rasborinae, the latter representing a key taxon for the understanding of early cyprinid evolution. Our supertree also proved useful for testing macro-evolutionary scenarios at a wide taxonomic scale. Ancestral reconstructions using linear parsimony confirmed that the Oriental tropical region was the centre of origin of Cyprinidae, and identified three Oriental-to-Palaearctic, two Palaearctic-to-Nearctic, and one Oriental-to-Afrotropical major migration events. On the other hand, we almost completely rejected the hypothesis of presence of barbels as a plesiomorphic condition within Cyprinidae (although ambiguous for maxillary barbels of the Barbinae-Cyprininae type). The supertree of Cyprinidae serves as a basis to discuss the applications and bias of the newly proposed (p)rQS, to provide future guidelines for a better achievement of cyprinid phylogeny, and to elaborate further on inter-continental migrations and the adaptive value of barbels.

We re-assess the value of morphological specific descriptors within the spring-snail genus Bythinella by sequencing mitochondrial COI and nuclear ITS1 gene fragments. Taxonomic coverage represents 16 nominal species sampled among 35 populations from France. Application of monophyly and cohesive haplotype networks as criteria to delineate species allow us to identify 10 mitochondrial species-level lineages, all but one of which are recovered by ITS1. COI species thresholds that are estimated from newly delimited species (ca. 1.5%) agree with values found among other hydrobioids. Our results strongly suggest that classical morphological descriptors may not constitute valid specific criteria within Bythinella. Our analyses support a complex scenario of invasions of subterranean habitats, as illustrated by the syntopy of several mitochondrial lineages or the conflicting evolutionary histories between COI and ITS1 in caves. In addition, morphological convergence related to subterranean ecological constraints that affect shell shape and size among the hypogean springsnails studied is suspected.

We reconstructed the phylogeny of the subfamily Viverrinae (Mammalia, Carnivora, Viverridae) using a approximately 3kb data set in order to reassess timing and patterns of faunal exchanges between Asia and Africa. Maximum parsimony, maximum likelihood, and Bayesian analyses of separated and combined matrices (cytochrome b, transthyretin intron I and IRBP exon 1 [IRBP1]) recovered all the well-supported relationships within feliformian lineages. In addition, IRBP1 supported paraphyly of genus Herpestes and contributed to the resolution of equivocal hypotheses within Viverridae, including (1) the monophyly of Viverrinae, and (2) Viverricula sister-group of the other terrestrial civets (Civettictis and Viverra). The combined analysis yielded a robust phylogeny, recovering monophyly of Prionodontidae and yielding high posterior probabilities for nodes (1) (Prionodontidae, Felidae) and (2) ((Felidae, Prionodontidae), ((Hyaenidae, (Herpestidae, Eupleridae)), Viverridae)). Using a fossil cross-validation method, we estimated the emergence of Viverridae at 34.29Myr, with a separation between the three traditional subfamilies Hemigalinae, Paradoxurinae, and Viverrinae during the Late Oligocene-Early Miocene. The terrestrial civets and the splits between (1) Civettictis and Viverra and (2) Poiana and Genetta were estimated to appear during the Middle Miocene. Parsimony- and maximum likelihood-based methods yielded unambiguous ancestral area reconstructions, including the Asian origin of the family Viverridae, the subfamily Viverrinae, the terrestrial civets and the clade (Civettictis, Viverra). On the grounds of genetic distances, morphological divergence, and divergence time estimates, we propose the erection of the subfamily Genettinae (including Genetta and Poiana). Our analyses suggested two independent migration events from Asia to Africa, during the Middle Miocene (Civettictis) and between the Late Oligocene and Middle Miocene (Genettinae). These results are in agreement with the hypothesis of Miocene routes from Asia to Africa-via the Arabian microplate-that would have involved several independent events of migrations. Couched in the context of the viverrid fossil record, our study calls for a revision of the paleontological data in order to fully appreciate the complexity of Afro-Asian faunal exchanges.

Adaptive convergence in morphological characters has not been thoroughly investigated, and the processes by which phylogenetic relationships may be misled by morphological convergence remains unclear. We undertook a case study on the morphological evolution of viverrid-like feliformians (Nandinia, Cryptoprocta, Fossa, Eupleres, Prionodon) and built the largest morphological matrix concerning the suborder Feliformia to date. A total of 349 characters grouped into four anatomical partitions were used for all species of Viverridae and viverrid-like taxa plus representatives of the Felidae, Hyaenidae, Herpestidae, and one Malagasy mongoose. Recent molecular phylogenetic analyses suggest that viverrid-like morphotypes appeared independently at least three times during feliformian evolution. We thus used a synthetic molecular tree to assess morphological evolutionary patterns characterizing the viverrid-like taxa. We examined phylogenetic signal, convergence and noise in morphological characters using (a) tree-length distribution (g1), (b) partitioned Bremer support, (c) RI values and their distribution, (d) respective contributions of diagnostic synapomorphies at the nodes for each partition, (e) patterns of shared convergences among viverrid-like taxa and other feliformian lineages, (f) tree-length differences among alternative hypotheses, and (g) the successive removal of convergent character states from the original matrix. In addition, the lability of complex morphological structures was assessed by mapping them onto the synthetic molecular tree. The unconstrained morphological analysis yielded phylogenetic groupings that closely reflected traditional classification. The use of a synthetic molecular tree (constraint) combined with our thorough morphological investigations revealed the mosaics of convergences likely to have contributed to part of the historical uncertainty over viverrid classification. It also showed that complex morphological structures could be subjected to reversible evolutionary trends. The morphological matrix proved useful in characterizing several feliformian clades with diagnostic synapomorphies. These results support the removal from the traditionally held Viverridae of several viverrid-like taxa into three distinct families: Nandiniidae (Nandinia), Prionodontidae (Prionodon), and the newly defined Eupleridae (including Cryptoprocta, Fossa, Eupleres plus all "mongoose-like" Malagasy taxa). No clearly "phylogenetically misleading" data subsets could be identified, and the great majority of morphological convergences appeared to be nonadaptive. The multiple approaches used in this study revealed that the most disruptive element with regards to morphological phylogenetic reconstruction was noise, which blured the expression of phylogenetic signal. This study demonstrates the crucial need to consider independent (molecular) phylogenies in order to produce reliable evolutionary hypotheses and should promote a new approach to the definition of morphological characters in mammals. [Constrained analysis; convergence; evolutionary scenario; Feliformia; morphology; noise; phylogenetic signal; phylogeny; Viverridae.].

The Herpestidae are small terrestrial carnivores comprising 18 African and Asian genera, currently split into two subfamilies, the Herpestinae and the Galidiinae. The aim of this work was to resolve intra-familial relationships and to test the origin of sociality in the group. For this purpose we analysed sequences of the complete cytochrome b gene for 18 species of Herpestidae. The results showed that the mongooses were split into three clades: (1) the Malagasy taxa (Galidiinae and Cryptoprocta), (2) the true social mongooses and (3) the solitary mongooses, each group being also supported by morphological and chromosomal data. Our results suggested unexpected phylogenetic relationships: (1) the genus Cynictis is included in the solitary mongoose clade, (2) the genera Liberiictis and Mungos are sister-group, and (3) the genus Herpestes is polyphyletic. We examined the evolution of the sociality in mongooses by combining behavioural traits with the cytochrome b data. Some of the behavioural traits provided good synapomorphies for characterizing the social species clade, showing the potential benefit of using such characters in phylogeny. The mapping of ecological and behavioural features resulted in hypothesizing solitary behavior and life in forest as the conditions at the base of the mongoose clade.

Although molecular studies have helped to clarify the phylogeny of the problematic family Viverridae, a recent phylogenetic investigation based on cytochrome b (cyt b) has excluded the Asiatic linsangs (genus Prionodon) from the family. To assess the phylogenetic position of the Asiatic linsangs within the Feliformia, we analysed an exhaustive taxonomic sample set with cyt b and newly produced transthyretin intron I sequences (TR-I-I). TR-I-I alone and cyt b +TR-I-I combined (maximum-likelihood analysis) highly support the position of Asiatic linsangs as sister-group of the Felidae. The estimation of minimum divergence dates from molecular data suggests a splitting event ca. 33.3 million years (Myr) ago, which lends support to historical assertions that the Asiatic linsangs are "living fossils" that share a plesiomorphic morphotype with the Oligocene feliform Paleoprionodon. The African linsang is estimated to appear more than 20 Myr later and represents the sister-group of the genus Genetta. Our phylogenetic results illustrate numerous morphological convergences of "diagnostic" characters among Feliformia that might be problematic for the identification of fossil taxa. The morphotype reappearance from the Asiatic to the African linsangs suggests that the genome of the Feliformia conserved its potential ability of expression for a peculiar adaptive phenotype throughout evolution, in this case arboreality and hypercarnivory in tropical forest.

We report our experience concerning the laparoscopic treatment of ectopic pregnancy (EP). One-hundred and nine women with EP were treated in our department over a 4 year period, between February 1988 (date of our first laparoscopic surgery for EP) and December 1991. Twenty-two these women underwent laparotomy and the remaining 87 laparoscopic surgery alone. Four therapeutic failures were noted in women treated by laparoscopy. Our results are compared with those of other series and the indications, as well as the modalities of laparoscopic treatment are detailed. It is concluded that laparoscopic surgery of EP is a reliable method which must always be considered, except for a few rare indications.

We report our experience concerning the laparoscopic treatment of ectopic pregnancy (EP). One-hundred and nine women with EP were treated in our department over a 4 year period, between February 1988 (date of our first laparoscopic surgery for EP) and December 1991. Twenty-two of these women underwent laparotomy and the remaining 87 laparoscopic surgery alone. Four therapeutic failures were noted in women treated by laparoscopy. Our results are compared with those of other series and the indications, as well as the modalities of laparoscopic treatment are detailed. It is concluded that laparoscopic surgery of EP is a reliable method which must always be considered, except for a few rare indications.