Non-invasive ancient DNA protocol for fluid-preserved specimens and phylogenetic systematics of the genus Orestias (Teleostei: Cyprinodontidae).

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.

Molecular systematics and origin of sociality in mongooses (Herpestidae, Carnivora).

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.