François Catzeflis

Phylogeny and biogeography of African Murinae based on mitochondrial and nuclear gene sequences, with a new tribal classification of the subfamily.

BackgroundWithin the subfamily Murinae, African murines represent 25% of species biodiversity, making this group ideal for detailed studies of the patterns and timing of diversification of the African endemic fauna and its relationships with Asia. Here we report the results of phylogenetic analyses of the endemic African murines through a broad sampling of murine diversity from all their distribution area, based on the mitochondrial cytochrome b gene and the two nuclear gene fragments (IRBP exon 1 and GHR).ResultsA combined analysis of one mitochondrial and two nuclear gene sequences consistently identified and robustly supported ten primary lineages within Murinae. We propose to formalize a new tribal arrangement within the Murinae that reflects this phylogeny. The diverse African murine assemblage includes members of five of the ten tribes and clearly derives from multiple faunal exchanges between Africa and Eurasia. Molecular dating analyses using a relaxed Bayesian molecular clock put the first colonization of Africa around 11 Mya, which is consistent with the fossil record. The main period of African murine diversification occurred later following disruption of the migration route between Africa and Asia about 7–9 Mya. A second period of interchange, dating to around 5–6.5 Mya, saw the arrival in Africa of Mus (leading to the speciose endemic Nannomys), and explains the appearance of several distinctive African lineages in the late Miocene and Pliocene fossil record of Eurasia.ConclusionOur molecular survey of Murinae, which includes the most complete sampling so far of African taxa, indicates that there were at least four separate radiations within the African region, as well as several phases of dispersal between Asia and Africa during the last 12 My. We also reconstruct the phylogenetic structure of the Murinae, and propose a new classification at tribal level for this traditionally problematic group.

The Sahara as a vicariant agent, and the role of Miocene climatic events, in the diversification of the mammalian order Macroscelidea (elephant shrews).

Although the Sahara is a major geographical feature of the African continent, its role in the diversification of animal species is not well understood. We present here a molecular phylogeny for members of the endemic African mammalian order Macroscelidea (elephant shrews) with molecular-clock calculations; this molecular phylogeny provides convincing evidence that the genus Elephantulus is diphyletic. Elephantulus rozeti, the only elephant shrew species that resides north of the Sahara, is the sister group of a species from a different genus (Petrodromus tetradactylus), which resides just south of the Sahara. The split between these taxa coincided with major Miocene climatic events, which triggered the cooling and aridification of midlatitude continental regions, and a shift in the Sahara from a tropical to an arid environment. Thus, the North African distribution of E. rozeti is not the result of dispersion from an eastern species of the genus, but instead the result of a vicariant event involving the formation of the Sahara. The splitting events involved with most Elephantulus species in our analysis appear to coincide with these climatic events. This coincidence suggests that the environmental consequences associated with this period played an important role in the radiation of this order of mammals. The strongly supported phylogeny provides compelling evidence for a complex history of mosaic evolution, including pronounced bradytelic morphological evolution in some lineages, accelerated morphological evolution in others, and a remarkably slow rate of evolution of the male reproductive structure.

Multiple geographic origins of commensalism and complex dispersal history of black rats

The Black Rat (Rattus rattus) spread out of Asia to become one of the worlds worst agricultural and urban pests, and a reservoir or vector of numerous zoonotic diseases, including the devastating plague. Despite the global scale and inestimable cost of their impacts on both human livelihoods and natural ecosystems, little is known of the global genetic diversity of Black Rats, the timing and directions of their historical dispersals, and the risks associated with contemporary movements. We surveyed mitochondrial DNA of Black Rats collected across their global range as a first step towards obtaining an historical genetic perspective on this socioeconomically important group of rodents. We found a strong phylogeographic pattern with well-differentiated lineages of Black Rats native to South Asia, the Himalayan region, southern Indochina, and northern Indochina to East Asia, and a diversification that probably commenced in the early Middle Pleistocene. We also identified two other currently recognised species of Rattus as potential derivatives of a paraphyletic R. rattus. Three of the four phylogenetic lineage units within R. rattus show clear genetic signatures of major population expansion in prehistoric times, and the distribution of particular haplogroups mirrors archaeologically and historically documented patterns of human dispersal and trade. Commensalism clearly arose multiple times in R. rattus and in widely separated geographic regions, and this may account for apparent regionalism in their associated pathogens. Our findings represent an important step towards deeper understanding the complex and influential relationship that has developed between Black Rats and humans, and invite a thorough re-examination of host-pathogen associations among Black Rats.

Variance of molecular datings, evolution of rodents and the phylogenetic affinities between Ctenodactylidae and Hystricognathi

The von Willebrand factor (vWF) gene has been used to understand the origin and timing of Rodentia evolution in the context of placental phylogeny. vWF exon 28 sequences of 15 rodent families and eight non–rodent eutherian clades are analysed with two different molecular dating methods (uniform clock on a linearized tree; quartet dating). Three main conclusions are drawn from the study of this nuclear exon. First, Ctenodactylidae (gundis) and Hystricognathi (e.g. porcupines, guinea–pigs, chinchillas) robustly cluster together in a newly recognized clade, named ‘Ctenohystrica’. The Sciurognathi monophyly is subsequently rejected. Pedetidae (springhares) is an independent and early diverging rodent lineage, suggesting a convergent evolution of the multiserial enamel of rodent incisors. Second, molecular date estimates are here more influenced by accuracy and choice of the palaeontological temporal references used to calibrate the molecular clock than by either characters analysed (nucleotides versus amino acids) or species sampling. The caviomorph radiation at 31 million years (Myr) and the pig–porpoise split at 63Myr appear to be reciprocally compatible dates. Third, during the radiation of Rodentia, at least three lineages (Gliridae, Sciuroidea and Ctenohystrica) emerged close to the Cretaceous–Tertiary boundary, and their common ancestor separated from other placental orders in the Late Cretaceous.

Molecular phylogeny and the historical biogeography of the warblers of the genus Sylvia (Aves)

A molecular phylogeny based on DNA/DNA hybridization revealed that the Sylvia‐Parisoma complex is monophyletic and includes three main groups of species, the “mid‐European” warblers, the genus Parisoma, and the “eu‐Mediterranean” Sylvia species sensu stricto. The latter can be assigned to three main clusters, a “West‐Mediterranean” group, a “Central‐Mediterranean group”, and an “East‐Mediterranean” group. The radiation of the whole complex is much more ancient than formerly believed. It started ca 12–13 Ma ago and the ancestors of the main extant groups differentiated during the Pliocene. Only speciation events within the “eu‐Mediterranean” lineages occurred during the Pleistocene. The paleoclimatical and paleoecological history of the Mediterranean region is too complicated to provide any evidence for direct relationships between past events and evolutionary steps of these taxa which did not leave any reliable fossil record. However, some major speciation events may be related to well documented climatical crises as well as paleobotanical data. The largely man‐induced extension of matorrals over several millenia presumably extended the range of several species that were formerly much more restricted, which complicates reconstruction of the spatio‐temporal course of speciation.

Muroid rodents: Phylogeny and evolution

The Muroidea, a group of rodents that includes mice, rats, gerbils, hamsters and others, encompasses a tremendous diversity of fairly recent geological origin. The taxonomy, systematics, phylogeny and paleontology of the muroid rodents have progressed enormously during the last two decades, and many hypotheses on their evolutionary biology have been formalized. Nevertheless, there still remain important unanswered questions - regarding, for example, local conflicts between molecular and paleontological data, or the origin of the fast rate of DNA change in rats and mice - that need more investigation.

Male sex determination in the spiny rat Tokudaia osimensis (Rodentia: Muridae) is not Sry dependent

In mammals, gender follows from the development of the embryonic gonads as testis or ovaries and is under genetic control. In males, testis development is dependent on the inheritance of the Y Chromosome-encoded Sry gene. It is believed, as a result of transgenic experiments, that Sry is the only gene encoded on the Y Chr that is necessary to switch the genital ridges towards the testicular developmental pathway (Koopman et al. 1990). An exception to this rule is described in a recent report describing the absence ofSry in two species of an arvicoline rodent, Ellobius, which have an unconventional gonosome complement (Just et al. 1995). In this context,Tokudaia osimensis osimensis, a small murine rodent from the Amami Island (part of the Ryukyu Archipelago in the south of Japan), is an intriguing mammal. Indeed, in this particular species, the Ryukyu Spiny Rat, males and females have an identical karyotype (2n4 25,XO; Honda et al. 1977). This small (head and body length from 12 to 18 cm) spiny rat is a rare mammal (Nowak 1991), believed by IUCN (1978) to be seriously endangered by the general destruction of natural arcas on the Ryukyu Islands.Tokudaia osimensisis fully protected in Japan. The presence or the absence of Sryon either the X Chr or on one of the autosomes of this particular mammal was, therefore, investigated. Using degenerate oligonucleotides designed from an alignment of all Sry HMG box sequences reported so far from rodent only or from mammal and a nested touch-down PCR strategy (Don et al. 1991), we were unable to amplify any Srysequence from genomic DNA of both male and female T. osimensis osimensis (Fig. 1). To ensure that our primers were able to amplify Sry sequences from either a closely related rodent or from an unrelated one, we also amplified DNA of other species such as two murines Apodemus agrarius, Apodemus flavicollis, and one cricetineMesocricetus auratus.In these cases we obtained PCR products of the expected sizes (Fig. 1), which we confirmed as being Sryby sequencing (not shown). This result suggests that the lack of Sryamplification inT. osimensiswas not simply the result of strong sequence divergence owing to the high evolutionary rate of the Sry gene observed in rodents (Tucker and Lundrigan 1993; Whitfield et al. 1993). The positive result observed in the two Apodemusspecies is particularly important because of their close relationship with Tokudaia, as revealed by morphological studies (Kawamura 1989) and also from phylogenetic inferences derived from albumin immunology (Watts and Baverstock 1994) and mitochondrial DNA sequences (S. Soullier and F. Catzeflis, unpublished data). Tokudaia osimensis DNA integrity was next checked by amplification of other genes, such as the androgen receptor AR, and two transcription factorsSox3andZfx (Fig. 1 and data not shown). These results, obtained with autosomal and X Chr encoded genes, clearly demonstrate that our conditions allow the detection of single-copy genes. Taken together, these data indicate that the Sry gene is absent from the T. osimensis osimensis genome. Nevertheless, our results should be tested by additional biological material (from testis tissues) and additional experiments such as Southern blotting. This species is thus the second rodent genus described so far as lacking the mammalian sex-determining factor Sry,since two species of voles in the genus Ellobius (E. lutescensand E. tancrei ) display the same unusual feature (Just et al. 1995). Phylogenetically, the arvicolineEllobius is not closely related to the murine Tokudaia(Fig. 2), as these genera belong to different subfamilies of Muridae, thought to have diverged from each other ca. 17 to 25 My ago (Catzeflis et al. 1993). As all other rodents so far analyzed possess aSry gene (Just et al. 1995; Tucker and Lundrigan 1993; Suzuki et al. 1997), we therefore conclude that the loss of Sry as a switch mechanism in sex determination among Tokudaiaand Ellobius rodents is the result of two independent events. So, even between mammals, a high plasticity of the sex determination mechanism is observed. Such a variety of genetic strategies in sex determination within the same taxon (in this case: the Muridae family) has been previously reported in the case of dipteran insects. In this case, eithe r a Y Chr located factor, an autosomal male dominant factor, or the X:A ratio can be used as the primary signal (Dübendorfer et al. 1992). What may be the genetic switch controlling sex determination in TokudaiaandEllobius?Although several hypotheses are possible at this stage, there is no indication that the same mechanism occurs in these two rodent genera. As a hypothesis, a gene under the control ofSry in the usual genetic pathway may be the master control gene. Recently, the discovery of an X Chr inactivation event occurring during embryonic development in the male mouse urogenital ridges coincident withSry gene expression raises the intriguing possibility of a connection between these two events (Jamieson et al. 1997). To decipher the mechanism(s) of sex determination in these particular species, it is important to know whether there is a genetic difference between males and females. If not, sex determination could be the result of an imprinting mechanism involving the X Chr, since a nonrandom X inactivation has already been reported in mouse extraembryonic tissues (Marahrens et al. 1997) and in somatic cells of X*XY wood lemmings (genusMyopus,another arvicoline rodent) leading to different sexual phenotypes (Schempp et al. 1985). The description of these mechanisms will now require a more precise genetic analysis of these species and will bring us new information on sex determination mechanisms, evolution, and plasticity. Correspondence to: P. Berta Mammalian Genome 9, 590–592 (1998).

The Virtues of Gaps: Xenarthran (Edentate) Monophyly Supported by a Unique Deletion in αA-Crystallin

Shared insertions or deletions (indels) in protein-coding DNA can be strong indicators of the monophyly of a taxon. A three-amino acid deletion had previously been noted in the eye lens protein alpha A-crystallin of two species of sloths and two species of anteaters, which represent the Pilosa, one of the two infraorders of Xenarthra (Edentata). This deletion has not been observed in 55 species from 16 other eutherian orders, or in 2 species of marsupials, or in 34 nonmammalian vertebrates, from birds to shark. At the genomic level, we have now detected this deletion in two species of armadillos of the second xenarthran infraorder, Cingulata, as well as in an additional species of anteater. Phylogenetic trees were constructed from a 145-bp sequence of the alpha A-crystallin gene of 39 tetrapod species, supporting xenarthran monophyly with values from 76% to 90%. To quantify the additional support for xenarthran monophyly, as given by the three-residue deletion, we computed the probabilities for the occurrence of this deletion per evolutionary time unit for alternative hypothetical tree topologies. In the estimates obtained, the six trees in which the xenarthran subgroups are unresolved or paraphyletic give an increasingly lower likelihood than do the two trees that assume xenarthran monophyly. For the monophyletic trees, the probability that the deletion observed in the xenarthrans is due to a single event is > 0.99. Thus, this deletion in alpha A-crystallin gives strong molecular support for the monophyly of this old and diverse order.

Molecular evolution of the mitochondrial 12S rRNA in Ungulata (mammalia)

The complete 12S rRNA gene has been sequenced in 4 Ungulata (hoofed eutherians) and 1 marsupial and compared to 38 available mammalian sequences in order to investigate the molecular evolution of the mitochondrial small-subunit ribosomal RNA molecule. Ungulata were represented by one artiodactyl (the collared peccary, Tayassu tajacu, suborder Suiformes), two perissodactyls (the Grevys zebra, Equus grevyi, suborder Hippomorpha; the white rhinoceros, Ceratotherium simum, suborder Ceratomorpha), and one hyracoid (the tree hyrax, Dendrohyrax dorsalis). The fifth species was a marsupial, the eastern gray kangaroo (Macropus giganteus). Several transition/transversion biases characterized the pattern of changes between mammalian 12S rRNA molecules. A bias toward transitions was found among 12S rRNA sequences of Ungulata, illustrating the general bias exhibited by ribosomal and protein-encoding genes of the mitochondrial genome. The derivation of a mammalian 12S rRNA secondary structure model from the comparison of 43 eutherian and marsupial sequences evidenced a pronounced bias against transversions in stems. Moreover, transversional compensatory changes were rare events within double-stranded regions of the ribosomal RNA. Evolutionary characteristics of the 12S rRNA were compared with those of the nuclear 18S and 28S rRNAs. From a phylogenetic point of view, transitions, transversions and indels in stems as well as transversional and indels events in loops gave congruent results for comparisons within orders. Some compensatory changes in double-stranded regions and some indels in single-stranded regions also constituted diagnostic events. The 12S rRNA molecule confirmed the monophyly of infraorder Pecora and order Cetacea and demonstrated the monophyly of suborder Suiformes. However, the monophyly of the suborder Ruminantia was not supported, and the branching pattern between Cetacea and the artiodactyl suborders Ruminantia and Suiformes was not established. The monophyly of the order Perissodactyla was evidenced, but the relationships between Artiodactyla, Cetacea, and Perissodactyla remained unresolved. Nevertheless, we found no support for a Perissodactyla + Hyracoidea clade, neither with distance approach, nor with parsimony reconstruction. The 12S rRNA was useful to solve intraordinal relationships among Ungulata, but it seemed to harbor too few informative positions to decipher the bushlike radiation of some Ungulata orders, an event which has most probably occurred in a short span of time between 55 and 70 MYA.

Retroviral envelope gene captures and syncytin exaptation for placentation in marsupials

Significance Syncytins are “captured” genes of retroviral origin, corresponding to the fusogenic envelope gene of endogenized retroviruses. They are present in a series of eutherian mammals, including humans and mice where they play an essential role in placentation. Here we show that marsupials—which diverged from eutherian mammals ∼190 Mya but still possess a primitive, short-lived placenta (rapidly left by the embryo for development in an external pouch)—have also captured such genes. The present characterization of the syncytin-Opo1 gene in the opossum placenta, together with the identification of two additional endogenous retroviral envelope gene captures, allow a recapitulation of the natural history of these unusual genes and definitely extends their “symbiotic niche” to all clades of placental mammals. Syncytins are genes of retroviral origin captured by eutherian mammals, with a role in placentation. Here we show that some marsupials—which are the closest living relatives to eutherian mammals, although they diverged from the latter ∼190 Mya—also possess a syncytin gene. The gene identified in the South American marsupial opossum and dubbed syncytin-Opo1 has all of the characteristic features of a bona fide syncytin gene: It is fusogenic in an ex vivo cell–cell fusion assay; it is specifically expressed in the short-lived placenta at the level of the syncytial feto–maternal interface; and it is conserved in a functional state in a series of Monodelphis species. We further identify a nonfusogenic retroviral envelope gene that has been conserved for >80 My of evolution among all marsupials (including the opossum and the Australian tammar wallaby), with evidence for purifying selection and conservation of a canonical immunosuppressive domain, but with only limited expression in the placenta. This unusual captured gene, together with a third class of envelope genes from recently endogenized retroviruses—displaying strong expression in the uterine glands where retroviral particles can be detected—plausibly correspond to the different evolutionary statuses of a captured retroviral envelope gene, with only syncytin-Opo1 being the present-day bona fide syncytin active in the opossum and related species. This study would accordingly recapitulate the natural history of syncytin exaptation and evolution in a single species, and definitely extends the presence of such genes to all major placental mammalian clades.