The name of the game: palaeoproteomics and radiocarbon dates further refine the presence and dispersal of caprines in eastern and southern Africa.

We report the first large-scale palaeoproteomics research on eastern and southern African zooarchaeological samples, thereby refining our understanding of early caprine (sheep and goat) pastoralism in Africa. Assessing caprine introductions is a complicated task because of their skeletal similarity to endemic wild bovid species and the sparse and fragmentary state of relevant archaeological remains. Palaeoproteomics has previously proved effective in clarifying species attributions in African zooarchaeological materials, but few comparative protein sequences of wild bovid species have been available. Using newly generated type I collagen sequences for wild species, as well as previously published sequences, we assess species attributions for elements originally identified as caprine or 'unidentifiable bovid' from 17 eastern and southern African sites that span seven millennia. We identified over 70% of the archaeological remains and the direct radiocarbon dating of domesticate specimens allows refinement of the chronology of caprine presence in both African regions. These results thus confirm earlier occurrences in eastern Africa and the systematic association of domesticated caprines with wild bovids at all archaeological sites. The combined biomolecular approach highlights repeatability and accuracy of the methods for conclusive contribution in species attribution of archaeological remains in dry African environments.

Molecular phylogeny of 42 species of Culicoides (Diptera, Ceratopogonidae) from three continents.

The genus Culicoides includes vectors of important animal diseases such as bluetongue and Schmallenberg virus (BTV and SBV). This genus includes 1300 species classified in 32 subgenera and 38 unclassified species. However, the phylogenetic relationships between different subgenera of Culicoides have never been studied. Phylogenetic analyses of 42 species belonging to 12 subgenera and 8 ungrouped species of genus Culicoides from Ecuador, France, Gabon, Madagascar and Tunisia were carried out using two molecular markers (28S rDNA D1 and D2 domains and COI mtDNA). Sequences were subjected to non-probabilistic (maximum parsimony) and probabilistic (Bayesian inference (BI)) approaches. The subgenera Monoculicoides, Culicoides, Haematomyidium, Hoffmania, Remmia and Avaritia (including the main vectors of bluetongue disease) were monophyletic, whereas the subgenus Oecacta was paraphyletic. Our study validates the subgenus Remmia (= Schultzei group) as a valid subgenus, outside of the subgenus Oecacta. In Europe, Culicoides obsoletus, Culicoides scoticus and Culicoides chiopterus should be part of the Obsoletus complex whereas Culicoides dewulfi should be excluded from this complex. Our study suggests that the current Culicoides classification needs to be revisited with modern tools.

Correction: What Do Pneumocystis Organisms Tell Us about the Phylogeography of Their Hosts? The Case of the Woodmouse Apodemus sylvaticus in Continental Europe and Western Mediterranean Islands.

[This corrects the article DOI: 10.1371/journal.pone.0120839.].

What do Pneumocystis organisms tell us about the phylogeography of their hosts? The case of the woodmouse Apodemus sylvaticus in continental Europe and western Mediterranean islands.

Pneumocystis fungi represent a highly diversified biological group with numerous species, which display a strong host-specificity suggesting a long co-speciation process. In the present study, the presence and genetic diversity of Pneumocystis organisms was investigated in 203 lung samples from woodmice (Apodemus sylvaticus) collected on western continental Europe and Mediterranean islands. The presence of Pneumocystis DNA was assessed by nested PCR at both large and small mitochondrial subunit (mtLSU and mtSSU) rRNA loci. Direct sequencing of nested PCR products demonstrated a very high variability among woodmouse-derived Pneumocystis organisms with a total number of 30 distinct combined mtLSU and mtSSU sequence types. However, the genetic divergence among these sequence types was very low (up to 3.87%) and the presence of several Pneumocystis species within Apodemus sylvaticus was considered unlikely. The analysis of the genetic structure of woodmouse-derived Pneumocystis revealed two distinct groups. The first one comprised Pneumocystis from woodmice collected in continental Spain, France and Balearic islands. The second one included Pneumocystis from woodmice collected in continental Italy, Corsica and Sicily. These two genetic groups were in accordance with the two lineages currently described within the host species Apodemus sylvaticus. Pneumocystis organisms are emerging as powerful tools for phylogeographic studies in mammals.

Phylogeny of fossil and extant glypheid and litogastrid lobsters (Crustacea, Decapoda) as revealed by morphological characters.

A phylogenetic analysis of a total of 31 species: 27 fossil species from seven families (Glypheidae, Litogastridae, Mecochiridae, Pemphicidae, Erymidae, Clytiopsidae, Chimaerastacidae), and four extant species from three families (Glypheidae, Nephropidae, Stenopodidae) is proposed. Most of the genera considered are coded exclusively based upon their type species and, as much as possible, based upon the type specimens. The cladistic analysis demonstrates that the glypheidean lobsters (infraorder Glypheidea) form a monophyletic group including two superfamilies: Glypheoidea and Pemphicoidea new status. Glypheoidea includes three families: Glypheidae, Mecochiridae and Litogastridae. Litogastridae is the sister group of the clade Glypheidae + Mecochiridae. Pemphicoidea includes a single family: Pemphicidae. A new classification of Glypheidea is proposed and currently known genera are rearranged based upon the phylogenetic analysis.

Characterizing Pneumocystis in the lungs of bats: understanding Pneumocystis evolution and the spread of Pneumocystis organisms in mammal populations.

Bats belong to a wide variety of species and occupy diversified habitats, from cities to the countryside. Their different diets (i.e., nectarivore, frugivore, insectivore, hematophage) lead Chiroptera to colonize a range of ecological niches. These flying mammals exert an undisputable impact on both ecosystems and circulation of pathogens that they harbor. Pneumocystis species are recognized as major opportunistic fungal pathogens which cause life-threatening pneumonia in severely immunocompromised or weakened mammals. Pneumocystis consists of a heterogeneous group of highly adapted host-specific fungal parasites that colonize a wide range of mammalian hosts. In the present study, 216 lungs of 19 bat species, sampled from diverse biotopes in the New and Old Worlds, were examined. Each bat species may be harboring a specific Pneumocystis species. We report 32.9% of Pneumocystis carriage in wild bats (41.9% in Microchiroptera). Ecological and behavioral factors (elevation, crowding, migration) seemed to influence the Pneumocystis carriage. This study suggests that Pneumocystis-host association may yield much information on Pneumocystis transmission, phylogeny, and biology in mammals. Moreover, the link between genetic variability of Pneumocystis isolated from populations of the same bat species and their geographic area could be exploited in terms of phylogeography.

[Biological evolution and ancient DNA]. / Evolution biologique et ADN ancien.

Twenty years after the advent of ancient DNA studies, this discipline seems to have reached the maturity formerly lacking to the fulfilment of its objectives. In its early development paleogenetics, as it is now acknowledged, had to cope with very limited data due to the technical limitations of molecular biology. It led to phylogenetic assumptions often limited in their scope and sometimes non-focused or even spurious results that cast the reluctance of the scientific community. This time seems now over and huge amounts of sequences have become available which overcome the former limitations and bridge the gap between paleogenetics, genomics and population biology. The recent studies over the charismatic woolly mammoth (independent sequencing of the whole mitochondrial genome and of millions of base pairs of the nuclear genome) exemplify the growing accuracy of ancient DNA studies thanks to new molecular approaches. From the earliest publications up to now, the number of mammoth nucleotides was multiplied by 100,000. Likewise, populational approaches of ice-age taxa provide new historical scenarios about the diversification and extinction of the Pleistocene megafauna on the one hand, and about the processes of domestication of animal and vegetal species by Man on the other. They also shed light on the differential structure of molecular diversity between short-term populational research (below 2 My) and long-term (over 2 My) phylogenetic approaches. All those results confirm the growing importance of paleogenetics among the evolutionary biology disciplines.

Phylogenetic systematics and evolution of primate-derived Pneumocystis based on mitochondrial or nuclear DNA sequence comparison.

Previous studies have demonstrated that the agent of Pneumocystis pneumonia (PcP), Pneumocystis carinii, is actually a complex of eukaryotic organisms, and cophylogeny could explain the distribution of the hosts and parasites. In the present work, we tested the hypothesis of cophylogeny between the primate-derived Pneumocystis group and their hosts. Specific strains isolated from 20 primate species, including humans, were used to produce a phylogeny of the parasites. Aligned sequences corresponding to DNA sequences of three genes (DHPS, mtSSU-rRNA, and mtLSU-rRNA) were separately analyzed and then combined in a single data set. The resulting parasite phylogeny was compared with different controversial phylogenies for the hosts. This comparison demonstrated that, depending upon which topology is accepted for the hosts, at least 61% and perhaps 77% of the homologous nodes of the respective cladograms of the hosts and parasites may be interpreted as resulting from codivergence events. This finding and the high specificity of these parasites suggests that cophylogeny may be considered the dominant pattern of evolution for Pneumocystis organisms, representing a new example of parallel evolution between primates and their specific parasites. Because the phylogeny of Pneumocystis followed very closely the differentiation of their hosts at the species level, the study of the parasites could provide valuable information on the phylogeny of their hosts. We used this information to explore controversial hypotheses of the phylogeny of the Platyrrhini by comparison with the phylogeny of their specific Pneumocystis parasites. If these organisms were closely associated as lung parasites with primates through the ages, the hypothesis of the Pneumocystis spp. being new pathogenic agents could be refuted. However, these organisms are opportunistic symbionts, becoming pathogenic whenever the immunological defences of their hosts decline. This study also provides support for the hypothesis that the different Pneumocystis species are genetically independent organisms, helping to clarify their taxonomic status.

Status of the so-called African pygmy elephant (Loxodonta pumilio (NOACK 1906)): phylogeny of cytochrome b and mitochondrial control region sequences.

Among the African elephants, it has been unanimously acknowledged that the forest elephants (cyclotis form) are peculiar, so that they have been elevated to the specific rank. The development of molecular analyses of extant Loxodonta has only focused on two forms yet: the savannah form (africana) and the forest form (cyclotis), disregarding the so-called pygmy elephants (pumilio or fransseni) the systematic status of which has been debated since their discovery. Therefore, we have sampled nine dwarfed-labelled specimens in collection and eight specimens of typical forest elephants that we compared to three savannah elephants and two Asian elephants. Because of the degraded nature of the nuclear DNA content in bone samples of old specimens, we assayed mitochondrial markers; 1961 bp of the mitochondrial genome were sequenced (over a continuous range spanning the cytochrome b gene, tRNA Thr, tRNA Pro, hypervariable region 1 and central conserved region of the control region). Pumilio and cyclotis are not sister-taxa: the phylogenetic analyses rather account for the inclusion of the so-called pygmy elephants within a monophyletic group of forest elephants sensu lato. The internal structure of this clade reveals to depend on isolation and remoteness between populations, characteristics that may have been extensively influenced by climatic variations during the Quaternary period. We conclude that the specific taxon Loxodonta pumilio (or Loxodonta fransseni) should be abandoned.

Mitochondrial cytochrome b of the Lyakhov mammoth (Proboscidea, Mammalia): new data and phylogenetic analyses of Elephantidae.

The phylogenetic relationships between recent Elephantidae (Proboscidea, Mammalia), that is to say extant elephants (Asian and African) and extinct woolly mammoth, have remained unclear to date. The prevailing morphological scheme (mammoth grouped with Asian elephant) is either supported or questioned by the molecular results. Recently, the monophyly of woolly mammoths on mitochondrial grounds has been demonstrated (Thomas, et al., 2000), but it conflicts with previous studies (Barriel et al., 1999; Derenko et al., 1997). Here, we report the partial sequencing of two mitochondrial genes: 128 bp of 12S rDNA and 561 bp of cytochrome b for the Lyakhov mammoth, a 49,000-year-old Siberian individual. We use the most comprehensive sample of mammoth (11 sequences) to determine whether the sequences achieved by former studies were congruent or not. The monophyly of a major subset of mammoths sequences (including ours) is recovered. Such a result is assumed to be a good criterion for ascertaining the origin of ancient DNA. Our sequence is incongruent with that of Yang et al. (1996), though obtained for the same individual. As far as the latter sequence is concerned, a contamination by non-identified exogenous DNA is suspected. The robustness and reliability of the sister group relation between Mammuthus primigenius and Loxodonta africana are examined: down-weighting saturated substitutions has no impact on the topology; analyzing data partitions proves that the support of this clade can be assigned to the most conservative phylogenetic signal; insufficient taxonomic and/or characters sampling contributed to former discordant conclusions. We therefore assume the monophyly of "real mammoth sequences" and the (Mammuthus, Loxodonta) clade.

Complete mitochondrial DNA of the hagfish, Eptatretus burgeri: the comparative analysis of mitochondrial DNA sequences strongly supports the cyclostome monophyly.

The phylogenetic position of cyclostomes, i.e., the relationships between hagfishes, lampreys, and jawed vertebrates is an unresolved problem. Anatomical data support the paraphyly of cyclostomes, whereas nuclear genes data support monophyly of cyclostomes. Previous results obtained using mitochondrial DNA are ambiguous, presumably due to a lack of informative sequences. By adding the complete mtDNA of a hagfish, Eptatretus burgeri, we have generated a novel data set for sequences of hagfishes and of lampreys. The addition of this mtDNA sequence to the 12 taxa we have already used becomes sufficient to obtain unambiguous results. This data set, which includes sequences of mtDNA of animals closely related to the lamprey/hagfish node, was used in a phylogenetic analysis with two independent statistical approaches and unequivocally supported the monophyly of cyclostomes. Thus molecular data, i.e., our results and those obtained using nuclear genes, conclude that hagfishes and lampreys form a clade.

Cladistic coding of genomic maps.

A new method of genomic maps analysis is described. The purpose of the method is to reconstruct phylogenetic relationships from the genomic organization of taxa. Our approach is based on gene order coding. This coding allows the description of genome topology without a prior hypothesis about evolutionary events and phylogenetic relationships. Different characters are used for each gene: (1) presence/absence, (2) orientation, and (3) relative position. The relative position of a particular gene inside the genome is the pair of genes surrounding it. The relative position character represents all the positions of a gene in the sampled genomes. It is coded as a multistate character. Our coding method has a priori variable cost implications on operators such as inversion, transposition, and gene loss/gain, which we discuss. The overall approach best fits the "duplication, random loss" evolutionary model. The coding method allows the reconstitution of a possible hypothetical common ancestor genome at each node of the tree. This reconstitution is based on the character states' optimization; it comes down to choosing, among all possible optimizations, the optimization compatible with a complete genome topology at each internal node. The multistate coding of gene relative position, which is an undeniable advantage of this method, permits this reconstitution.

Rooting with Multiple Outgroups: Consensus Versus Parsimony.

Using outgroup(s) is the most frequent method to root trees. Rooting through unconstrained simultaneous analysis of several outgroups is a favoured option because it serves as a test of the supposed monophyly of the ingroup. When contradiction occurs among the characters of the outgroups, the branching pattern of basal nodes of the rooted tree is dependent on the order of the outgroups listed in the data matrix, that is, on the prime outgroup (even in the case of exhaustive search). Different equally parsimonious rooted trees (=cladograms) can be obtained by permutation of prime outgroups. An alternative to a common implicit practice (select one outgroup to orientate the tree) is that the accepted cladogram is the strict consensus of the different equally parsimonious rooted trees. The consensus tree is less parsimonious but is not hampered with extra assumption such as the choice of one outgroup (or more) among the initial number of outgroup terminals. It also does not show sister-group relations that are ambiguously resolved or not resolved at all.