The evolution of armadillos, anteaters and sloths depicted by nuclear and mitochondrial phylogenies: implications for the status of the enigmatic fossil Eurotamandua.

The mammalian order Xenarthra (armadillos, anteaters and sloths) is one of the four major clades of placentals, but it remains poorly studied from the molecular phylogenetics perspective. We present here a study encompassing most of the order's diversity in order to establish xenarthrans' intra-ordinal relationships, discuss the evolution of their morphological characters, search for their extant sister group and specify the timing of their radiation with special emphasis on the status of the controversial fossil Eurotamandua. Sequences of three genes (nuclear exon 28 of the Von Willebrand factor and mitochondrial 12S and 16S rRNAs) are compared for eight of the 13 living genera. Phylogenetic analyses confirm the order's monophyly and that of its three major lineages: armadillos (Cingulata), anteaters (Vermilingua) and sloths ('Tardigrada', renamed in 'Folivora'), and our results strongly support the grouping of hairy xenarthrans (anteaters and sloths) into Pilosa. Within placentals, Afrotheria might be the first lineage to branch off, followed by Xenarthra. The morphological adaptative convergence between New World xenarthrans and Old World pangolins is confirmed. Molecular datings place the early emergence of armadillos around the Cretaceous/Tertiary boundary, followed by the divergence between anteaters and sloths in the Early Eocene era. These Tertiary dates contradict the concept of a very ancient origin of modern xenarthran lineages. They also question the placement of the purported fossil anteater (Eurotamandua) from the Middle Eocene period of Europe with the Vermilingua and instead suggest the independent and convergent evolution of this enigmatic taxon.

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 63 Myr 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.

Armadillos exhibit less genetic polymorphism in North America than in South America: nuclear and mitochondrial data confirm a founder effect in Dasypus novemcinctus (Xenarthra).

Heterozygosity at eight nuclear enzymatic loci and mitochondrial DNA control region (D-loop) sequence polymorphism was compared between North and South American nine-banded armadillos (Dasypus novemcinctus: Xenarthra, Dasypodidae). All markers revealed a striking genetic homogeneity amongst Texas, Louisiana, and Mississippi individuals, vs. the usual level of polymorphism for the French Guiana population. This may reflect a founder effect during colonization of North America. Occurrence of polymorphism in the D-loop microsatellite motif of North American armadillos suggests a recent recovery of mitochondrial variability. Phylogeographic analyses using Dasypus kappleri as outgroup provides evidence for a clear separation between North and South American control region haplotypes.

The phylogenetic position of "Acomyinae" (Rodentia, Mammalia) as sister group of a Murinae + Gerbillinae clade: evidence from the nuclear ribonuclease gene.

The phylogenetic relationships of Acomys and Uranomys within Muridae were investigated using nuclear pancreatic ribonuclease A gene sequences. The various kinds of substitutions in the data matrix (15 taxa x 375 nucleotides) were examined for saturation, in order to apply a weighted parsimony approach. Phylogenies were derived by maximum parsimony (weighted and unweighted) and maximum likelihood procedures, using a dormouse (Gliridae) as outgroup. Maximum likelihood gave the most robust results. All analyses cluster some traditional taxa with a strong robustness, such as three species of the genus Mus, two South-East Asian rats, and two genera in each of the gerbil and vole families. When analyzed with those of other murid rodents representing Murinae, Gerbillinae, Arvicolinae, Cricetinae, and Sigmodontinae, sequences of the ribonuclease gene suggest that Acomys and Uranomys constitute a monophyletic clade at the subfamily level, denoted "Acomyinae." The relationships between the six subfamilies of Muridae appear poorly resolved, except for a clade uniting Murinae, Acomyinae, and Gerbillinae. Within this clade, the sister group of Acomyinae could not be identified, as the branch length defining a Gerbillinae + Murinae cluster is extremely short. The poor resolution of our phylogenetic inferences is probably the result of two confounding factors, namely the limited size of the pancreatic ribonuclease sequence and the probable short time intervals during the radiation of the six murid subfamilies involved in this study.

Molecular evolution of the nuclear von Willebrand factor gene in mammals and the phylogeny of rodents.

Nucleotide sequences of exon 28 of the von Willebrand Factor (vWF) were analyzed for a representative sampling of rodent families and eutherian orders, with one marsupial sequence as outgroup. The aim of this study was to test if inclusion of an increased taxonomic diversity in molecular analyses would shed light on three uncertainties concerning rodent phylogeny: (1) relationships between rodent families, (2) Rodentia monophyly, and (3) the sister group relationship of rodents and lagomorphs. The results did not give evidence of any particular rodent pattern of molecular evolution relative to a general eutherian pattern. Base compositions and rates of evolution of vWF sequences of rodents were in the range of placental variation. The 10 rodent families studied here cluster in five clades: Hystricognathi, Sciuridae and Aplodontidae (Sciuroidea), Muridae, Dipodidae, and Gliridae. Among hystricognaths, the following conclusions are drawn: a single colonization event in South America by Caviomorpha, a paraphyly of Old World and New World porcupines, and an African origin for Old World porcupines. Despite a broader taxonomic sampling diversity, we did not obtain a robust answer to the question of Rodentia monophyly, but in the absence of any other alternative, we cannot reject the hypothesis of a single origin of rodents. Moreover, the phylogenetic position of Lagomorpha remains totally unsettled.

Phylogenetic relationships of artiodactyls and cetaceans as deduced from the comparison of cytochrome b and 12S rRNA mitochondrial sequences.

A data set of complete mitochondrial cytochrome b and 12S rDNA sequences is presented here for 17 representatives of Artiodactyla and Cetacea, together with potential outgroups (two Perissodactyla, two Carnivora, two Tethytheria, four Rodentia, and two Marsupialia). We include seven sequences not previously published from Hippopotamidae (Ancodonta) and Camelidae (Tylopoda), yielding a total of nearly 2.1 kb for both genes combined. Distance and parsimony analyses of each gene indicate that 11 clades are well supported, including the artiodactyl taxa Pecora, Ruminantia (with low 12S rRNA support), Tylopoda, Suina, and Ancodonta, as well as Cetacea, Perissodactyla, Carnivora, Tethytheria, Muridae, and Caviomorpha. Neither the cytochrome b nor the 12S rDNA genes resolve the relationships between these major clades. The combined analysis of the two genes suggests a monophyletic Cetacea +Artiodactyla clade (defined as "Cetartiodactyla"), whereas Perissodactyla, Carnivora, and Tethytheria fall outside this clade. Perissodactyla could represent the sister taxon of Cetartiodactyla, as deduced from resampling studies among outgroup lineages. Cetartiodactyla includes five major lineages: Ruminantia, Tylopoda, Suina, Ancodonta, and Cetacea, among which the phylogenetic relationships are not resolved. Thus, Suiformes do not appear to be monophyletic, justifying their split into the Suina and Ancodonta infraorders. An association between Cetacea and Hippopotamidae is supported by the cytochrome b gene but not by the 12S rRNA gene. Calculation of divergence dates suggests that the Cetartiodactyla could have diverged from other Ferungulata about 60 MYA.

Genome sizes of the entomopathogenic nematodes Steinernema carpocapsae and Heterorhabditis bacteriophora (Nematoda:Rhabditida).

There is currently no information on the genome size and complexity in the entomoparasitic nematodes Steinernema and Heterorhabditis. DNA reassociation kinetics were used to determine the genome size and complexity in 2 species: Steinernema carpocapsae and Heterorhabditis bacteriophora. C0t curves derived from renaturation kinetics of denatured DNA indicate that the genomes of these entomoparasitic rhabditids are different both in size and complexity. Genome sizes were estimated at 2.3 x 10(8) bp for S. carpocapsae and 3.9 x 10(7) bp for H. bacteriophora and repetitive DNA contents were found to represent 39% and 51% of these respective genomes.

Molecular relationships between closely related species of Bothriocephalus (Cestoda:Platyhelminthes).

DNA/DNA hybridization has been carried out to establish the evolutionary relationships among several host-specific bothriocephalid tapeworms species (Cestoda: Platyhelminthes). Comparative anatomy is not informative for deciphering the relationships among these sibling morphological taxa, and clearcut genetic differences have previously identified seven biological species which are each highly specific to a single host. We show that two species (Bothriocephalus gregarius and B. renaudii) infesting the same host (turbot: Psetta maxima) in two different geographic areas are not sister-taxa. Moreover, a strong decrease in the amount of DNA change is observed in one species of tapeworm, documenting a marked heterogeneity of rates of nucleotidic substitution among these very closely related organisms. Based on these observations, different hypotheses are developed for understanding the evolutionary history of this assemblage of parasites, suggesting that host-switching has played an important role in the recent past.

Interordinal mammalian relationships: evidence for paenungulate monophyly is provided by complete mitochondrial 12S rRNA sequences.

The complete mitochondrial 12S rRNA sequences of 5 placental mammals belonging to the 3 orders Sirenia, Proboscidea, and Hyracoidea are reported together with phylogenetic analyses (distance and parsimony) of a total of 51 mammalian orthologues. This 12S rRNA database now includes the 2 extant proboscideans (the African and Asiatic elephants Loxodonta africana and Elephas maximus), 2 of the 3 extant sirenian genera (the sea cow Dugong dugon and the West Indian manatee Trichechus manatus), and 2 of the 3 extant hyracoid genera (the rock and tree hyraxes Procavia capensis and Dendrohyrax dorsalis). The monophyly of the 3 orders Sirenia, Proboscidea, and Hyracoidea is supported by all kinds of analysis. There are 23 and 3 diagnostic subsitutions shared by the 2 proboscideans and the 2 hyracoids, respectively, but none by the 2 sirenians. The 2 proboscideans exhibit the fastest rates of 12S rRNA evolution among the 11 placental orders studied. Based on various taxonomic sampling methods among eutherian orders and marsupial outgroups, the most strongly supported clade in our comparisons clusters together the 3 orders Sirenia, Proboscidea, and Hyracoidea in the superorder Paenungulata. Within paenungulates, the grouping of sirenians and proboscideans within the mirorder Tethytheria is observed. This branching pattern is supported by all analyses by high bootstrap percentages (BPs) and decay indices. When only one species is selected per order or suborder, the taxonomic sampling leads to a relative variation in bootstrap support of 53% for Tethytheria (BPs ranging from 44 to 93%) and 7% for Paernungulata (92-99%). When each order or suborder is represented by two species, this relative variation decreased to 10% for Tethytheria (78-87%) and 3% for Paenungulata (96-99%). Two nearly exclusive synapomorphies for paenungulates are identified in the form of one transitional compensatory change, but none were detected for tethytherians. Such a robust and reliable resolution of the paenungulate node implies a long history of the common ancestors, allowing time for synapomorphies to accumulate. This observation suggests a Late Cretaceous/Early Paleocene origin for the Paenungulata.

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 Grevy's 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 the suborder Ruminantia was not supported and the branching pattern between Cetacea and the artiodacytyl 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.

L1 (LINE-1) retrotransposable elements provide a "fossil" record of the phylogenetic history of murid rodents.

The single most difficult problem in phylogenetic analysis is deciding whether a shared taxonomic character is due to common ancestry or one that appeared independently due to convergence, parallelism, or reversion to an ancestral state. Mammalian L1 retrotransposons undergo periodic amplifications in which multiple copies of the elements are interspersed in the genome. Because these elements apparently are transmitted only by inheritance and are retained in the genome, a shared L1 amplification event can only be an inherited ancestral character. We propose that L1 amplification events can be an excellent tool for analyzing mammalian evolution and demonstrate here how we addressed several refractory problems in rodent systematics using L1 DNA as a taxonomic character.

Molecular evidence that the spiny mouse (Acomys) is more closely related to gerbils (Gerbillinae) than to true mice (Murinae).

Spiny mice of the genus Acomys traditionally have been classified as members of the Murinae, a subfamily of rodents that also includes rats and mice with which spiny mice share a complex set of morphological characters, including a unique molar pattern. The origin and evolution of this molar pattern, documented by many fossils from Southern Asia, support the hypothesis of the monophyly of Acomys and all other Murinae. This view has been challenged by immunological studies that have suggested that Acomys is as distantly related to mice (Mus) as are other subfamilies (e.g., hamsters: Cricetinae) of the muroid rodents. We present molecular evidence derived from DNA.DNA hybridization data that indicate that the spiny mouse Acomys and two African genera of Murinae, Uranomys and Lophuromys, constitute a monophyletic clade, a view that was recently suggested on the basis of dental characters. However, our DNA.DNA hybridization data also indicate that the spiny mice (Acomys) are more closely related to gerbils (Gerbillinae) than to the true mice and rats (Murinae) with which they have been classified. Because Acomys and the brush-furred mice Uranomys and Lophuromys share no derived morphological characters with the Gerbillinae, their murine morphology must have evolved by convergence, including the molar pattern previously considered to support the monophyly of the Murinae.

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.

DNA reassociation kinetics and genome complexity of a fish (Psetta maxima: Teleostei) and its gut parasite (Bothriocephalus gregarius: Cestoda).

1. The genomic structure of a fish (Psetta maxima) and of a Tapeworm (Bothriocephalus), who form a close host-parasite association, was determined by reassociation kinetics experiments. 2. Spectrophotometric readings of single-stranded versus double-stranded DNA separated on hydroxylapatite columns after reassociation at Cot values ranging from 0.0001 to 10(5) allowed the drawing of the reassociation curves of both genomes. 3. Different fractions according to their degree of repetitivity were evidenced, and the relative amounts of repetitive versus single-copy sequences, as well as their complexity, were calculated. 4. It appears that the amount of non-repetitive DNA is lower in the Tapeworm than in its vertebrate host, although the complexity of these single-copy sequences is the same.

DNA hybridization as a guide to phylogenies: raw data in muroid rodents.

A strong criticism of raw data analysis in DNA/DNA hybridization studies (Marks et al., 1988; Sarich et al., 1989) states that a widely used statistic, T50H, can in some cases lead to spurious conclusions, as exemplified by some hominoid and bird DNA/DNA results found in Sibley and Ahlquist (1987a, b). To test this criticism and compare the phylogenetic information deduced from several alternative methods of analysis, I have re-analysed the melting curves of more than 150 hybrid comparisons among muroid rodents, including almost all of my previously published data (Catzeflis et al. 1987). Four major statistics have been compared: Mode, Tm, T50H and NPH (for definitions of terms: see Sibley and Ahlquist 1981). Although the T50H statistic was found to be more variable when compared to Mode and Tm, confirming the claims of Sarich et al. (1989), it nevertheless appeared to be, under certain technical conditions, the method of choice for analyzing distantly related hybrids. The Mode and Tm appeared to be the best indexes of genomic dissimilarity for analyzing more similar genomes. The use of Tm is not very useful for comparing dissimilar genomes, as its range of efficient use was shown to be much more limited towards the lower temperatures (i.e., -larger delta-values) than for the Mode. Mode was found to be the statistic of choice because of i) its large range of application (up to delta-Mode values of 18-20 degrees C), ii) its reproducibility, and iii) its consistency between experiments and in reciprocal comparisons. For the data set on which our earlier conclusions concerning the very rapid rate of DNA change in muroid rodents (Catzeflis et al., 1987) were based, it appears that Mode, Tm and T50H do yield similar conclusions as far as the evolutionary tree of the muroid rodents is concerned. The fourth index, which is the Normalized Percent of Hybridization, appears to be strongly affected by experimental conditions, and should be used with caution.