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Molecular evidence from retroposons that whales form a clade within even-toed ungulates.

The origin of whales and their transition from terrestrial life to a fully aquatic existence has been studied in depth. Palaeontological, morphological and molecular studies suggest that the order Cetacea (whales, dolphins and porpoises) is more closely related to the order Artiodactyla (even-toed ungulates, including cows, camels and pigs) than to other ungulate orders. The traditional view that the order Artiodactyla is monophyletic has been challenged by molecular analyses of variations in mitochondrial and nuclear DNA. We have characterized two families of short interspersed elements (SINEs) that were present exclusively in the genomes of whales, ruminants and hippopotamuses, but not in those of camels and pigs. We made an extensive survey of retropositional events that might have occurred during the divergence of whales and even-toed ungulates. We have characterized nine retropositional events of a SINE unit, each of which provides phylogenetic resolution of the relationships among whales, ruminants, hippopotamuses and pigs. Our data provide evidence that whales, ruminants and hippopotamuses form a monophyletic group.

Animals↗

Biodiversity of multiple Pregnancy-Associated Glycoprotein (PAG) family: gene cloning and chorionic protein purification in domestic and wild eutherians (Placentalia)--a review.

This review presents a broad overview of chorionic glycoproteins encoded by the Pregnancy-Associated Glycoprotein (PAG) gene family and also serves to illustrate how the recent discovery of the PAG family has contributed to our general knowledge of genome evolution, placental transcription and placental protein expression. The complex and large PAG family is restricted to the Artiodactyla order, although single PAG-like genes have also been identified in species outside the Artiodactyla. The PAGs are members of the aspartic proteinase (AP) superfamily. Unexpectedly, however, some members of the PAG family possess amino acid substitutions within and around the active site that likely render them unable to act as proteinases. This paper summarises the available information regarding biodiversity of PAG gene expression based on cDNA cloning, mRNA localisation studies and the structural organisation of the PAG genes with a particular emphasis on PAG promoters. It also compares available data regarding PAG protein purifications, sequencing and their N-glycodiversity. Finally, it discusses the scientific relevance, possible functional roles of the PAGs and describes possible profitable applications related to the detection of PAG proteins in the blood of pregnant domestic and wild species.

Animals↗

The diversity and evolutionary relationships of the pregnancy-associated glycoproteins, an aspartic proteinase subfamily consisting of many trophoblast-expressed genes.

The pregnancy-associated glycoproteins (PAGs) are structurally related to the pepsins, thought to be restricted to the hooved (ungulate) mammals and characterized by being expressed specifically in the outer epithelial cell layer (chorion/trophectoderm) of the placenta. At least some PAGs are catalytically inactive as proteinases, although each appears to possess a cleft capable of binding peptides. By cloning expressed genes from ovine and bovine placental cDNA libraries, by Southern genomic blotting, by screening genomic libraries, and by using PCR to amplify portions of PAG genes from genomic DNA, we estimate that cattle, sheep, and most probably all ruminant Artiodactyla possess many, possibly 100 or more, PAG genes, many of which are placentally expressed. The PAGs are highly diverse in sequence, with regions of hypervariability confined largely to surface-exposed loops. Nonsynonymous (replacement) mutations in the regions of the genes coding for these hypervariable loop segments have accumulated at a higher rate than synonymous (silent) mutations. Construction of distance phylograms, based on comparisons of PAG and related aspartic proteinase amino acid sequences, suggests that much diversification of the PAG genes occurred after the divergence of the Artiodactyla and Perissodactyla, but that at least one gene is represented outside the hooved species. The results also suggest that positive selection of duplicated genes has acted to provide considerable functional diversity among the PAGs, whose presence at the interface between the placenta and endometrium and in the maternal circulation indicates involvement in fetal-maternal interactions.

Amino Acid Sequence↗

Phylogenetic assessment of molecular and morphological data for eutherian mammals.

The interordinal relationships of eutherian (placental) mammals were evaluated by a phylogenetic analysis of four published data sets (three sequences and one morphological). The nature and degree of support and conflict for particular groups were assessed by separate bootstrap and homogeneity tests, which were followed by combined analyses of the sequence and morphological data. Between orders, strong support (i.e., > or = 95% bootstrap scores) was found for a paraphyletic Artiodactyla (relative to Cetacea) and a monophyletic Cetartiodactyla (Artiodactyla and Cetacea) and Paenungulata (Hyracoidea, Proboscidea, and Sirenia). In turn, some reasonable to strong evidence (> or = 85%) was obtained for Hyracoidea with Sirenia, Dermoptera with Scandentia, Glires (Lagomorpha with Rodentia), and Afrotheria (Amblysomus, Macroscelidea, Paenungulata, and Tubulidentata). Otherwise, no other interordinal clades were supported at these reasonable to strong levels. This overall lack of resolution for eutherian interordinal clusters agrees with other studies that suggest further progress will continue to be slow and difficult. Further resolution will require the integration of more recently published data, the continued sampling of taxa and characters, and the use of more powerful methods of data analysis.

Animals↗

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.

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Analyses of mitochondrial genomes strongly support a hippopotamus-whale clade.

Although the sister-group relationship between Cetacea and Artiodactyla is widely accepted, the actual artiodactyl group which is closest to Cetacea has not been conclusively identified. In the present study, we have sequenced the complete mitochondrial genome of the hippopotamus, Hippopotamus amphibius, and included it in phylogenetic analyses together with 15 other placental mammals. These analyses separated the hippopotamus from the other suiform included, the pig, and identified the hippopotamus as the artiodactyl sister group of the cetaceans, thereby making both. Artiodactyla and the suborder. Suiformes paraphyletic. The divergence between the hippopotamid and cetacean lineages was calculated using this molecular data and was estimated at ca. 54 Ma BP.

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Mining the mammalian genome for artiodactyl systematics.

A total of 7,806 nucleotide positions derived from one mitochondrial and eight nuclear DNA segments were used to provide a robust phylogeny for members of the order Artiodactyla. Twenty-four artiodactyl and two cetacean species were included, and the horse (order Perissodactyla) was used as the outgroup. Limited rate heterogeneity was observed among the nuclear genes. The partition homogeneity tests indicated no conflicting signal among the nuclear genes fragments, so the sequence data were analyzed together and as separate loci. Analyses based on the individual nuclear DNA fragments and on 34 unique indels all produced phylogenies largely congruent with the topology from the combined data set. In sharp contrast to the nuclear DNA data, the mtDNA cytochrome b sequence data showed high levels of homoplasy, failed to produce a robust phylogeny, and were remarkably sensitive to taxon sampling. The nuclear DNA data clearly support the paraphyletic nature of the Artiodactyla. Additionally, the family Suidae is diphyletic, and the nonruminating pigs and peccaries (Suiformes) were the most basal cetartiodactyl group. The morphologically derived Ruminantia was always monophyletic; within this group, all taxa with paired bony structures on their skulls clustered together. The nuclear DNA data suggest that the Antilocaprinae account for a unique evolutionary lineage, the Cervidae and Bovidae are sister taxa, and the Giraffidae are more primitive.

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[Morphological and histological study of neocortex of bovides (Antilopinae, Cephalophinae) and Tragulidae with comments on evolutionary development].

1. Macroscopical and histological studies on the neocortex of the cerebrum of small Artiodactyla (Tragulidae, Cephalophinae, Antilopinae) lead to some informations on the level of evolution of these animals. 2. A big part of the macroscopical investigations (pattern of sulci, relations between bodyweight and brainweight on the one hand and surface of neocortex on the other hand) already had been realized by HAARMANN and OBOUSSIER (1972). Therefore in this paper only the results for the Tragulidae and Cephalophus sylvicultor are added. The first are relatively primitive, while the latter obviously is a higher developed animal. C. sylvicultor fits into the general scheme of the brain of the Cephalophinae. 3. The investigations are complemented by microscopical investigations (histology of the neocortex: number of nerve cells per volume unit, gray cell and cortex coefficient, thickness of the neocortex). The knowledge of the evolution level obtained by macroscopical methods is confirmed. The Tragulidae are clearly separated from the other Artiodactyla as primitive animals, whereas the Cephalophinae measured with the cortex coefficient are the most developed, most "intelligent" Bovidae so far analyzed in this paper. The Antilopinae reach only lower coefficients. This subfamily may be separated into three groups (Antilopini, Neotragini, Oreotragus). The differences between the Antilopinae and the Cephalophinae are not so striking as between these Bovidae and the Tragulidae.

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The paleoenvironment of Sivapithecus parvada.

Remains of the hominoid Sivapithecus parvada and a diversity of mammalian taxa are preserved at locality Y311 (ca. 10 Ma) in the Siwalik Nagri Formation of northern Pakistan. Bovids (Bovidae, Artiodactyla) are the most abundant mammals next to tragulids (Tragulidae, Artiodactyla) at locality Y311 and provide a means for reconstructing the paleoenvironments that would have been available to Sivapithecus parvada. A functional model indicates a linkage between habitat and several femoral characters among extant bovids. Based on this model, we infer that forested habitats predominated at locality Y311 but that some less densely covered areas may also have been present. Paleoenvironments in the earlier Chinji Formation appear comparable to those at locality Y311, although the presence of a continuous canopy in the former is more certain. Thus, adaptive changes in the bovid fauna from the Chinji through the Nagri Formations appear to have preceded the shift to predominantly C4 grasslands which, based on other lines of evidence, occurred locally (and possibly globally) between 8 and 6 Ma. The paleoenvironments of locality Y311 and the Chinji Formation localities appear different from the paleoenvironment of Kenyapithecus at Fort Ternan in Kenya, where the presence of continuous canopy is unlikely. The Fort Ternan fauna is dominated by two genera of bovids. One of these is adapted to light cover while the other appears better adapted to heavy cover. Sivapithecus and Kenyapithecus lived in different ecological settings probably characterized by varying degrees of vegetative cover.

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Further report on Cryptosporidium in Barcelona zoo mammals.

The prevalence of fecal shedding of Cryptosporidium in 36 primates (21 species and subspecies) and 62 herbivores (36 species and subspecies) housed at the Barcelona zoo was studied. Cryptosporidial oocysts were found in stool samples of 14 Primate, 18 Artiodactyla, 2 Perissodactyla, and 1 Proboscidea species. None of them showed symptoms related to the parasite. Neither the sex nor the group condition (alone or in a group) of the animals studied appeared to be correlated with parasitic prevalence. The results extend the host species range of the protozoan to 18 new animals (6 Primate, 10 Artiodactyla, 1 Perissodactyla, and 1 Proboscidea species) and confirm the endemic status of Cryptosporidium at the Barcelona zoo. We conclude that maintenance of the parasitic endemic status is probably due to the presence of animal carriers as well as to the physical features of some facilities where oocysts could remain viable and infectious.

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Identification of fetuin in human and rat fetuses and in other species.

The fetal protein, fetuin, has previously only been identified in species belonging to the order Artiodactyla. Samples of fetal, newborn and adult human (Homo sapiens) and rat (Rattus norvegicus) plasma and tissues have been studied using three techniques: (a) crossed immunoelectrophoresis of plasma against each of four different anti-fetuin antisera (two anti-cattle, one anti-pig and one anti-sheep); (b) the peroxidase-antiperoxidase technique applied to agarose gels containing plasma spots; (c) the indirect immunoperoxidase technique applied to human fetal tissue sections. In human fetal samples all three methods gave evidence for the presence of fetuin except late in gestation and in the newborn. Adult plasma was negative. In rat fetuses only plasma was tested, by methods (a) and (b). Positive reactions were obtained for both fetal and adult samples; the fetal samples cross-reacted with several of the anti-fetuins, adult samples reacted with only one. All the fetal and embryonal plasma samples tested with the peroxidase-antiperoxidase method were positive for fetuin except for the chicken. Thus fetuin appears to be distributed in at least five mammalian orders (Artiodactyla, Primates, Rodentia, Carnivora and Perissodactyla).

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Mitogenomic analyses provide new insights into cetacean origin and evolution.

The evolution of the order Cetacea (whales, dolphins, porpoises) has, for a long time, attracted the attention of evolutionary biologists. Here we examine cetacean phylogenetic relationships on the basis of analyses of complete mitochondrial genomes that represent all extant cetacean families. The results suggest that the ancestors of recent cetaceans had an explosive evolutionary radiation 30-35 million years before present. During this period, extant cetaceans divided into the two primary groups, Mysticeti (baleen whales) and Odontoceti (toothed whales). Soon after this basal split, the Odontoceti diverged into the four extant lineages, sperm whales, beaked whales, Indian river dolphins and delphinoids (iniid river dolphins, narwhals/belugas, porpoises and true dolphins). The current data set has allowed test of two recent morphological hypotheses on cetacean origin. One of these hypotheses posits that Artiodactyla and Cetacea originated from the extinct group Mesonychia, and the other that Mesonychia/Cetacea constitutes a sister group to Artiodactyla. The current results are inconsistent with both these hypotheses. The findings suggest that the claimed morphological similarities between Mesonychia and Cetacea are the result of evolutionary convergence rather than common ancestry.

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Cross reactivities among some mammalian haptoglobins studied by a monoclonal antibody.

Mouse monoclonal antibody antihuman-Hp, product of clone 2.36.71.41 was found to recognize, however, with different affinities, the immunological determinant on haptoglobin of some mammals (goat, sheep, cow, horse, rabbit). The following differences in immunological cross reactivities were noticed: (i) haptoglobins present in sera of goat, sheep and cow (Artiodactyla, Bovidae) react in enzyme-linked immunosorbent assay (ELISA) and form precipitates in agarose gel; (ii) horse (Perissodactyla) and rabbit (Lagomorpha) haptoglobins react in ELISA, but they do not form precipitates; and (iii) haptoglobins of dog, fox, cat (Carnivora) and pig (Artiodactyla, Suidae) are not recognized by the tested monoclonal antibody either in ELISA or in agarose gel. This study suggests that monoclonal antibody, clone 2.36.71.41, recognizes the structure on haptoglobin around the disulphide bond linking two alpha chains. Antigenic structure of Bovidae haptoglobins is rather similar to human haptoglobin 2-2 (circular polymers) than to 2-1 type (linear polymers). Monoclonal antibody 2.36.71.41 could be used for classification of mammalian haptoglobins by epitope structure. It can distinguish polymeric haptoglobins similar to human type 2-2 from other mammalian haptoglobins.

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Cattle breed-variation in infestation by the horn fly Haematobia irritans.

A study was carried out to assess the resistance of pure and cross-bred groups of cattle to the horn fly Haematobia irritans (Linnaeus) (Diptera: Muscidae) in northern Argentina. Pure-bred cattle were Criolla, Iberian Bos taurus Linnaeus (Artiodactyla: Bovidae) and Nellore, Bos indicus Linnaeus (Artiodactyla: Bovidae). Cross-bred cattle were Hereford, British B. taurus (34%) X Nellore (66%) and Hereford (66%) X Nellore (34%). All were heifers and animals were maintained in two groups, each containing a mixture of pure and cross-breeds. The lowest fly numbers were found on Criolla heifers and the highest on Hereford X Nellore cross-breeds. However, it could not be determined from this study whether this was a consequence of breed and/or size, as Criolla heifers were lighter than the corresponding Hereford X Nellore heifers. Fly numbers on the heifers followed an approximately negative binomial distribution. However, the ranking of individual animals in their level of infestation within subgroups was not consistent. Hence, culling the most infested heifers on any given date would at best give only a small improvement in H. irritans control.

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Role of small mammals in the persistence of Louping-ill virus: field survey and tick co-feeding studies.

Louping-ill (LI) is a tick-borne viral disease of red grouse, Lagopus lagopus scoticus Lath. (Tetraonidae: Galliformes), and sheep, Ovis aries L. (Bovidae: Artiodactyla), that causes economic loss to upland farms and sporting estates. Unvaccinated sheep, grouse and mountain hares, Lepus timidus L. (Leporidae: Lagomorpha), are known to transmit LI virus, whereas red deer, Cenrus elaphus L. (Cervidae: Artiodactyla), and rabbits, Oryctolagus cuniculus L. (Leporidae: Lagomorpha), do not. However, the role of small mammals is unknown. Here, we determine the role of small mammals, in particular field voles, Microtus agrestis L. (Muridae: Rodentia), in the persistence of LI virus on upland farms and sporting estates in Scotland, using field sampling and non-viraemic transmission trials. Small mammals were not abundant on the upland sites studied, few ticks were found per animal and none of the caught animals tested seropositive to LI virus. Laboratory trials provided no evidence that small mammals (field voles, bank voles, Clethrionomys glareolus L. (Muridae: Rodentia), and wood mice, Apodemus sylvaticus L. (Muridae: Rodentia), can transmit LI virus between cofeeding ticks and, in the field, LI virus was prevalent only in areas with known LI virus competent hosts (grouse, mountain hares or unvaccinated sheep) and absent elsewhere. In contrast to the case of tick-borne encephalitis (TBE) virus in Europe, it is concluded that small mammals seem to be relatively unimportant in LI virus persistence.

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Maximum likelihood analysis of gene-based and structure-based process partitions, using mammalian mitochondrial genomes.

Aligned protein-coding genes from 19 completely sequenced mammalian mitochondrial genomes were examined by parsimony and maximum likelihood analyses. Particular attention is given to a comparison between gene-based and structure-based data partitions. Because actual structures are not known for most of the mitochondrially encoded proteins, three different surrogate partitioning schemes were examined, each based on the identity of the consensus amino acid at a specific homologous position. One of the amino-acid-based partitioning schemes gave the highest likelihood, but that scheme was based on concordance with a well-corroborated phylogeny from an earlier parsimony analysis. The gene-based partitioning scheme gave a significantly higher likelihood compared to the only structure-based scheme examined that could be generated without prior assumptions about the phylogeny. Two contrasting phylogenetic inferences were supported by the analyses. Both unpartitioned analyses and analyses in which all partitions were constrained to have identical patterns of branch lengths supported ((Artiodactyla, Cetacea) (Perissodactyla, Carnivora)), whereas all analyses with that constraint relaxed supported (((Artiodactyla, Cetacea) Carnivora) Perissodactyla).

Amino Acid Sequence↗

Identification of a new aspartic proteinase expressed by the outer chorionic cell layer of the equine placenta.

The pregnancy-associated glycoproteins (PAGs) are placental antigens that were initially characterized as pregnancy markers in the maternal circulation of domestic ruminant species. They are members of the aspartic proteinase gene family, having greatest sequence identity with pepsinogens. However, some are not capable of functioning as enzymes. The PAGs are associated with a large gene family within the Artiodactyla order (cattle, camels, pigs). So far, no members of this family have been characterized in species outside this order. This report describes the cloning and initial characterization of a PAG-like protein (equine PAG or ePAG) expressed in the placenta of the horse and zebra (order Perrisodactyla). Equine PAG is a proteinase capable of degrading 14C-hemoglobin and catalyzing the removal of its own pro-peptide. The ePAG mRNA is restricted to the chorion both prior to implantation and in the term placenta. Equine PAG is secreted from cultured placental tissue as both a processed (mature) and unprocessed (zymogen) form. Equine PAG shares similar identity with the PAGs and pepsinogens and probably arose from a pepsinogen-like precursor that gained the ability to be expressed in the placenta. The promoter of the ePAG gene shares sequence identity with the promoter from a bovine PAG gene but not with promoters of other aspartic proteinases. Therefore, we hypothesize that ePAG is a remnant of the pepsinogen-like progenitor gene that was expanded within the Artiodactyla to create the large and highly diverse PAG family.

Amino Acid Sequence↗

A higher-level MRP supertree of placental mammals.

BACKGROUND: The higher-level phylogeny of placental mammals has long been a phylogenetic Gordian knot, with disagreement about both the precise contents of, and relationships between, the extant orders. A recent MRP supertree that favoured 'outdated' hypotheses (notably, monophyly of both Artiodactyla and Lipotyphla) has been heavily criticised for including low-quality and redundant data. We apply a stringent data selection protocol designed to minimise these problems to a much-expanded data set of morphological, molecular and combined source trees, to produce a supertree that includes every family of extant placental mammals. RESULTS: The supertree is well-resolved and supports both polyphyly of Lipotyphla and paraphyly of Artiodactyla with respect to Cetacea. The existence of four 'superorders'--Afrotheria, Xenarthra, Laurasiatheria and Euarchontoglires--is also supported. The topology is highly congruent with recent (molecular) phylogenetic analyses of placental mammals, but is considerably more comprehensive, being the first phylogeny to include all 113 extant families without making a priori assumptions of suprafamilial monophyly. Subsidiary analyses reveal that the data selection protocol played a key role in the major changes relative to a previously published higher-level supertree of placentals. CONCLUSION: The supertree should provide a useful framework for hypothesis testing in phylogenetic comparative biology, and supports the idea that biogeography has played a crucial role in the evolution of placental mammals. Our results demonstrate the importance of minimising poor and redundant data when constructing supertrees.

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