Paleogenomics illuminates the evolutionary history of the extinct Holocene "horned" crocodile of Madagascar, Voay robustus.

Ancient DNA is transforming our ability to reconstruct historical patterns and mechanisms shaping modern diversity and distributions. In particular, molecular data from extinct Holocene island faunas have revealed surprising biogeographic scenarios. Here, we recovered partial mitochondrial (mt) genomes for 1300-1400 year old specimens (n = 2) of the extinct "horned" crocodile, Voay robustus, collected from Holocene deposits in southwestern Madagascar. Phylogenetic analyses of partial mt genomes and tip-dated timetrees based on molecular, fossil, and stratigraphic data favor a sister group relationship between Voay and Crocodylus (true crocodiles). These well supported trees conflict with recent morphological systematic work that has consistently placed Voay within Osteolaeminae (dwarf crocodiles and kin) and provide evidence for likely homoplasy in crocodylian cranial anatomy and snout shape. The close relationship between Voay and Crocodylus lends additional context for understanding the biogeographic origins of these genera and refines competing hypotheses for the recent extinction of Voay from Madagascar.

The evolution of HOM-C homeoboxes in the Dipteran family Drosophilidae.

Forty-five new Homeotic Complex (HOM-C) homeobox sequences from six species of Drosophilidae (Drosophila heteroneura, D. adiastola, Zaprionus vittiger, Chymomyza amoena, Scaptodrosophila pattersoni and Hirtodrosophila pictiventris) were obtained using a PCR-cloning method. These new homeoboxes are from the labial, proboscipedia, Deformed, Sex combs reduced, fushi tarazu, Antennapedia, Ultrabithorax, abdominal-A and Abdominal-B genes. Phylogenetic signal in the homeobox sequences was assessed and several aspects of sequence evolution were examined. In particular, codon bias was examined and found to exist between the drosophilid species examined here and Anopheles gambiae outgroup sequences. In addition, different patterns of codon bias were detected in homeoboxes interrupted with introns when compared to homeoboxes that are uninterrupted.

Extreme diversity, conservation, and convergence of spider silk fibroin sequences.

Spiders (Araneae) spin high-performance silks from liquid fibroin proteins. Fibroin sequences from basal spider lineages reveal mosaics of amino acid motifs that differ radically from previously described spider silk sequences. The silk fibers of Araneae are constructed from many protein designs. Yet, the repetitive sequences of fibroins from orb-weaving spiders have been maintained, presumably by stabilizing selection, over 125 million years of evolutionary history. The retention of these conserved motifs since the Mesozoic and their convergent evolution in other structural superproteins imply that these sequences are central to understanding the exceptional mechanical properties of orb weaver silks.

Hidden morphological support for the phylogenetic placement of Pseudoryx nghetinhensis with bovine bovids: a combined analysis of gross anatomical evidence and DNA sequences from five genes.

The saola, Pseudoryx nghetinhensis, was unknown to science until its formal description in 1993. This endangered species is a member of the ruminant artiodactyl family Bovidae (cattle, sheep, goats, and antelopes). However, given its puzzling combination of morphological traits, the specific affinities of Pseudoryx within Bovidae are controversial. A preliminary genetic investigation suggested that Pseudoryx should be placed in the subfamily Bovinae (cattle, buffaloes, spiral-horned antelopes, and nilgai), but a recent cladistic analysis of skeletal and dental characters allied Pseudoryx with caprine bovids (sheep, goats, musk oxen, goat antelopes, and Pantholops). The morphological and molecular hypotheses differ in assigning the saola to either of the two most divergent clades of Bovidae. In this report, phylogenetic analyses of DNA sequences from five genes are used to test these alternatives. Protein coding regions, introns, and ribosomal DNAs from the nuclear and mitochondrial genomes discount the hypothesis that Pseudoryx is a close relative of Caprinae. Instead, combined analyses of the DNA data and published morphological evidence place Pseudoryx with Bovini (cattle and buffaloes), a subclade of Bovinae. In a separate analysis, the matrix of morphological characters links Pseudoryx with caprine bovids, but in the context of the molecular data, the gross anatomical evidence strongly supports a grouping of Pseudoryx with Bovinae. Surprisingly, the morphological partition provides the most character support in the combined analysis. This striking result is obscured by separate analyses of the individual data sets and the taxonomic congruence approach.

Stability of cladistic relationships between Cetacea and higher-level artiodactyl taxa.

Over the past 10 years, the phylogenetic relationships among higher-level artiodactyl taxa have been examined with multiple data sets. Many of these data sets suggest that Artiodactyla (even-toed ungulates) is paraphyletic and that Cetacea (whales) represents a highly derived "artiodactyl" subgroup. In this report, phylogenetic relationships between Cetacea and artiodactyls are tested with a combination of 15 published data sets plus new DNA sequence data from two nuclear loci, interphotoreceptor retinoid-binding protein (IRBP) and von Willebrand factor (vWF). The addition of the IRBP and vWF character sets disrupts none of the relationships supported by recent cladistic analyses of the other 15 data sets. Simultaneous analyses support three critical clades: (Cetacea + Hippopotamidae), (Cetacea + Hippopotamidae + Ruminantia), and (Cetacea + Hippopotamidae + Ruminantia + Suina). Perturbations of the combined matrix show that the above clades are stable to a variety of disturbances. A chronicle of phylogenetic results over the past 3 years suggests that cladistic relationships between Cetacea and artiodactyls have been stable to increased taxonomic sampling and to the addition of more than 1,400 informative characters from 15 data sets.

A cladistic analysis of mitochondrial ribosomal DNA from the Bovidae.

There is a huge data base of genetic information for the domestic artiodactyl species Bos taurus (cow), Ovis aries (sheep), and Capra hircus (goat). However, the phylogenetic relationships of these economically critical taxa and their close relatives, family Bovidae, remain for the most part unresolved. In this report, we aligned new mitochondrial (mt) 12S and 16S ribosomal (r) DNA sequences from 26 bovid taxa with published sequences. Phylogenetic analyses of the more than 64 kilobases of mt rDNA from 57 taxa support a basal division in the Bovidae that separates Bos and its close relatives from Capra, Ovis, and their kin. As suggested by previous molecular and morphological studies, "antelopes" are a paraphyletic assemblage. Caprinae (sheep, goats, goat antelopes, and musk oxen) groups consistently with hippotragine and alcelaphine antelopes, while Bovini (cattle and buffaloes) clusters with tragelaphine and boselaphine antelopes. The traditional tribal subdivisions of Bovidae are supported in most cases, but there are exceptions within Caprinae and Antilopinae (gazelles and close relatives). The rDNA data consistently place the enigmatic genera Pelea, Pantholops, and Saiga, but the origin of Aepyceros, the impala, remains obscure. Combined phylogenetic analyses of the rDNA data with the skeletal characters of Gentry (1992) were used to assess the stability of the molecular results.

More DNA support for a Cetacea/Hippopotamidae clade: the blood-clotting protein gene gamma-fibrinogen.

Recent phylogenetic analyses of DNA sequences suggest that cetaceans (whales) and hippopotamid artiodactyls (hippos) are extant sister taxa. Consequently, the shared aquatic specializations of these taxa may be synapomorphies. This molecular view is contradicted by paleontological data that overwhelmingly support a monophyletic Artiodactyla (even-toed ungulates) and a close relationship between Cetacea and extinct mesonychian ungulates. According to the fossil evidence, molecular, behavioral, and anatomical resemblances between hippos and whales are interpreted as convergences or primitive retentions. In this report, competing interpretations of whale origins are tested through phylogenetic analyses of the blood-clotting protein gene gamma-fibrinogen from cetaceans, artiodactyls, perissodactyls (odd-toed ungulates), and carnivores (cats, dogs, and kin). In combination with published DNA sequences, the gamma-fibrinogen data unambiguously support a hippo/whale clade and are inconsistent with the paleontological perspective. If the phylogeny favored by fossil evidence is accepted, the convergence at the DNA level between Cetacea and Hippopotamidae is remarkable in its distribution across three genetic loci: gamma-fibrinogen, the linked milk casein genes, and mitochondrial cytochrome b.

Evidence from milk casein genes that cetaceans are close relatives of hippopotamid artiodactyls.

The inferred transition from terrestrial hoofed mammal to fully aquatic cetacean has been intensively studied with fossil evidence. However, large sections of this remarkable evolutionary sequence are missing. Phylogenetic analysis of extant taxa may help to fill in some of these gaps. In this report, kappa-casein (exon 4) and beta-casein (exon 7) milk protein genes from cetaceans and other placental mammals were PCR-amplified, sequenced, and aligned to previously published sequences. Phylogenetic analyses of the casein data suggest that hippopotamid artiodactyls are more closely related to cetaceans than to other artiodactyls (even-toed hoofed mammals). An analysis of the nuclear casein sequences combined with published mitochondrial cytochrome b DNA sequences also supports the Cetacea/Hippopotamidae sister group. This affinity implies that some of the aquatic traits of cetaceans were derived in the common ancestor of Cetacea and Hippopotamidae. An extant "missing link" to Cetacea may have been overlooked by science since the description of the semiaquatic Hippopotamus in 1758. Paleontological information is grossly inconsistent with this hypothesis. If the casein phylogeny is accurate, large gaps in the fossil record as well as extensive morphological reversals and convergences must be acknowledged.

Elision: a method for accommodating multiple molecular sequence alignments with alignment-ambiguous sites.

The process of multiple sequence alignment provides homology statements for the phylogenetic analysis of molecular data. Unfortunately, multiple alignments are frequently nonunique. Two sources of these multiple alignments are analysis based on different sets of alignment parameter values (gap:change cost ratios) and nonunique equally costly alignments based on a single set of alignment parameters. By "eliding" these individual alignments into a single grand alignment, phylogeny that is weighted toward those positions that align more consistently can be reconstructed. Positions that show greater variation among alignments will be relatively downweighted. The technique results in a weighting procedure that is a posteriori and based on the evidence established from the original sequence alignments.

PCR assays of variable nucleotide sites for identification of conservation units.

A number of authors have recently suggested that the best approach for identifying units of conservation is to follow a systematics model of character analysis (Amato, 1991; Cracraft, 1991; Vogler and DeSalle, 1994). This approach necessitates the use of an operational, typological, evolutionary species concept. The use of the phylogenetic species concept has the utility and philosophical logic appropriate for this task. Additionally, there is a large body of literature that uses this framework, along with a parsimony based character analysis to identify patterns of phylogeny (Cracraft, 1983; Nelson and Platnick, 1981; Nixon and Wheeler, 1990). While we advocate this approach, we recognize that one of its limiting factors is sample size. We propose that by selective direct sequencing plus rapid sampling of variable target characters by polymerase chain reaction (PCR) assays of specific sites, sufficiently large numbers of individuals can be accurately, inexpensively, and quickly surveyed for diagnostic characters. This procedure is demonstrated by a survey of variable nucleotide sites in the Caiman crocodilus complex.

Alignment-ambiguous nucleotide sites and the exclusion of systematic data.

Molecular systematists generally rely on computer algorithms to establish the alignment of DNA sequences. However, when alignment regions are characterized by multiple insertions and deletions, these gap-filled stretches of DNA are often excised before phylogenetic reconstruction. This exclusion of systematic data is generally determined by subjective criteria. We explore a replicable methodology in which the comparison of several multiple sequence alignments can be used to eliminate regions of unstable sequence alignment. Using crocodilian and insect mitochondrial (mt) ribosomal (r) DNA as examples, we caution against the removal of sequence data prior to phylogenetic reconstruction.

DNA sequences from a fossil termite in Oligo-Miocene amber and their phylogenetic implications.

DNA was extracted from the fossil termite Mastotermes electrodominicus preserved in Oligo-Miocene amber (25 million to 30 million years old). Fragments of mitochondrial [16S ribosomal DNA (rDNA)] and nuclear (18S rDNA) genes were amplified by polymerase chain reaction. Phylogenetic analysis of fossil and extant 18S rDNA confirmed morphological cladistic analyses of living dictyopterans (termites, cockroaches, and mantids). The fossil termite shares several sequence attributes with Mastotermes darwiniensis. Addition of this fossil to living-species phylogeny is required to substantiate Mastotermes monophyly and affects molecular phylogenetic hypotheses of termites in this, the oldest DNA yet characterized.

Phylogeny of the Bovidae (Artiodactyla, Mammalia), based on mitochondrial ribosomal DNA sequences.

Portions of the 12S and 16S mitochondrial ribosomal genes for 16 species representing nine tribes in the mammal family Bovidae were compared with six previously published orthologous sequences. Phylogenetic analysis of variable nucleotide positions under different constraints and weighting schemes revealed no robust groupings among tribes. Consensus trees support previous hypotheses of monophyly for four clades, including the traditional subfamily Bovinae. However, the basal diversification of bovid tribes, which was largely unresolved by morphological, immunodiffusion, allozyme, and protein sequence data, remains unresolved with the addition of DNA sequence data. The intractability of this systematic problem is consistent with a rapid radiation of the major bovid groups. Several analyses of our data show that monophyly of the Bovidae, which was weakly supported by previous morphological and molecular work, is questionable.

Induction and characterization of an interface tissue by implantation of methylmethacrylate cement into the posterior part of the cervical spine of the dog.

After the implantation of methylmethacrylate cement into the posterior part of the cervical spine of the dog, a thick layer of connective tissue forms at the bone-cement interface. The tissue is six to eight millimeters thick and in all animals it surrounds the dorsal and lateral aspects of the masses of implanted cement, grows between the undersurface of the cement and the bone of the posterior elements, and completely covers that bone. This tissue was examined by light and electron microscopy and its collagenous components were extracted and analyzed biochemically by gel electrophoresis. Specific extracellular matrix proteins in the tissue at the bone-cement interface were also localized by immunohistochemistry. The tissue at the host-cement interface contained zones of fibrocytes and plump and teardrop-shaped cells within a collagenous matrix. Type-I, Type-III, and Type-V collagen were extracted and were identified by gel electrophoresis. Type-V collagen and fibronectin were localized predominantly around the plump and teardrop-shaped cells. Type-IV collagen and laminin were localized predominantly in an area just beneath the teardrop-shaped cells at the surface of the tissue overlying the cement, suggesting that a basement-membrane-like tissue had formed in this area.