The idea that the pattern of point mutation in Drosophila has remained constant during the evolution of the genus has recently been challenged. A study of the nucleotide composition focused on the Drosophila saltans group has evidenced unsuspected nucleotide composition differences among lineages. Compositional differences are associated with an accelerated rate of amino acid replacement in functionally less constrained regions. Here we reassess this issue from a different perspective. Adopting a maximum-likelihood estimation approach, we focus on the different predictions that mutation and selection make about the nonsynonymous-to-synonymous rate ratio. We investigate two gene regions, alcohol dehydrogenase (Adh) and xanthine dehydrogenase (Xdh), using a balanced data set that comprises representatives from the melangaster, obscura, saltans, and willistoni groups. We also consider representatives of the Hawaiian picture-winged group. These Hawaiian species are known to have experienced repeated bottlenecks and are included as a reference for comparison. Our results confirm patterns previously detected. The branch ancestral to the fast-evolving willistoni/saltans lineage, where most of the change in GC content has occurred, exhibits an excess of synonymous substitutions. The shift in mutation bias has affected the extent of the rate variation among sites in Xdh.

Lateral gene transfer (LGT) is a major evolutionary mechanism in prokaryotes. Knowledge about LGT— particularly, multicellular— eukaryotes has only recently started to accumulate. A widespread assumption sees the gene as the unit of LGT, largely because little is yet known about how LGT chances are affected by structural/functional features at the subgenic level. Here we trace the evolutionary trajectory of VEin Patterning 1, a novel gene family known to be essential for plant development and defense. At the subgenic level VEP1 encodes a dinucleotide-binding Rossmann-fold domain, in common with members of the short-chain dehydrogenase/reductase (SDR) protein family. We found: i) VEP1 likely originated in an aerobic, mesophilic and chemoorganotrophic α-proteobacterium, and was laterally propagated through nets of ecological interactions, including multiple LGTs between phylogenetically distant green plant/fungi-associated bacteria, and five independent LGTs to eukaryotes. Of these latest five transfers, three are ancient LGTs, implicating an ancestral fungus, the last common ancestor of land plants and an ancestral trebouxiophyte green alga, and two are recent LGTs to modern embryophytes. ii) VEP1's rampant LGT behavior was enabled by the robustness and broad utility of the dinucleotide-binding Rossmann-fold, which provided a platform for the evolution of two unprecedented departures from the canonical SDR catalytic triad. iii) The fate of VEP1 in eukaryotes has been different in different lineages, being ubiquitous and highly conserved in land plants, whereas fungi underwent multiple losses. And iv) VEP1-harboring bacteria include non-phytopathogenic and phytopathogenic symbionts which are non-randomly distributed with respect to the type of harbored VEP1 gene. Our findings suggest that VEP1 may have been instrumental for the evolutionary transition of green plants to land, and point to a LGT-mediated ‘Trojan Horse’ mechanism for the evolution of bacterial pathogenesis against plants. VEP1 may serve as tool for revealing microbial interactions in plant/fungi-associated environments.

Rooting is frequently the most precarious step in any phylogenetic analysis. Outgroups can become useless for rooting if they are too distantly related to the ingroup. Specifically, little attention has been paid to scenarios where outgroups have evolved different nucleotide frequencies from the ingroup. We investigate one empirical example that arose seeking to determine the phylogenetic relationship between the saltans and the willistoni groups of Drosophila (subgenus Sophophora). We have analyzed 2085 coding nucleotides from the xanthine dehydrogenase (Xdh) gene in 14 species, 6 from the saltans group and 8 from the willistoni group. We adopt a two-step strategy: (1) we investigate the phylogeny without outgroups, rooting the network by the midpoint method; (2) we reinvestigate the rooting of this phylogeny using predefined outgroups in both a parsimony- and a model-based maximum-likelihood framework. A satisfactory description of the substitution process along the Xdh region calls for six substitution types and substitution rate variation among codon positions. When the ingroup sequences are considered alone, the phylogeny obtained using this description corroborates the known relationships derived from anatomical criteria. Inclusion of the outgroups makes the root unstable, apparently because of differences between ingroups and outgroups in the substitution processes; these differences are better accounted for by a simplified model of evolution than by more complex, realistic descriptions of the substitution process.

The time of origin of the animal phyla is controversial. Abundant fossils from the major animal phyla are found in the Cambrian, starting 544 million years ago. Many paleontologists hold that these phyla originated in the late Neoproterozoic, during the 160 million years preceding the Cambrian fossil explosion. We have analyzed 18 protein-coding gene loci and estimated that protostomes (arthropods, annelids, and mollusks) diverged from deuterostomes (echinoderms and chordates) about 670 million years ago, and chordates from echinoderms about 600 million years ago. Both estimates are consistent with paleontological estimates. A published analysis of seven gene loci that concludes that the corresponding divergence times are 1,200 and 1,000 million years ago is shown to be flawed because it extrapolates from slow-evolving vertebrate rates to faster-evolving invertebrate rates, as well as in other ways.

The neutrality theory predicts that the rate of neutral molecular evolution is constant over time, and thus that there is a molecular clock for timing evolutionary events. It has been observed that the variance of the rate of evolution is generally larger than expected according to the neutrality theory, which has raised the question of how reliable the molecular clock is or, indeed, whether there is a molecular clock at all. We have carried out an extensive investigation of three proteins, glycerol-3-phosphate dehydrogenase (GPDH), superoxide dismutase (SOD), and xanthine dehydrogenase (XDH). We have observed that (i) the three proteins evolve erratically through time and across lineages and (ii) the erratic patterns of acceleration and deceleration differ from locus to locus, so that one locus may evolve faster in one than another lineage, whereas the opposite may be the case for another locus. The observations are inconsistent with the predictions made by various subsidiary hypotheses proposed to account for the overdispersion of the molecular clock.

We investigated the evolution of xanthine dehydrogenase (Xdh) in 34 species from the three multicellular kingdoms, including one plant, two fungi, and three animal phyla, two classes of vertebrates, four orders of mammals, and two orders of insects. We adopted a model-based maximum-likelihood framework of inference. After accounting for among-site rate variation and heterogeneous nucleotide composition of the sequences using the discrete gamma distribution, and using nonhomogeneous nonstationary representations of the substitution process, the rate of amino acid replacement is 30.4 x 10(-10)/site/year when Drosophila species are compared but only approximately 18 x 10(-10)/site/year when comparisons are made between mammal orders, between insect orders, or between different animal phyla and approximately 11 x 10(-10)/site/year when comparisons are made between birds and mammals, between fungi, or between the three multicellular kingdoms. To account for these observations, the rate of amino acid replacement must have been eight or more times higher in some lineages and at some times than in others. Spastic evolution of Xdh appears to be related to the particularities of the genomes in which the locus is embedded.

Pneumonia is a significant disease of horses. Although pneumonia has traditionally been studied in racehorses, little information is available for non-racing horses. Non-racing horses that died with pulmonary lesions (n = 156) were available from cases submitted for autopsy from January 2015 to June 2020. Bronchopneumonia (35%), interstitial pneumonia (29%), embolic pneumonia (21%), granulomatous pneumonia (13%), and pleuritis (2%) were observed in the examined horses. Seventy-four horses died or were euthanized because of pulmonary diseases, and 82 horses died or were euthanized because of non-pulmonary causes but had lung lesions. Of the horses that died from pulmonary causes, the most common finding was bronchopneumonia, with abscesses and/or necrosis in the cranioventral aspect of the lung. Bacteria isolated from cases of bronchopneumonia were Streptococcus equi subsp. zooepidemicus (48.5%), Klebsiella pneumoniae (12.1%), and Actinobacillus equuli subsp. haemolyticus (9.1%). The most common extrapulmonary lesions responsible for death in horses that also had lesions in the lung were mainly in the gastrointestinal system (30%), multiple systems (septicemia and/or toxemia; 27%), and musculoskeletal system (12%). The main postmortem findings in cases of bronchopneumonia of non-racing horses were similar to those reported previously in racehorses. However, some non-racing horses also had interstitial and granulomatous pneumonia, patterns not described previously in racehorses in California, likely as a result of the inclusion of extended age categories for non-racing horses. We also found that the equine lung was frequently affected in cases of sepsis and gastrointestinal problems of infectious origin.

We investigated the nucleotide composition of five genes, Xdh, Adh, Sod, Per, and 28SrRNA, in nine species of Drosophila (subgenus Sophophora) and one of Scaptodrosophila. The six species of the Drosophila saltans group markedly differ from the others in GC content and codon use bias. The GC content in the third codon position, and to a lesser extent in the first position and the introns, is higher in the D. melanogaster and D. obscura groups than in the D. saltans group (in Scaptodrosophila it is intermediate but closer to the melanogaster and obscura species). Differences are greater for Xdh than for Adh, Sod, Per, and 28SrRNA, which are functionally more constrained. We infer that rapid evolution of GC content in the saltans lineage is largely due to a shift in mutation pressure, which may have been associated with diminished natural selection due to smaller effective population numbers rather than reduced recombination rates. The rate of GC content evolution impacts the rate of protein evolution and may distort phylogenetic inferences. Previous observations suggesting that GC content evolution is very limited in Drosophila may have been distorted due to the restricted number of genes and species (mostly D. melanogaster) investigated.

We have analyzed the phylogenetic distribution of introns in the gene coding for xanthine dehydrogenase in 37 species, including 31 dipterans sequenced by us. We have discovered three narrowly distributed novel introns, one in the medfly Ceratitis capitata, the second in the willistoni and saltans groups of Drosophila, and the third in two sibling species of the willistoni group. The phylogenetic distribution of these introns favors the "introns-late" theory of the origin of genes. Analysis of the nucleotide sequences indicates that all three introns have arisen by duplication of a preexisting intron, which is pervasive in Drosophila and other dipterans (and has a homologous position as an intron found in humans and other diverse organisms).

Drosophila nuclear introns are commonly assumed to change according to a single rate of substitution, yet little is known about the evolution of these non-coding sequences. The hypothesis of a uniform substitution rate for introns seems to be at odds with recent findings that the nucleotide composition of introns varies at a scale unknown before, and that their base content variation is correlated with that of the adjacent exons. However, no direct attempt at comparing substitution rates in introns seems to have been addressed so far. We have studied the rate of nucleotide substitution over a region of the Xdh gene containing two adjacent short, constitutively spliced introns, in several species of Drosophila and related genera. The two introns differ significantly in base composition and substitution rate, with one intron evolving at least twice as fast as the other. In addition, the substitution pattern of the introns is positively associated with that of the surrounding coding regions, evidencing that the molecular evolution of these introns is impacted by the region in which they are embedded. The observed differences cannot be attributed to selection acting differently at the level of the secondary structure of the pre-mRNA. Rather, they are better accounted for by locally heterogeneous patterns of mutation.