Approaches to the resolution of contradictions between phylogenetic reconstructions based on morphofunctional and genetic data

Essential differences between genetic and morphofunctional characteristics as sources of information about evolutionary development are discussed using examples of mammal taxa. The approaches to combined analysis of data characterizing different levels of biological organization in phylogenetic reconstructions are considered using examples of certain mammalian taxa.     

Molecular evolution of rDNA in early diverging Metazoa: First comparative analysis and phylogenetic application of complete SSU rRNA secondary structures in Porifera

Background  

The cytoplasmic ribosomal small subunit (SSU, 18S) ribosomal RNA (rRNA) is the most frequently-used gene for molecular phylogenetic studies. However, information regarding its secondary structure is neglected in most phylogenetic analyses. Incorporation of this information is essential in order to apply specific rRNA evolutionary models to overcome the problem of co-evolution of paired sites, which violates the basic assumption of the independent evolution of sites made by most phylogenetic methods. Information about secondary structure also supports the process of aligning rRNA sequences across taxa. Both aspects have been shown to increase the accuracy of phylogenetic reconstructions within various taxa.     

The primates of the Neotropics: genomes and chromosomes

The classification of neotropical primates has been controversial. Different arrangements have been proposed, depending on taxonomic criteria and on the traits selected for phylogenetic reconstructions. These include gross morphologic characters, karyotypic attributes and DNA sequence data of nuclear and mitochondrial genes and of repetitive genomic components. These approaches have substantially clarified the main intergeneric relationships although several intrageneric arrangements still remain to be elucidated. In this review, we compare karyologic and molecular data of this speciose group.     

A phylogeny of the damselfly genus calopteryx (Odonata) using mitochondrial 16S rDNA markers

We seek to reconstruct the phylogenetic relationships of the damselfly genus Calopteryx, for which extensive behavioral and morphological knowledge already exists. To date, analyses of the evolutionary pathways of different life history traits have been hampered by the absence of a robust phylogeny based on morphological data. In this study, we concentrate on establishing phylogenetic information from parts of the 16S rDNA gene, which we sequenced for nine Calopteryx species and five outgroup species. The mt 16S rDNA data set did not show signs of saturated variation for ingroup taxa, and phylogenetic reconstructions were insensitive to variation of outgroup taxa. Parsimony, neighbor-joining, and maximum-likelihood reconstructions agreed on parts of the tree. A consensus tree summarizes the significant results and indicates problematic nodes. The 16S rDNA sequences support monophyly of the genera Mnais, Matrona, and Calopteryx. However, the genus Calopteryx may not be monophyletic, since Matrona basilaris and Calopteryx atrata are sister taxa under every parameter setting. The North American and European taxa each appear as monophyletic clades, while the Asian Calopteryx atrata and Calopteryx cornelia are not monophyletic. Our data implies a different paleobiogeographic history of the Eurasian and North American species, with extant Eurasian species complexes shaped by glacial periods, in contrast to extant North American species groups.     

Comprehensive phylogenetic reconstruction of amoebozoa based on concatenated analyses of SSU-rDNA and actin genes

Evolutionary relationships within Amoebozoa have been the subject of controversy for two reasons: 1) paucity of morphological characters in traditional surveys and 2) haphazard taxonomic sampling in modern molecular reconstructions. These along with other factors have prevented the erection of a definitive system that resolves confidently both higher and lower-level relationships. Additionally, the recent recognition that many protosteloid amoebae are in fact scattered throughout the Amoebozoa suggests that phylogenetic reconstructions have been excluding an extensive and integral group of organisms. Here we provide a comprehensive phylogenetic reconstruction based on 139 taxa using molecular information from both SSU-rDNA and actin genes. We provide molecular data for 13 of those taxa, 12 of which had not been previously characterized. We explored the dataset extensively by generating 18 alternative reconstructions that assess the effect of missing data, long-branched taxa, unstable taxa, fast evolving sites and inclusion of environmental sequences. We compared reconstructions with each other as well as against previously published phylogenies. Our analyses show that many of the morphologically established lower-level relationships (defined here as relationships roughly equivalent to Order level or below) are congruent with molecular data. However, the data are insufficient to corroborate or reject the large majority of proposed higher-level relationships (above the Order-level), with the exception of Tubulinea, Archamoebae and Myxogastrea, which are consistently recovered. Moreover, contrary to previous expectations, the inclusion of available environmental sequences does not significantly improve the Amoebozoa reconstruction. This is probably because key amoebozoan taxa are not easily amplified by environmental sequencing methodology due to high rates of molecular evolution and regular occurrence of large indels and introns. Finally, in an effort to facilitate future sampling of key amoebozoan taxa, we provide a novel methodology for genome amplification and cDNA extraction from single or a few cells, a method that is culture-independent and allows both photodocumentation and extraction of multiple genes from natural samples.     

The chorda tympani and its significance for rodent phylogeny

The topographical relationship of the chorda tympani to the tensor tympani muscle in the middle ear region of rodents was recorded, finding that this character provides new phylogenetic information. For this purpose we examined histological serial sections of 63 rodent species representing most families. Two lagomorph species were used as an outgroup. Computed 3D models of middle ear structures of several rodent taxa were prepared for the first time. We observed two different character states concerning the topography of the chorda tympani and the insertion tendon of the tensor tympani muscle: hypotensoric and epitensoric; the latter displays three subtypes. We assume that the shift from a plesiomorphic hypotensoric chorda tympani to an apomorphic epitensoric condition occurred at least six times independently in rodent phylogeny; however, this derived state is bound to higher taxonomic levels. The anomalurids under study show an epitensoric course of the chorda tympani through a mallear channel above the insertion of the tendon – just as in Pedetes capensis. Therefore, we suggest a new synapomorphy for these two taxa, confirming other new evidence as to their monophyly. The principal quality of the character used in this study is discussed.     

VITCOMIC2: visualization tool for the phylogenetic composition of microbial communities based on 16S rRNA gene amplicons and metagenomic shotgun sequencing

Background

The 16S rRNA gene-based amplicon sequencing analysis is widely used to determine the taxonomic composition of microbial communities. Once the taxonomic composition of each community is obtained, evolutionary relationships among taxa are inferred by a phylogenetic tree. Thus, the combined representation of taxonomic composition and phylogenetic relationships among taxa is a powerful method for understanding microbial community structure; however, applying phylogenetic tree-based representation with information on the abundance of thousands or more taxa in each community is a difficult task. For this purpose, we previously developed the tool VITCOMIC (VIsualization tool for Taxonomic COmpositions of MIcrobial Community), which is based on the genome-sequenced microbes’ phylogenetic information. Here, we introduce VITCOMIC2, which incorporates substantive improvements over VITCOMIC that were necessary to address several issues associated with 16S rRNA gene-based analysis of microbial communities.

Results

We developed VITCOMIC2 to provide (i) sequence identity searches against broad reference taxa including uncultured taxa; (ii) normalization of 16S rRNA gene copy number differences among taxa; (iii) rapid sequence identity searches by applying the graphics processing unit-based sequence identity search tool CLAST; (iv) accurate taxonomic composition inference and nearly full-length 16S rRNA gene sequence reconstructions for metagenomic shotgun sequencing; and (v) an interactive user interface for simultaneous representation of the taxonomic composition of microbial communities and phylogenetic relationships among taxa. We validated the accuracy of processes (ii) and (iv) by using metagenomic shotgun sequencing data from a mock microbial community.

Conclusions

The improvements incorporated into VITCOMIC2 enable users to acquire an intuitive understanding of microbial community composition based on the 16S rRNA gene sequence data obtained from both metagenomic shotgun and amplicon sequencing.

     

Conserved sequence motifs, alignment, and secondary structure for the third domain of animal 12S rRNA

Secondary structure models are an important step for aligning sequences, understanding probabilities of nucleotide substitutions, and evaluating the reliability of phylogenetic reconstructions. A set of conserved sequence motifs is derived from comparative sequence analysis of 184 invertebrate and vertebrate taxa (including many taxa from the same genera, families, and orders) with reference to a secondary structure model for domain III of animal mitochondrial small subunit (12S) ribosomal RNA. A template is presented to assist with secondary structure drawing. Our model is similar to previous models but is more specific to mitochondrial DNA, fitting both invertebrate and vertebrate groups, including taxa with markedly different nucleotide compositions. The second half of the domain III sequence can be difficult to align precisely, even when secondary structure information is considered. This is especially true for comparisons of anciently diverged taxa, but well-conserved motifs assist in determining biologically meaningful alignments. Patterns of conservation and variability in both paired and unpaired regions make differential phylogenetic weighting in terms of "stems" and "loops" unsatisfactory. We emphasize looking carefully at the sequence data before and during analyses, and advocate the use of conserved motifs and other secondary structure information for assessing sequencing fidelity.      

Phylogenetic relationships of Nembrothinae (Mollusca: Doridacea: Polyceridae) inferred from morphology and mitochondrial DNA

Within the Polyceridae, Nembrothinae includes some of the most striking and conspicuous sea slugs known, although several features of their biology and phylogenetic relationships remain unknown. This paper reports a phylogenetic analysis based on partial sequences of two mitochondrial genes (cytochrome c oxidase subunit I and 16S rRNA) and morphology for most species included in Nembrothinae. Our phylogenetic reconstructions using both molecular and combined morphological and molecular data support the taxonomic splitting of Nembrothinae into several taxa. Excluding one species (Tambja tentaculata), the monophyly of Roboastra was supported by all the phylogenetic analyses of the combined molecular data. Nembrotha was monophyletic both in the morphological and molecular analyses, always with high support. However, Tambja was recovered as para- or polyphyletic, depending on the analysis performed. Our study also rejects the monophyly of "phanerobranch" dorids based on molecular data.     

Evolution of the genomic recombination rate in murid rodents

Although very closely related species can differ in their fine-scale patterns of recombination hotspots, variation in the average genomic recombination rate among recently diverged taxa has rarely been surveyed. We measured recombination rates in eight species that collectively represent several temporal scales of divergence within a single rodent family, Muridae. We used a cytological approach that enables in situ visualization of crossovers at meiosis to quantify recombination rates in multiple males from each rodent group. We uncovered large differences in genomic recombination rate between rodent species, which were independent of karyotypic variation. The divergence in genomic recombination rate that we document is not proportional to DNA sequence divergence, suggesting that recombination has evolved at variable rates along the murid phylogeny. Additionally, we document significant variation in genomic recombination rate both within and between subspecies of house mice. Recombination rates estimated in F(1) hybrids reveal evidence for sex-linked loci contributing to the evolution of recombination in house mice. Our results provide one of the first detailed portraits of genomic-scale recombination rate variation within a single mammalian family and demonstrate that the low recombination rates in laboratory mice and rats reflect a more general reduction in recombination rate across murid rodents.     

Examination of hemiplasy, homoplasy and phylogenetic discordance in chromosomal evolution of the Bovidae

Robertsonian chromosomal fusions predominate in shaping the genomes of many species of Bovidae. These and other cytogenetic data (from 52 taxa representing 51 species and 9 tribes of Bovidae) were (i) examined for usefulness in defining phylogenetic relationships and (ii) subsequently mapped to a consensus tree based on mitochondrial and nuclear DNA gene sequences with divergence dates of the corresponding species calculated from cytochrome b sequences. This permitted persistence time estimates for the various rearrangements. The chromosomal data resulted in an unsupported higher-level topology, but with recognition of the monophyly of some genera and tribes within Bovidae. The distribution and temporal spread of character states on the species tree is suggestive of a restricted role for hemiplasy (the retention of an ancestral chromosomal polymorphism through multiple speciation events) and for introgression (resulting from secondary contact among taxa), processes that can potentially lead to phylogenetic discordance. We conclude that the most probable interpretation for these data is that genuine karyotypic homoplasy predominates, but that hemiplasy (and/or introgression) is a realistic hypothesis for the observed patterns of several shared characters in Bovidae.     

Evolutionary systematics in African gerbilline rodents of the genus Gerbilliscus: inference from mitochondrial genes

Gerbilliscus has recently been proposed as an endemic African rodent genus distinct from the Asian Tatera. A molecular phylogeny of the genus, including nine species from southern, western and eastern Africa, is presented here based on the analysis of the cytochrome b and 16S mitochondrial genes. With an adequate taxonomic sampling over a wide geographic range, we here provide a clear picture of the phylogenetic relationships between species and species groups in this genus. Three distinct clades were resolved, corresponding to major geographical subdivisions: an eastern clade that possibly diverged first, then a southern and a western clades which appeared later. We suggest two possible hypotheses concerning the dispersal of the genus across Africa, considering also the patterns of karyotypic variation. Finally, we discuss the taxonomic status of G. gambianus and the relationships between Gerbillurus and Gerbilliscus, as previous studies have suggested that the former should be included in the latter. Our data seem to support the synonymy of the two taxa and suggest that Gerbillurus and Gerbilliscus lineages diverged from a common ancestor appeared in eastern Africa.     

A genealogical view of chromosomal evolution and species delimitation in the Drosophila virilis species subgroup

Chromosomal arrangement was a historically important character used for defining taxonomic boundaries. The Drosophila virilis species group exhibits a series of chromosomal rearrangements, and the resulting differences among karyotypes were primary characters originally used to define taxa within the group. However, some chromosomally divergent forms have not been sufficiently resolved in phylogenetic reconstructions of DNA sequences from several nuclear genes. Sequences of mitochondrial regions have the potential for finer-scale resolution of closely related taxa; therefore, sequences of two mitochondrial genes were used to examine phylogenetic relationships within the chromosomally variable virilis subgroup. Sequences were obtained from multiple strains of the Palearctic species, D. virilis and D. lummei, and the Nearctic species, D. novamexicana and two chromosomal forms of D. americana. Analyses support the recent emergence of the different chromosomal forms in North America. However, none of these chromosomally divergent forms exhibit reciprocal monophyly of their mtDNA sequences, which is the requirement for attaining genealogical species status.     

Evolution of trnF(GAA) pseudogenes in cruciferous plants

In several studies we used the 5′-trnL(UAA)–trnF(GAA) region of the chloroplast DNA for phylogeographic reconstructions, gene diversity calculations and phylogenetic analyses among the genera Arabidopsis and Boechera. Despite the fact that extensive gene duplications are rare within the chloroplast genome of higher plants, within several genera of the Brassicaceae the anticodon domain of the trnF(GAA) gene exhibit extensive gene duplications with 1–12 tandemly repeated copies in close 5′-proximity of the functional gene. A recent re-examination and additional analysis of trnL(UAA)–trnF(GAA) regions from numerous cruciferous taxa not only reveal extensive trnF gene duplications, but also favour the hypothesis that in cruciferous taxa at least four independent phylogenetic lineages are characterized by these pseudogenes. Among these lineages there is one major clade of taxa carrying pseudogenes indicating an ancient split in crucifer evolution. In two case studies, Boechera and Arabidopsis, intra- and inter-molecular recombinations have been shown to be the reason for the reciprocal exchange of several similar motifs. However, functional constraints might favour two to three or five to six copies as shown for Arabidopsis and Boechera. Herein, we compare the occurrence and distribution of pseudogene copy number in the framework of a comprehensive survey of cpDNA haplotype variation in Boechera, the former genus Cardaminopsis and Arabidopsis thaliana and comment on the value of such kind of mutations in phylogenetic and evolutionary reconstructions.     

c-mos variation in songbirds: molecular evolution, phylogenetic implications, and comparisons with mitochondrial differentiation

Nucleotide sequences from the c-mos proto-oncogene have previously been used to reconstruct the phylogenetic relationships between distantly related vertebrate taxa. To explore c-mos variation at shallower levels of avian divergence, we compared c-mos sequences from representative passerine taxa that span a range of evolutionary differentiation, from basal passerine lineages to closely allied genera. Phylogenetic reconstructions based on these c-mos sequences recovered topologies congruent with previous DNA-DNA hybridization-based reconstructions, with many nodes receiving high support, as indicated by bootstrap and reliability values. One exception was the relationship of Acanthisitta to the remaining passerines, where the c-mos-based searches indicated a three-way polytomy involving the Acanthisitta lineage and the suboscine and oscine passerine clades. We also compared levels of c-mos and mitochondrial differentiation across eight oscine passerine taxa and found that c-mos nucleotide substitutions accumulate at a rate similar to that of transversion substitutions in mitochondrial protein-coding genes. These comparisons suggest that nuclear-encoded loci such as c-mos provide a temporal window of phylogenetic resolution that overlaps the temporal range where mitochondrial protein-coding sequences have their greatest utility and that c-mos substitutions and mtDNA transversions can serve as complementary, informative, and independent phylogenetic markers for the study of avian relationships.     

Molecular systematics and time-scale for the evolution of Timarcha, a leaf-beetle genus with a disjunct Holarctic distribution

In recent years we have investigated the evolution of the Holarctic leaf-beetle genus Timarcha using molecular approaches, but to date several important questions remained unanswered, including its systematic arrangement in a temporal context, or the phylogenetic placement of the Nearctic taxa. Here I present a reanalysis of available genetic data together with newly generated data for key taxa (markers 16S rDNA, CO2, ITS-2, and 18S rDNA), including the Nearctic species (subgenus Americanotimarcha), using direct optimization-based phylogenetic reconstructions. Lineage ages are estimated using maximum likelihood branch-length estimates and the molecular clock calibration derived from several presumed vicariance events in the Mediterranean. Phylogenetic analyses and 18S rDNA divergences suggest the ancient divergence of the Nearctic and Palaearctic lineages, related to the North Atlantic opening in the middle Eocene. The diversification of the Palaearctic Timarcha seems closely related to the geological evolution of the Mediterranean area during the Tertiary, with Pleistocenic climate changes affecting species ranges and lineage extinction, but not resulting in extensive speciation.     

The Evolution of a High Copy Gene Array in Arabidopsis

Local gene duplication is a prominent mechanism of gene copy number expansion. Elucidating the mechanisms by which local duplicates arise is necessary in understanding the evolution of genomes and their host organisms. Chromosome one of Arabidopsis thaliana contains an 81-gene array subdivided into 27 triplet units (t-units), with each t-unit containing three pre-transfer RNA genes. We utilized phylogenetic tree reconstructions and comparative genomics to order the events leading to the array’s formation, and propose a model using unequal crossing-over as the primary mechanism of array formation. The model is supported by additional phylogenetic information from intergenic spacer sequences separating each t-unit, comparative analysis to an orthologous array of 12 t-units in the sister taxa Arabidopsis lyrata, and additional modeling using a stochastic simulation of orthologous array divergence. Lastly, comparative phylogenetic analysis demonstrates that the two orthologous t-unit arrays undergo concerted evolution within each taxa and are likely fluctuating in copy number under neutral evolutionary drift. These findings hold larger implications for future research concerning gene and genome evolution.     

Phylogenomic analysis resolves the interordinal relationships and rapid diversification of the laurasiatherian mammals

Although great progress has been made in resolving the relationships of placental mammals, the position of several clades in Laurasiatheria remain controversial. In this study, we performed a phylogenetic analysis of 97 orthologs (46,152 bp) for 15 taxa, representing all laurasiatherian orders. Additionally, phylogenetic trees of laurasiatherian mammals with draft genome sequences were reconstructed based on 1608 exons (2,175,102 bp). Our reconstructions resolve the interordinal relationships within Laurasiatheria and corroborate the clades Scrotifera, Fereuungulata, and Cetartiodactyla. Furthermore, we tested alternative topologies within Laurasiatheria, and among alternatives for the phylogenetic position of Perissodactyla, a sister-group relationship with Cetartiodactyla receives the highest support. Thus, Pegasoferae (Perissodactyla + Carnivora + Pholidota + Chiroptera) does not appear to be a natural group. Divergence time estimates from these genes were compared with published estimates for splits within Laurasiatheria. Our estimates were similar to those of several studies and suggest that the divergences among these orders occurred within just a few million years.     

Karyotypic evolution in Aotus

Great karyotypic diversity exists within the platyrrhine genus Aotus. Primarily by comparing banded karyograms of different forms of Aotus, the pattern of karyotypic evolution can be assessed. Out-group comparisons are used to establish primitive and derived states of particular chromosomes, and a parsimonious cladogram is constructed. Other karyotypic changes are then positioned at appropriate nodes of the cladogram. The resulting phylogenetic hypothesis is entirely self-consistent, is in accord with out-group comparisons, does not invoke hybridization between ancestral forms, and, importantly, indicates a single origin for each rearrangement of euchromatic segments. Moreover, it is consistent with the hypothesis, derived from pelage studies, that the more southerly Aotus taxa constitute a holophyletic group. The reconstructed ancestral karyotype had a diploid number of 54. There has been little loss of euchromatic material during Aotus evolution.