A mitogenomic timescale for birds detects variable phylogenetic rates of molecular evolution and refutes the standard molecular clock

Current understanding of the diversification of birds is hindered by their incomplete fossil record and uncertainty in phylogenetic relationships and phylogenetic rates of molecular evolution. Here we performed the first comprehensive analysis of mitogenomic data of 48 vertebrates, including 35 birds, to derive a Bayesian timescale for avian evolution and to estimate rates of DNA evolution. Our approach used multiple fossil time constraints scattered throughout the phylogenetic tree and accounts for uncertainties in time constraints, branch lengths, and heterogeneity of rates of DNA evolution. We estimated that the major vertebrate lineages originated in the Permian; the 95% credible intervals of our estimated ages of the origin of archosaurs (258 MYA), the amniote-amphibian split (356 MYA), and the archosaur-lizard divergence (278 MYA) bracket estimates from the fossil record. The origin of modern orders of birds was estimated to have occurred throughout the Cretaceous beginning about 139 MYA, arguing against a cataclysmic extinction of lineages at the Cretaceous/Tertiary boundary. We identified fossils that are useful as time constraints within vertebrates. Our timescale reveals that rates of molecular evolution vary across genes and among taxa through time, thereby refuting the widely used mitogenomic or cytochrome b molecular clock in birds. Moreover, the 5-Myr divergence time assumed between 2 genera of geese (Branta and Anser) to originally calibrate the standard mitochondrial clock rate of 0.01 substitutions per site per lineage per Myr (s/s/l/Myr) in birds was shown to be underestimated by about 9.5 Myr. Phylogenetic rates in birds vary between 0.0009 and 0.012 s/s/l/Myr, indicating that many phylogenetic splits among avian taxa also have been underestimated and need to be revised. We found no support for the hypothesis that the molecular clock in birds "ticks" according to a constant rate of substitution per unit of mass-specific metabolic energy rather than per unit of time, as recently suggested. Our analysis advances knowledge of rates of DNA evolution across birds and other vertebrates and will, therefore, aid comparative biology studies that seek to infer the origin and timing of major adaptive shifts in vertebrates.     

Dating the early evolution of plants: detection and molecular clock analyses of orthologs

Orthologs generally are under selective pressure against loss of function, while paralogs usually accumulate mutations and finally die or deviate in terms of function or regulation. Most ortholog detection methods contaminate the resulting datasets with a substantial amount of paralogs. Therefore we aimed to implement a straightforward method that allows the detection of ortholog clusters with a reduced amount of paralogs from completely sequenced genomes. The described cross-species expansion of the reciprocal best BLAST hit method is a time-effective method for ortholog detection, which results in 68% truly orthologous clusters and the procedure specifically enriches single-copy orthologs. The detection of true orthologs can provide a phylogenetic toolkit to better understand evolutionary processes. In a study across six photosynthetic eukaryotes, nuclear genes of putative mitochondrial origin were shown to be over-represented among single copy orthologs. These orthologs are involved in fundamental biological processes like amino acid metabolism or translation. Molecular clock analyses based on this dataset yielded divergence time estimates for the red/green algae (1,142 MYA), green algae/land plant (725 MYA), mosses/seed plant (496 MYA), gymno-/angiosperm (385 MYA) and monocotyledons/core eudicotyledons (301 MYA) divergence times. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

SuperTRI: A new approach based on branch support analyses of multiple independent data sets for assessing reliability of phylogenetic inferences

Supermatrix and supertree are two methods for constructing a phylogenetic tree by using multiple data sets. However, these methods are not a panacea, as conflicting signals between data sets can lead to misinterpret the evolutionary history of taxa. In particular, the supermatrix approach is expected to be misleading if the species-tree signal is not dominant after the combination of the data sets. Moreover, most current supertree methods suffer from two limitations: (i) they ignore or misinterpret secondary (non-dominant) phylogenetic signals of the different data sets; and (ii) the logical basis of node robustness measures is unclear.To overcome these limitations, we propose a new approach, called SuperTRI, which is based on the branch support analyses of the independent data sets, and where the reliability of the nodes is assessed using three measures: the supertree Bootstrap percentage and two other values calculated from the separate analyses: the mean branch support (mean Bootstrap percentage or mean posterior probability) and the reproducibility index.The SuperTRI approach is tested on a data matrix including seven genes for 82 taxa of the family Bovidae (Mammalia, Ruminantia), and the results are compared to those found with the supermatrix approach. The phylogenetic analyses of the supermatrix and independent data sets were done using four methods of tree reconstruction: Bayesian inference, maximum likelihood, and unweighted and weighted maximum parsimony. The results indicate, firstly, that the SuperTRI approach shows less sensitivity to the four phylogenetic methods, secondly, that it is more accurate to interpret the relationships among taxa, and thirdly, that interesting conclusions on introgression and radiation can be drawn from the comparisons between SuperTRI and supermatrix analyses. To cite this article: A. Ropiquet et al., C. R. Biologies 332 (2009).     

Multi-locus phylogenetic analyses support the monophyly and the Miocene diversification of Iksookimia (Teleostei: Cypriniformes: Cobitidae)

The genus Iksookimia contains six species of primary freshwater fishes that are endemic to South Korea. Previous phylogenetic studies, based on DNA sequence data from three or fewer loci, have suggested non-monophyly of the genus, providing inconsistent resolutions of the relationships of Iksookimia. Our coalescent and concatenation-based phylogenetic analyses, utilizing seven unlinked nuclear-encoded genes, strongly supported Iksookimia as a monophyletic group, emphasizing the importance of multi-locus data in investigating complicated phylogenetic relationships. A relaxed molecular clock analysis using fossil calibrations, indicated that the origin of the major lineages of Iksookimia occurred between ～12 to 5 Ma, which is consistent with the Miocene uplift of the Taebaek and Sobaek Mountains and the Miocene activation of the major south-eastern faults. These palaeogeographic events may have served as vicariant events in the diversification of Iksookimia.     

Serotonin immunoreactive interneurons in the brain of the Remipedia: new insights into the phylogenetic affinities of an enigmatic crustacean taxon

ABSTRACT: BACKGROUND: Remipedia, a group of homonomously segmented, cave-dwelling, eyeless arthropods have been regarded as basal crustaceans in most early morphological and taxonomic studies. However, molecular sequence information together with the discovery of a highly differentiated brain led to a reconsideration of their phylogenetic position. Various conflicting hypotheses have been proposed including the claim for a basal position of Remipedia up to a close relationship with Malacostraca or Hexapoda. To provide new morphological characters that may allow phylogenetic insights, we have analyzed the architecture of the remipede brain in more detail using immunocytochemistry (serotonin, acetylated alpha-tubulin, synapsin) combined with confocal laser-scanning microscopy and image reconstruction techniques. This approach allows for a comprehensive neuroanatomical comparison with other crustacean and hexapod taxa. RESULTS: The dominant structures of the brain are the deutocerebral olfactory neuropils, which are linked by the olfactory globular tracts to the protocerebral hemiellipsoid bodies. The olfactory globular tracts form a characteristic chiasm in the center of the brain. In Speleonectes tulumensis, each brain hemisphere contains about 120 serotonin immunoreactive neurons, which are distributed in distinct cell groups supplying fine, profusely branching neurites to 16 neuropilar domains. The olfactory neuropil comprises more than 300 spherical olfactory glomeruli arranged in sublobes. Eight serotonin immunoreactive neurons homogeneously innervate the olfactory glomeruli. In the protocerebrum, serotonin immunoreactivity revealed several structures, which, based on their position and connectivity resemble a central complex comprising a central body, a protocerebral bridge, W-, X-, Y-, Z-tracts, and lateral accessory lobes. CONCLUSIONS: The brain of Remipedia shows several plesiomorphic features shared with other Mandibulata, such as deutocerebral olfactory neuropils with a glomerular organization, innervations by serotonin immunoreactive interneurons, and connections to protocerebral neuropils. Also, we provided tentative evidence for W-, X-, Y-, Z-tracts in the remipedian central complex like in the brain of Malacostraca, and Hexapoda. Furthermore, Remipedia display several synapomorphies with Malacostraca supporting a sister group relationship between both taxa. These homologies include a chiasm of the olfactory globular tract, which connects the olfactory neuropils with the lateral protocerebrum and the presence of hemiellipsoid bodies. Even though a growing number of molecular investigations unites Remipedia and Cephalocarida, our neuroanatomical comparison does not provide support for such a sister group relationship.     

Optimizing stem cells for cardiac repair: Current status and new frontiers in regenerative cardiology

Cell therapy has the potential to improve healing of ischemic heart, repopulate injured myocardium and restore cardiac function. The tremendous hope and potential of stem cell therapy is well understood, yet recent trials involving cell therapy for cardiovascular diseases have yielded mixed results with inconsistent data thereby readdressing controversies and unresolved questions regarding stem cell efficacy for ischemic cardiac disease treatment. These controversies are believed to arise by the lack of uniformity of the clinical trial methodologies, uncertainty regarding the underlying reparative mechanisms of stem cells, questions concerning the most appropriate cell population to use, the proper delivery method and timing in relation to the moment of infarction, as well as the poor stem cell survival and engraftment especially in a diseased microenvironment which is collectively acknowledged as a major hindrance to any form of cell therapy. Indeed, the microenvironment of the failing heart exhibits pathological hypoxic, oxidative and inflammatory stressors impairing the survival of transplanted cells. Therefore, in order to observe any significant therapeutic benefit there is a need to increase resilience of stem cells to death in the transplant microenvironment while preserving or better yet improving their reparative functionality. Although stem cell differentiation into cardiomyocytes has been observed in some instance, the prevailing reparative benefits are afforded through paracrine mechanisms that promote angiogenesis, cell survival, transdifferentiate host cells and modulate immune responses. Therefore, to maximize their reparative functionality, ex vivo manipulation of stem cells through physical, genetic and pharmacological means have shown promise to enable cells to thrive in the postischemic transplant microenvironment. In the present work, we will overview the current status of stem cell therapy for ischemic heart disease, discuss the most recurring cell populations employed, the mechanisms by which stem cells deliver a therapeutic benefit andstrategies that have been used to optimize and increase survival and functionality of stem cells including ex vivo preconditioning with drugs and a novel pharmacooptimizer as well as genetic modifications.     

Combined molecular and morphological phylogenetic analyses of the New Zealand wolf spider genus Anoteropsis (Araneae: Lycosidae)

Datasets from the mitochondrial gene regions NADH dehydrogenase subunit I (ND1) and cytochrome c oxidase subunit I (COI) of the 20 species in the New Zealand wolf spider (Lycosidae) genus Anoteropsis were generated. Sequence data were phylogenetically analysed using parsimony and maximum likelihood analyses. The phylogenies generated from the ND1 and COI sequence data and a previously generated morphological dataset were significantly congruent (p<0.001). Sequence data were combined with morphological data and phylogenetically analysed using parsimony. The ND1 region sequenced included part of tRNA(Leu(CUN)), which appears to have an unstable amino-acyl arm and no TpsiC arm in lycosids. Analyses supported the existence of five species groups within Anoteropsis and the monophyly of species represented by multiple samples. A radiation of Anoteropsis species within the last five million years is inferred from the ND1 and COI likelihood phylograms, habitat and geological data, which also indicates that Anoteropsis arrived in New Zealand some time after it separated from Gondwana.     

Molecular phylogenetic analyses reveal three new Ceratocystis species and provide evidence for geographic differentiation of the genus in Africa

The emergence of wattle wilt disease on non-native Acacia mearnsii trees in Africa, caused by the indigenous fungus Ceratocystis albifundus, has highlighted a need to better understand the diversity, ecology and distribution of Ceratocystis species in natural African environments. In this study we applied phylogenetic inference to identify and characterize isolates of Ceratocystis collected in a natural savanna ecosystem in South Africa. Three new species were recognized and are described as C. cryptoformis sp. nov. in the C. moniliformis complex, as well as C. thulamelensis sp. nov. and C. zambeziensis sp. nov., both residing in the C. fimbriata complex. Incorporating the new species into global phylogenies of Ceratocystis provided insights into the patterns of evolution and biogeography of this group of fungi. Notably, the African continent was identified as an important centre of diversification of Ceratocystis spp., from which several lineages of these fungi were shown to have radiated.     

Fomitiporia in sub-Saharan Africa: morphology and multigene phylogenetic analysis support three new species from the Guineo-Congolian rainforest

Fomitiporia nobilissima sp. nov., F. gabonensis sp. nov. and F. ivindoensis sp. nov., three species from the rainforest of the Guineo-Congolian phytogeographic region in Gabon, are described and illustrated. These species share a pileate basidiome, small basidiospores and an absence of setae. The critical morphological features that differentiate them are the pileus habit or shape, pore surface color, pore diameter and possibly ecology. Each new species forms distinct but closely related clades in phylogenetic analysis of DNA sequences from nuclear ribosomal LSU, ITS and translation elongation factor 1-α. Other species in sub-Saharan Africa are discussed briefly. A key to six species of Fomitiporia from sub-Saharan Africa is provided. The new combination Fomitiporia apiahyna is proposed.     

Nomimoscolex suspectus n. sp. (Eucestoda: Proteocephalidea: Zygobothriinae) with morphological and molecular phylogenetic analyses of the genus

We describe a new species of Nomimoscolex from the Amazon siluriform fishes Brachyplatystoma filamentosum, B. flavicans and B. vaillanti. It differs from N. piraeeba in a lower mean number of testes, the paramuscular position of the vitelline follicles, the ovarian width/proglottis width ratio and the cirrus-pouch length/proglottis width ratio. Protein electrophoresis assays performed for 25 enzymatic systems showed that specimens of N. suspectus n. sp. from the three host species form a homogenous population which was genetically isolated from N. piraeeba and N. dorad. Moreover, the latter two species, synonymised by Rego (1991) because of their close morphological similarity, could be separated at eight loci. We thus restore N. dorad as a valid species. We finally examined the composition of the genus Nomimoscolex using DNA sequences from the 5.8S rRNA, ITS-2 and 28S rRNA nuclear ribosomal genes and a matrix of 24 morphological characters. Phylogenetic relationships were inferred for nine species of the genus, five members of other monticelliid genera and two outgroup species. The results of the phylogenetic analyses performed on morphological and molecular characters converged with those from allozyme studies and showed that N. suspectus, N. piraeeba and N. dorad clustered in a distinct clade that excluded other members of the genus. We therefore recognised them as an aggregate of species to reflect an isolation supported by both morphological and genetic data. Because relationships among the remaining Nomimoscolex representatives and other genera were generally poorly resolved, regardless of the database analysed, no action was taken to reorganise them into alternative groupings.     

Kakusan4 and Aminosan: two programs for comparing nonpartitioned, proportional and separate models for combined molecular phylogenetic analyses of multilocus sequence data

Proportional and separate models able to apply different combination of substitution rate matrix (SRM) and among-site rate variation model (ASRVM) to each locus are frequently used in phylogenetic studies of multilocus data. A proportional model assumes that branch lengths are proportional among partitions and a separate model assumes that each partition has an independent set of branch lengths. However, the selection from among nonpartitioned (i.e., a common combination of models is applied to all-loci concatenated sequences), proportional and separate models is usually based on the researcher's preference rather than on any information criteria. This study describes two programs, 'Kakusan4' (for DNA sequences) and 'Aminosan' (for amino-acid sequences), which allow the selection of evolutionary models based on several types of information criteria. The programs can handle both multilocus and single-locus data, in addition to providing an easy-to-use wizard interface and a noninteractive command line interface. In the case of multilocus data, SRMs and ASRVMs are compared at each locus and at all-loci concatenated sequences, after which nonpartitioned, proportional and separate models are compared based on information criteria. The programs also provide model configuration files for mrbayes, paup*, phyml, raxml and Treefinder to support further phylogenetic analysis using a selected model. When likelihoods are optimized by Treefinder, the best-fit models were found to differ depending on the data set. Furthermore, differences in the information criteria among nonpartitioned, proportional and separate models were much larger than those among the nonpartitioned models. These findings suggest that selecting from nonpartitioned, proportional and separate models results in a better phylogenetic tree. Kakusan4 and Aminosan are available at http://www.fifthdimension.jp/. They are licensed under gnugpl Ver.2, and are able to run on Windows, MacOS X and Linux.     

Taxonomic revision of three species of the genus Genarchopsis (Digenea: Hemiuroidea: Derogenidae) in Japan by molecular phylogenetic analyses

The taxonomic status of three nominal species of Genarchopsis (G. goppo Ozaki, 1925; G. gigi Yamaguti 1939; and G. fellicola Shimazu, 1995) (Digenea: Hemiuroidea: Derogenidae) was investigated by molecular phylogenetic analyses using partial sequences of the genomic ITS-1 region and the mitochondrial COI. The analyzed samples were divided into four groups: Lake Biwa, West Japan, Central Japan and G. fellicola. The Lake Biwa group, a sister taxon to the other three groups, was interpreted as G. gigi, so we concluded that G. gigi is valid; thus, this species is resurrected taxonomically. The specimens from the type host caught near the type locality of G. goppo were included in the West Japan group, so this group was regarded as G. goppo sensu stricto. Because the phylogenetic position of the Central Japan group could not be confirmed, it was identified tentatively as G. goppo, even though this species thus becomes paraphyletic. The taxonomic validity of G. fellicola was reconfirmed. The divergence time of G. gigi is discussed in relation to the geological history of Lake Biwa and the origin of host species.     

Evaluating molecular support for and against the monophyly of the Peritrichia and phylogenetic relationships within the Mobilida (Ciliophora,Oligohymenophorea)

Zhan, Z., Xu, K. & Dunthorn, M. (2012). Evaluating molecular support for and against the monophyly of the Peritrichia and phylogenetic relationships within the Mobilida (Ciliophora, Oligohymenophorea). —Zoologica Scripta,00, 000–000. An ongoing debate in ciliate molecular phylogenetic analyses is whether the Peritrichia – composed of the Sessilida and the Mobilida – are monophyletic. To further investigate this group, here we increase taxon sampling with four further mobilids: Trichodinella sp., Trichodina pectenis, Urceolaria serpularum and Urceolaria korschelti. Results show that support for and against monophyly of the peritrichs depends on different methods of alignment, and different methods of masking ambiguously aligned nucleotide positions. Results from constrained analyses also are dependent on different alignment and masking methods. We propose that there is no well‐supported SSU‐rDNA evidence supporting for and against the removal of the mobilids from the peritrichs. Within the Mobilida, we find that Trichodinella nest within Trichodina, Urceolaria branch basally, and Trichodina are non‐monophyletic, and suggest that Leiotrocha be formally transferred into Urceolaria. In addition, the data support that Urceolaria‐like denticles and Urceolaria‐/Trichodina‐like adoral ciliary spiral are plesiomorphic conditions within the mobilids.     

Mitochondrial cytochrome b of the Lyakhov mammoth (Proboscidea,Mammalia): new data and phylogenetic analyses of Elephantidae

The phylogenetic relationships between recent Elephantidae (Proboscidea, Mammalia), that is to say extant elephants (Asian and African) and extinct woolly mammoth, have remained unclear to date. The prevailing morphological scheme (mammoth grouped with Asian elephant) is either supported or questioned by the molecular results. Recently, the monophyly of woolly mammoths on mitochondrial grounds has been demonstrated (Thomas, et al., 2000), but it conflicts with previous studies (Barriel et al., 1999; Derenko et al., 1997). Here, we report the partial sequencing of two mitochondrial genes: 128 bp of 12S rDNA and 561 bp of cytochrome b for the Lyakhov mammoth, a 49,000-year-old Siberian individual. We use the most comprehensive sample of mammoth (11 sequences) to determine whether the sequences achieved by former studies were congruent or not. The monophyly of a major subset of mammoths sequences (including ours) is recovered. Such a result is assumed to be a good criterion for ascertaining the origin of ancient DNA. Our sequence is incongruent with that of Yang et al. (1996), though obtained for the same individual. As far as the latter sequence is concerned, a contamination by non-identified exogenous DNA is suspected. The robustness and reliability of the sister group relation between Mammuthus primigenius and Loxodonta africana are examined: down-weighting saturated substitutions has no impact on the topology; analyzing data partitions proves that the support of this clade can be assigned to the most conservative phylogenetic signal; insufficient taxonomic and/or characters sampling contributed to former discordant conclusions. We therefore assume the monophyly of "real mammoth sequences" and the (Mammuthus, Loxodonta) clade.     

Testing the molecular clock: molecular and paleontological estimates of divergence times in the Echinoidea (Echinodermata)

The phylogenetic relationships of 46 echinoids, with representatives from 13 of the 14 ordinal-level clades and about 70% of extant families commonly recognized, have been established from 3 genes (3,226 alignable bases) and 119 morphological characters. Morphological and molecular estimates are similar enough to be considered suboptimal estimates of one another, and the combined data provide a tree that, when calibrated against the fossil record, provides paleontological estimates of divergence times and completeness of their fossil record. The order of branching on the cladogram largely agrees with the stratigraphic order of first occurrences and implies that their fossil record is more than 85% complete at family level and at a resolution of 5-Myr time intervals. Molecular estimates of divergence times derived from applying both molecular clock and relaxed molecular clock models are concordant with estimates based on the fossil record in up to 70% of cases, with most concordant results obtained using Sanderson's semiparametric penalized likelihood method and a logarithmic-penalty function. There are 3 regions of the tree where molecular and fossil estimates of divergence time consistently disagree. Comparison with results obtained when molecular divergence dates are estimated from the combined (morphology + gene) tree suggests that errors in phylogenetic reconstruction explain only one of these. In another region the error most likely lies with the paleontological estimates because taxa in this region are demonstrated to have a very poor fossil record. In the third case, morphological and paleontological evidence is much stronger, and the topology for this part of the molecular tree differs from that derived from the combined data. Here the cause of the mismatch is unclear but could be methodological, arising from marked inequality of molecular rates. Overall, the level of agreement reached between these different data and methodological approaches leads us to believe that careful application of likelihood and Bayesian methods to molecular data provides realistic divergence time estimates in the majority of cases (almost 80% in this specific example), thus providing a remarkably well-calibrated phylogeny of a character-rich clade of ubiquitous marine benthic invertebrates.     

Sequence evolution in mitochondrial ribosomal and ND-1 genes in lepidoptera: implications for phylogenetic analyses.

A 2,256-bp sequence of the mitochondrial genome of a lepidopteran (Spodoptera frugiperda) contains tRNAs for valine and leucine, the 16S rRNA, and three-quarters of the ND-1 presumptive protein-coding gene. A 64-bp stretch of unknown function was located between the rRNA and leucine tRNA. Sequence divergence in the 16S rRNA obtained from alignment with published insect sequences is consistent with phylogenetic hypotheses, in that Diptera and Lepidoptera are more closely related to each other (24% sequence divergence) than either is to Hymenoptera (31%). Within the ND-1 gene, sequences for four additional Lepidoptera were generated for a 314-bp region and contrasted with published sequences for the locust and Drosophila. Sequence divergence in this region was consistent with accepted phylogenetic relationships, but results of parsimony analyses were not. Cladograms consistently recovered accepted higher level relationships (monophyly of Lepidoptera), despite high homoplasy, but were unable to resolve superfamily and family relationships within Lepidoptera, regardless of the outgroup or character subset analyzed. Character analysis indicated that homoplasy was decreased at higher levels when first- and second-codon sites were used exclusively. At the lowest level (families), resolution was enhanced by inclusion of third-codon sites. Inability of molecular data to recover a well-established phylogeny may be rectified by additional characters or taxa, but it is clear that homoplasy is sufficiently high to caution against the acceptance of relationships generated with this molecular region that are not extremely robust.