Varpu Vahtera

Comparative description of ten transcriptomes of newly sequenced invertebrates and efficiency estimation of genomic sampling in non-model taxa

IntroductionTraditionally, genomic or transcriptomic data have been restricted to a few model or emerging model organisms, and to a handful of species of medical and/or environmental importance. Next-generation sequencing techniques have the capability of yielding massive amounts of gene sequence data for virtually any species at a modest cost. Here we provide a comparative analysis of de novo assembled transcriptomic data for ten non-model species of previously understudied animal taxa.ResultscDNA libraries of ten species belonging to five animal phyla (2 Annelida [including Sipuncula], 2 Arthropoda, 2 Mollusca, 2 Nemertea, and 2 Porifera) were sequenced in different batches with an Illumina Genome Analyzer II (read length 100 or 150 bp), rendering between ca. 25 and 52 million reads per species. Read thinning, trimming, and de novo assembly were performed under different parameters to optimize output. Between 67,423 and 207,559 contigs were obtained across the ten species, post-optimization. Of those, 9,069 to 25,681 contigs retrieved blast hits against the NCBI non-redundant database, and approximately 50% of these were assigned with Gene Ontology terms, covering all major categories, and with similar percentages in all species. Local blasts against our datasets, using selected genes from major signaling pathways and housekeeping genes, revealed high efficiency in gene recovery compared to available genomes of closely related species. Intriguingly, our transcriptomic datasets detected multiple paralogues in all phyla and in nearly all gene pathways, including housekeeping genes that are traditionally used in phylogenetic applications for their purported single-copy nature.ConclusionsWe generated the first study of comparative transcriptomics across multiple animal phyla (comparing two species per phylum in most cases), established the first Illumina-based transcriptomic datasets for sponge, nemertean, and sipunculan species, and generated a tractable catalogue of annotated genes (or gene fragments) and protein families for ten newly sequenced non-model organisms, some of commercial importance (i.e., Octopus vulgaris). These comprehensive sets of genes can be readily used for phylogenetic analysis, gene expression profiling, developmental analysis, and can also be a powerful resource for gene discovery. The characterization of the transcriptomes of such a diverse array of animal species permitted the comparison of sequencing depth, functional annotation, and efficiency of genomic sampling using the same pipelines, which proved to be similar for all considered species. In addition, the datasets revealed their potential as a resource for paralogue detection, a recurrent concern in various aspects of biological inquiry, including phylogenetics, molecular evolution, development, and cellular biochemistry.

Evaluating Topological Conflict in Centipede Phylogeny Using Transcriptomic Data Sets

Relationships between the five extant orders of centipedes have been considered solved based on morphology. Phylogenies based on samples of up to a few dozen genes have largely been congruent with the morphological tree apart from an alternative placement of one order, the relictual Craterostigmomorpha, consisting of two species in Tasmania and New Zealand. To address this incongruence, novel transcriptomic data were generated to sample all five orders of centipedes and also used as a test case for studying gene-tree incongruence. Maximum likelihood and Bayesian mixture model analyses of a data set composed of 1,934 orthologs with 45% missing data, as well as the 389 orthologs in the least saturated, stationary quartile, retrieve strong support for a sister-group relationship between Craterostigmomorpha and all other pleurostigmophoran centipedes, of which the latter group is newly named Amalpighiata. The Amalpighiata hypothesis, which shows little gene-tree incongruence and is robust to the influence of among-taxon compositional heterogeneity, implies convergent evolution in several morphological and behavioral characters traditionally used in centipede phylogenetics, such as maternal brood care, but accords with patterns of first appearances in the fossil record.

Evolution of blindness in scolopendromorph centipedes Chilopoda Scolopendromorpha insight from an expanded sampling of molecular data

Relative to its diversity (34 genera, 700 species), Scolopendromorpha has been undersampled in molecular phylogenetic analyses compared with the other chilopod orders. Previous analyses based on morphology have not resolved several key controversies in systematics and evolutionary morphology unambiguously. Here we apply new molecular and morphological data to scolopendromorph phylogenetics, with a focus on the evolution of blindness. The taxonomic sample includes 19 genera, many lacking previous molecular data, and diverse, cosmopolitan genera of Scolopendridae are sampled by multiple species. Phylogenetic analysis with Direct Optimization used 94 morphological characters and ca. 4.5 kb of sequence data from two nuclear (18S and 28S rRNA) and two mitochondrial (16S rRNA and COI) loci. A single most‐parsimonious cladogram selected after sensitivity analyses resolves Scolopendromorpha as monophyletic, and divides it into a blind clade of three families (Plutoniumidae, Cryptopidae, Scolopocryptopidae) and its ocellate sister group, Scolopendridae. Some species‐rich, cosmopolitan genera (Cormocephalus, Otostigmus, Scolopendra) in Scolopendridae are non‐monophyletic, and in several instances (e.g. New and Old World Scolopendra) relationships are more congruent with geographical distributions than with traditional classifications. The tribe Asanadini is particularly subject to parameter‐sensitivity, nesting in the combined analysis within Scolopendrini but as sister to all other Scolopendrinae for molecular data alone. The total‐evidence tree unambiguously optimizes trunk segmentation: a 23‐segmented trunk has a single origin in the blind clade.

Running WILD: the case for exploring mixed parameter sets in sensitivity analysis

The robustness of clades to parameter variation may be a desirable quality or even a goal in phylogenetic analyses. Sensitivity analyses used to assess clade stability have invoked the incongruence length difference (ILD or WILD) metric, a measure of congruence among datasets, to compare a series of most‐parsimonious results from re‐running analyses under different analytical conditions. It is also common practice to select a single “optimal” parameter set that minimizes WILD across all parameter sets. However, the divergent molecular evolution of ribosomal genes and protein‐encoding genes—specifically the bias against transversion events in coding genes of conserved function—suggests that deployment of multiple parameter sets could outperform the use of a single parameter set applied to all molecules. We explored congruence in five published datasets by including mixed parameter sets in our sensitivity analysis. In four cases, mixed parameter sets outperformed the previously reported, single optimal parameter set. Conversely, multiple parameter sets did not outperform a single optimal parameter set in a case in which actual strong topological conflict exists between data partitions. Exploration of mixed parameter sets may prove useful when combining ribosomal and protein‐encoding genes, due to the relatively higher frequency of single‐ and double‐base pair indel events in the former, and the relatively lower frequency of transversions in the latter.
© The Willi Hennig Society 2010.

Phylogenetics of scolopendromorph centipedes: Can denser taxon sampling improve an artificial classification?

Abstract. Previous phylogenetic analyses of the centipede order Scolopendromorpha indicated a fundamental division into blind and ocellate clades. These analyses corroborated the monophyly of most families and tribes but suggested that several species-rich, cosmopolitan genera in traditional and current classifications are polyphyletic. Denser taxon sampling is applied to a dataset of 122 morphological characters and sequences for four nuclear and mitochondrial loci. Phylogenetic analyses including 98 species and subspecies of Scolopendromorpha employ parsimony under dynamic and static homology schemes as well as maximum likelihood and Bayesian inference of multiple sequence alignments. The monotypic Australian genera Notiasemus and Kanparka nest within Cormocephalus and Scolopendra, respectively, and the New Caledonian Campylostigmus is likewise a clade within Cormocephalus. New World Scolopendra are more closely related to Hemiscolopendra and Arthrorhabdus than to Scolopendra s.s., which is instead closely allied to Asanada; the tribe Asanadini nests within Scolopendrini for molecular and combined datasets. The generic classification of Otostigmini has a poor fit to phylogenetic relationships, although nodal support within this tribe is weak. New synonymies are proposed for Ectonocryptopinae Shelley & Mercurio, 2005 (= Newportiinae Pocock, 1896), Asanadini Verhoeff, 1907 (= Scolopendrini Leach, 1814), and Kanparka Waldock & Edgecombe, 2012 (= Scolopendra Linnaeus, 1758). Scolopendrid systematics largely depicts incongruence between phylogeny and classification rather than between morphology and molecules.

Anischia , Perothops and the phylogeny of Elateroidea (Coleoptera: Elateriformia)

The larvae of Anischia Fleutiaux and Perothops Laporte are described. Cladistic analyses based on adult and larval morphology, as well as CO1 sequence data, place both genera in the Eucnemidae clade within the Elateroidea (sensu stricto). The subfamily Anischiinae Fleutiaux, 1936 is placed in the family Eucnemidae in a clade containing the more derived eucnemid subfamilies (Melasinae, Eucneminae and Macraulacinae), while Perothops and Phyllocerus Lepeletier & Serville represent subfamilies basal to the remaining eucnemid taxa. The genus Afranischia Basilewsky, 1955 is synonymized with Anischia Fleutiaux, 1896, and Anischia boliviana Fleutiaux is selected as the type species of the latter. Three new species are described: Anischia bicolor (New Caledonia), Anischia kuscheli (New Caledonia) and Anischia monteithi (NE Australia), and Anischia stupenda Fleutiaux, 1897 is recorded from Australia. Anischia crassicornis Champion, 1897 is synonymized with Anischia mexicana Fleutiaux, 1896. One new combination is made, Anischia ruandana (Basilewsky, 1955).

Phylogeny of the Thylacosterninae (Coleoptera, Elateridae)

The phylogeny of the Elateridae subfamily Thylacosterninae was studied using morphological characters and mitochondrial 16S rDNA sequences. Five monophyletic groups were recovered: the Asian Cussolenis, the American Pterotarsus, Balgus and Thylacosternus, and the African “Cussolenis”, which is described here as Lumumbaia new genus Muona & Vahtera (type‐species Cussolenis attenuatus Fleutiaux, 1925). The following new combinations are made: Lumumbaia africanus (Fleutiaux, 1897) [Soleniscus], Lumumbaia praeustus (Fleutiaux, 1926) [Cussolenis], Lumumbaia nigripes (Fleutiaux, 1926) [Cussolenis], Lumumbaia robustus (Fleutiaux, 1925) [Cussolenis], Lumumbaia attenuatus (Fleutiaux, 1925) [Cussolenis] and Lumumbaia notabilis (Fleutiaux, 1926) [Cussolenis]. A key to the genera of the subfamily is provided. The data were efficient in recovering generic limits within the group. At species level the mitochondrial 16S rDNA data seemed to work unpredictably, either agreeing or not with traditional species‐level limits based on male genitalia and body structure. The evolution of bioluminescence is optimized as a feature originating in the ancestors of clicking elateroids. © The Willi Hennig Society 2009.

Spiracle structure in scolopendromorph centipedes (Chilopoda: Scolopendromorpha) and its contribution to phylogenetics

The spiracles of scolopendromorph centipedes have long been a source of systematic characters based on their segmental distribution and gross morphology, but microscopic investigations to date have documented only a small number of species. A scanning electron microscopic survey of 34 species that samples the major groups of Scolopendromorpha reveals variability in such features as the structure of the peritremal margin, specific kinds of sensilla and glandular pores on the peritrema, projections on the valves that subdivide the atrium (in Scolopendrinae), and the form of the trichomes around the tracheal openings. Adding new characters from the spiracles to recent morphological datasets for phylogenetic inference reinforces the monophyly of major groups of Scolopendridae and is particularly informative for relationships within Scolopendrini. A bowl-like atrium with the tracheae opening between humps in its floor is more widespread in Scolopendromorpha than previously reported. Shared presence of spiracle muscles in Cryptopidae and Scolopendrinae may reflect convergent evolution of a subatrial cavity in these groups rather than being an apomorphic character for Scolopendromorpha as a whole.

The molecular phylogeny of the Miarus campanulae (Coleoptera: Curculionidae) species group inferred from CO1 and ITS2 sequences

Miarus is a Holarctic weevil genus with morphologically very similar species, all breeding on Campanula plants or their close relatives. Two European members of this genus, Miarus campanulae (L.), the type species, and Miarus graminis (Bohemann) have recently been split into several new species on the basis of slight external variations. The separation of these new forms has proved impossible and new data was needed. Molecular data were gathered from specimens from a number of locations in Finland, Estonia, Denmark and Sweden. The regions sequenced were mitochondrial CO1 and nuclear ITS2. Both combined and separate datasets were analyzed using the optimization alignment program POY, with parsimony as the optimality criterion. The recently separated Miarus species was found to be indistinguishable from the traditionally recognized form on the basis of this sequence data. On the other hand, the traditionally recognized species were characterized by numerous synapomorphies. Our data suggest that recent studies have underestimated the morphological variation in this genus. We propose that this may also be true for many taxonomically problematic beetle complexes in well‐studied European regions. The idea that molecular evidence will inevitably reveal unnoticed cryptic variation may only apply to poorly known regions. Miarus fennicus Kangas, 1978 is placed as a junior synonym of Miarus campanulae (Linnaeus, 1767) syn. nov.

First Molecular Data and the Phylogenetic Position of the Millipede-Like Centipede Edentistoma octosulcatum Tömösváry, 1882 (Chilopoda: Scolopendromorpha: Scolopendridae)

Edentistoma octosulcatum Tömösváry, 1882, is a rare, superficially millipede-like centipede known only from Borneo and the Philippines. It is unique within the order Scolopendromorpha for its slow gait, robust tergites, and highly modified gizzard and mandible morphology. Not much is known about the biology of the species but it has been speculated to be arboreal with a possibly vegetarian diet. Until now its phylogenetic position within the subfamily Otostigminae has been based only on morphological characters, being variably ranked as a monotypic tribe (Arrhabdotini) or classified with the Southeast Asian genus Sterropristes Attems, 1934. The first molecular data for E. octosulcatum sourced from a newly collected specimen from Sarawak were analysed with and without morphology. Parsimony analysis of 122 morphological characters together with two nuclear and two mitochondrial loci resolves Edentistoma as sister group to three Indo-Australian species of Rhysida, this clade in turn grouping with Ethmostigmus, whereas maximum likelihood and parsimony analyses of the molecular data on their own ally Edentistoma with species of Otostigmus. A position of Edentistoma within Otostigmini (rather than being its sister group as predicted by the Arrhabdotini hypothesis) is consistently retrieved under different analytical conditions, but support values within the subfamily remain low for most nodes. The species exhibits strong pushing behaviour, suggestive of burrowing habits. Evidence against a suggested vegetarian diet is provided by observation of E. octosulcatum feeding on millipedes in the genus Trachelomegalus.