Molecular phylogeny of Gastrotricha on the basis of a comparison of the 18S rRNA genes: Rejection of the hypothesis of a relationship between Gastrotricha and Nematoda

Gastrotricha are the small meiobenthic acoelomate worms whose phylogenetic relationships between themselves and other invertebrates remain unclear, despite all attempts to clarify them on the basis of both morphological and molecular analyses. The complete sequences of the 18S rRNA genes (8 new and 7 known) were analyzed in 15 Gastrotricha species to test different hypotheses on the phylogeny of this taxon and to determine the reasons for the contradictions in earlier results. The data were analyzed using both maximum likelihood and Bayesian methods. Based on the results, it was assumed that gastrotrichs form a monophyletic group within the Spiralia clade, which also includes Gnathostomulida, Plathelminthes, Syndermata (Rotifera + Acanthocephala), Nemertea, and Lophotrochozoa. Statistical tests rejected a phylogenetic hypotheses considering Gastrotricha to be closely related to Nematoda and other Ecdysozoa or placing them at the base of the Bilateria tree, close to Acoela or Nemertodermatida. Among gastrotrichs, species belonging to the orders Chaetonotida and Macrodasyida form two well-supported clades. The analysis confirmed monophyly of the families Chaetonotidae and Xenotrichulidae from the order Chaetonida, as well as the families Turbanellidae and Thaumastodermatidae from the order Macrodasyida. Lepidodasyidae is a polyphyletic family, because the genus Mesodasys forms a sister group for Turbanellidae; genus Cephalodasys forms a separate branch at the base of Macrodasyida; and Lepidodasys groups with Neodasys between Thaumastodermatidae and Turbanellidae. To confirm these conclusions and to get an authentic view of the phylogeny of Gastrotricha, it is necessary to study more Gastrotricha species and to analyze some other genes.     

Gastrotricha: a marine sister for a freshwater puzzle

Background

Within an evolutionary framework of Gastrotricha Marinellina flagellata and Redudasys fornerise bear special interest, as they are the only Macrodasyida that inhabit freshwater ecosystems. Notwithstanding, these rare animals are poorly known; found only once (Austria and Brazil), they are currently systematised as incertae sedis. Here we report on the rediscovery of Redudasys fornerise, provide an account on morphological novelties and present a hypothesis on its phylogenetic relationship based on molecular data.

Methodology/Principal Findings

Specimens were surveyed using DIC microscopy and SEM, and used to obtain the 18 S rRNA gene sequence; molecular data was analyzed cladistically in conjunction with data from 42 additional species belonging to the near complete Macrodasyida taxonomic spectrum. Morphological analysis, while providing new information on taxonomically relevant traits (adhesive tubes, protonephridia and sensorial bristles), failed to detect elements of the male system, thus stressing the parthenogenetic nature of the Brazilian species. Phylogenetic analysis, carried out with ML, MP and Bayesian approaches, yielded topologies with strong nodal support and highly congruent with each other. Among the supported groups is the previously undocumented clade showing the alliance between Redudasys fornerise and Dactylopodola agadasys; other strongly sustained clades include the densely sampled families Thaumastodermatidae and Turbanellidae and most genera.

Conclusions/Significance

A reconsideration of the morphological traits of Dactylopodola agadasys in light of the new information on Redudasys fornerise makes the alliance between these two taxa very likely. As a result, we create Anandrodasys gen. nov. to contain members of the previously described D. agadasys and erect Redudasyidae fam. nov. to reflect this novel relationship between Anandrodasys and Redudasys. From an ecological perspective, the derived position of Redudasys, which is deeply nested within the Macrodasyida clade, unequivocally demonstrates that invasion of freshwater by gastrotrichs has taken place at least twice, in contrast with the single event hypothesis recently put forward.     

Molecular phylogeny of gastrotricha based on 18S rRNA genes comparison: rejection of hypothesis of relatedness with nematodes

Gastrotrichs are meiobenthic free-living aquatic worms whose phylogenetic and intra-group relationships remain unclear despite some attempts to resolve them on the base of morphology or molecules. In this study we analysed complete sequences of the 18S rRNA gene of 15 taxa (8 new and 7 published) to test numerous hypotheses on gastrotrich phylogeny and to verify whether controversial interrelationships from previous molecular data could be due to the short region available for analysis and the poor taxa sampling. Data were analysed using both maximum likelihood and Bayesian inference. Results obtained suggest that gastrotrichs, together with Gnathostomulida, Plathelminthes, Syndermata (Rotifera + Acanthocephala), Nemertea and Lophotrochozoa, comprise a clade Spiralia. Statistical tests reject phylogenetic hypotheses regarding Gastrotricha as close relatives of Nematoda and other Ecdysozoa or placing them at the base of bilaterian tree close to acoels and nemertodermatides. Within Gastrotricha, Chaetonotida and Macrodasyida comprise two well supported clades. Our analysis confirmed the monophyly of the Chaetonotidae and Xenotrichulidae within Chaetonida as well as Turbanellidae and Thaumastodermatidae within Macrodasyida. Mesodasys is a sister group of the Turbanellidae, and Lepidodasyidae appears to be a polyphyletic group as Cephalodasys forms a separate lineage at the base of macrodasyids, whereas Lepidodasys groups with Neodasys between Thaumastodermatidae and Turbanellidae. To infer a more reliable Gastrotricha phylogeny many species and additional genes should be involved in future analyses.     

Shrimps down under: evolutionary relationships of subterranean crustaceans from Western Australia (Decapoda: Atyidae: Stygiocaris)

Background

We investigated the large and small scale evolutionary relationships of the endemic Western Australian subterranean shrimp genus Stygiocaris (Atyidae) using nuclear and mitochondrial genes. Stygiocaris is part of the unique cave biota of the coastal, anchialine, limestones of the Cape Range and Barrow Island, most of whose nearest evolutionary relations are found in coastal caves of the distant North Atlantic. The dominance of atyids in tropical waters and their food resources suggest they are pivotal in understanding these groundwater ecosystems.

Methodology/Principle Findings

Our nuclear and mitochondrial analyses all recovered the Mexican cave genus Typhlatya as the sister taxon of Stygiocaris, rather than any of the numerous surface and cave atyids from Australia or the Indo-Pacific region. The two described Stygiocaris species were recovered as monophyletic, and a third, cryptic, species was discovered at a single site, which has very different physiochemical properties from the sites hosting the two described species.

Conclusions/Significance

Our findings suggest that Stygiocaris and Typhlatya may descend from a common ancestor that lived in the coastal marine habitat of the ancient Tethys Sea, and were subsequently separated by plate tectonic movements. This vicariant process is commonly thought to explain the many disjunct anchialine faunas, but has rarely been demonstrated using phylogenetic techniques. The Cape Range''s geological dynamism, which is probably responsible for the speciation of the various Stygiocaris species, has also led to geographic population structure within species. In particular, Stygiocaris lancifera is split into northern and southern groups, which correspond to population splits within other sympatric subterranean taxa.     

A world-wide perspective on crucifer speciation and evolution: phylogenetics,biogeography and trait evolution in tribe Arabideae

Background and Aims

Tribe Arabideae are the most species-rich monophyletic lineage in Brassicaceae. More than 500 species are distributed in the majority of mountain and alpine regions worldwide. This study provides the first comprehensive phylogenetic analysis for the species assemblage and tests for association of trait and characters, providing the first explanations for the enormous species radiation since the mid Miocene.

Methods

Phylogenetic analyses of DNA sequence variation of nuclear encoded loci and plastid DNA are used to unravel a reliable phylogenetic tree. Trait and ancestral area reconstructions were performed and lineage-specific diversification rates were calculated to explain various radiations in the last 15 Myr in space and time.

Key Results

A well-resolved phylogenetic tree demonstrates the paraphyly of the genus Arabis and a new systematic concept is established. Initially, multiple radiations involved a split between lowland annuals and mountain/alpine perennial sister species. Subsequently, increased speciation rates occur in the perennial lineages. The centre of origin of tribe Arabideae is most likely the Irano-Turanian region from which the various clades colonized the temperate mountain and alpine regions of the world.

Conclusions

Mid Miocene early diversification started with increased speciation rates due to the emergence of various annual lineages. Subsequent radiations were mostly driven by diversification within perennial species during the Pliocene, but increased speciation rates also occurred during that epoch. Taxonomic concepts in Arabis are still in need of a major taxonomic revision to define monophyletic groups.     

Phylogeny of Gastrotricha: a morphology-based framework of gastrotrich relationships

Currently, the phylum Gastrotricha is divided into the orders Macrodasyida and Chaetonotida, with the structure of the myoepithelial pharynx being an important distinguishing feature. Macrodasyida currently has six recognized families, and Chaetonotida comprises seven families. However, within-group relationships are poorly understood. To arrive at a better understanding of gastrotrich systematics and phylogeny, we performed the first cladistic analysis of nearly all known gastrotrich genera using 71 morphological characters. Results suggest that the Gastrotricha is a monophyletic group (supported by 82% of bootstrap replications) with its most primitive taxa distributed among the families Dactylopodolidae and Neodasyidae. Monophyly of Macrodasyida and Chaetonotida was supported by 90% and 52% bootstrap replications, respectively. Within the Macrodasyida, the families Dactylopodolidae, Turbanellidae, Macrodasyidae, and Thaumastodermatidae all formed monophyletic clades. The families Planodasyidae and Lepidodasyidae were paraphyletic. Among the Chaetonotida, the marine family Xenotrichulidae was monophyletic, supported by 51% of bootstrap replications. A second clade containing all freshwater families was supported by 62% bootstrap values. However, Chaetonotidae were paraphyletic. Using this analysis as a framework, we now can explore possible patterns of evolution within it, and arrive at a consensus of the gastrotrich ground pattern. Moreover, in future molecular studies of metazoan phylogeny, we will be able to select gastrotrich species that are more appropriate representatives of the phylum.     

Evolutionary lineages of nickel hyperaccumulation and systematics in European Alysseae (Brassicaceae): evidence from nrDNA sequence data

Background and Aims

Nickel (Ni) hyperaccumulation is a rare form of physiological specialization shared by a small number of angiosperms growing on ultramafic soils. The evolutionary patterns of this feature among European members of tribe Alysseae (Brassicaceae) are investigated using a phylogenetic approach to assess relationships among Ni hyperaccumulators at the genus, species and below-species level.

Methods

Internal transcribed spacer (ITS) sequences were generated for multiple accessions of Alysseae. Phylogenetic trees were obtained for the genera of the tribe and Alyssum sect. Odontarrhena. All accessions and additional herbarium material were tested for Ni hyperaccumulation with the dimethylglyoxime colorimetric method.

Key Results

Molecular data strongly support the poorly known hyperaccumulator endemic Leptoplax (Peltaria) emarginata as sister to hyperaccumulator species of Bornmuellera within Alysseae. This is contrary to current assumptions of affinity between L. emarginata and the non-hyperaccumulator Peltaria in Thlaspideae. The lineage Bornmuellera–Leptoplax is, in turn, sister to the two non-hyperaccumulator Mediterranean endemics Ptilotrichum rupestre and P. cyclocarpum. Low ITS sequence variation was found within the monophyletic Alyssum sect. Odontarrhena and especially in A. murale sensu lato. Nickel hyperaccumulation was not monophyletic in any of three main clades retrieved, each consisting of hyperaccumulators and non-hyperaccumulators of different geographical origin.

Conclusions

Nickel hyperaccumulation in Alysseae has a double origin, but it did not evolve in Thlaspideae. In Bornmuellera–Leptoplax it represents an early synapomorphy inherited from an ancestor shared with the calcicolous, sister clade of Mediterranean Ptilotrichum. In Alyssum sect. Odontarrhena it has multiple origins even within the three European clades recognized. Lack of geographical cohesion suggests that accumulation ability has been lost or gained over the different serpentine areas of south Europe through independent events of microevolutionary adaptation and selection. Genetic continuity and strong phenotypic plasticity in the A. murale complex call for a reduction of the number of Ni hyperaccumulator taxa formally recognized.     

Phylogenetic relationships among NE Atlantic Plocamionida Topsent (1927) (Porifera, Poecilosclerida): under-estimated diversity in reef ecosystems

Background

Small and cryptic sponges associated with cold-water coral reefs are particularly numerous and challenging to identify, but their ecological and biochemical importance is likely to compete with megabenthic specimens.

Methodology/Principal Findings

Here we use a combination of the standard M1M6 and I3M11 partitions of the COI fragment, partial rDNA 28S sequences and morphology to delineate small encrusting Plocamionida species. In total, 46 specimens were retrieved from seven shallow to deep-water coral locations, crossing 3,000 km along the European margins. Our work provides evidence that the Plocamionida ambigua f. tylotata and f. grandichelata can be considered valid species, whereas Plocamionida ambigua f. tornata corresponds to the species P. ambigua. Within the monophyletic group of Plocamionida, P. microcionides is shown as really divergent from the other taxa, and four putative new Plocamionida species are suggested.

Conclusions/Significance

This study shows that the use of molecular and morphological information in an integrative approach is a powerful tool for the identification of sponge species, and suggests that an under-estimated biodiversity of sponges occurs in cold-water coral reefs.     

Phylogenetic utility of the nuclear genes AGAMOUS 1 and PHYTOCHROME B in palms (Arecaceae): an example within Bactridinae

Background and Aims

Molecular phylogenetic studies of palms (Arecaceae) have not yet provided a fully resolved phylogeny of the family. There is a need to increase the current set of markers to resolve difficult groups such as the Neotropical subtribe Bactridinae (Arecoideae: Cocoseae). We propose the use of two single-copy nuclear genes as valuable tools for palm phylogenetics.

Methods

New primers were developed for the amplification of the AGAMOUS 1 (AG1) and PHYTOCHROME B (PHYB) genes. For the AGAMOUS gene, the paralogue 1 of Elaeis guineensis (EgAG1) was targeted. The region amplified contained coding sequences between the MIKC K and C MADS-box domains. For the PHYB gene, exon 1 (partial sequence) was first amplified in palm species using published degenerate primers for Poaceae, and then specific palm primers were designed. The two gene portions were sequenced in 22 species of palms representing all genera of Bactridinae, with emphasis on Astrocaryum and Hexopetion, the status of the latter genus still being debated.

Key Results

The new primers designed allow consistent amplification and high-quality sequencing within the palm family. The two loci studied produced more variability than chloroplast loci and equally or less variability than PRK, RPBII and ITS nuclear markers. The phylogenetic structure obtained with AG1 and PHYB genes provides new insights into intergeneric relationships within the Bactridinae and the intrageneric structure of Astrocaryum. The Hexopetion clade was recovered as monophyletic with both markers and was weakly supported as sister to Astrocaryum sensu stricto in the combined analysis. The rare Astrocaryum minus formed a species complex with Astrocaryum gynacanthum. Moreover, both AG1 and PHYB contain a microsatellite that could have further uses in species delimitation and population genetics.

Conclusions

AG1 and PHYB provide additional phylogenetic information within the palm family, and should prove useful in combination with other genes to improve the resolution of palm phylogenies.     

Geographically widespread swordfish barcode stock identification: a case study of its application

Background

The swordfish (Xiphias gladius) is a cosmopolitan large pelagic fish inhabiting tempered and tropical waters and it is a target species for fisheries all around the world. The present study investigated the ability of COI barcoding to reliably identify swordfish and particularly specific stocks of this commercially important species.

Methodology

We applied the classical DNA barcoding technology, upon a 682 bp segment of COI, and compared swordfish sequences from different geographical sources (Atlantic, Indian Oceans and Mediterranean Sea). The sequences of the 5′ hyper-variable fragment of the control region (5′dloop), were also used to validate the efficacy of COI as a stock-specific marker.

Case Report

This information was successfully applied to the discrimination of unknown samples from the market, detecting in some cases mislabeled seafood products.

Conclusions

The NJ distance-based phenogram (K2P model) obtained with COI sequences allowed us to correlate the swordfish haplotypes to the different geographical stocks. Similar results were obtained with 5′dloop. Our preliminary data in swordfish Xiphias gladius confirm that Cytochrome Oxidase I can be proposed as an efficient species-specific marker that has also the potential to assign geographical provenance. This information might speed the samples analysis in commercial application of barcoding.     

Reproductive isolation and ecological niche partition among larvae of the morphologically cryptic sister species Chironomus riparius and C. piger

Background

One of the central issues in ecology is the question what allows sympatric occurrence of closely related species in the same general area? The non-biting midges Chironomus riparius and C. piger, interbreeding in the laboratory, have been shown to coexist frequently despite of their close relatedness, similar ecology and high morphological similarity.

Methodology/Principal Findings

In order to investigate factors shaping niche partitioning of these cryptic sister species, we explored the actual degree of reproductive isolation in the field. Congruent results from nuclear microsatellite and mitochondrial haplotype analyses indicated complete absence of interspecific gene-flow. Autocorrelation analysis showed a non-random spatial distribution of the two species. Though not dispersal limited at the scale of the study area, the sister species occurred less often than expected at the same site, indicating past or present competition. Correlation and multiple regression analyses suggested the repartition of the available habitat along water chemistry gradients (nitrite, conductivity, CaCO₃), ultimately governed by differences in summer precipitation regime.

Conclusions

We show that these morphologically cryptic sister species partition their niches due to a certain degree of ecological distinctness and total reproductive isolation in the field. The coexistence of these species provides a suitable model system for the investigation of factors shaping the distribution of closely related, cryptic species.     

Cryptic species in tropic sands--interactive 3D anatomy, molecular phylogeny and evolution of meiofaunal Pseudunelidae (Gastropoda, Acochlidia)

Background

Towards realistic estimations of the diversity of marine animals, tiny meiofaunal species usually are underrepresented. Since the biological species concept is hardly applicable on exotic and elusive animals, it is even more important to apply a morphospecies concept on the best level of information possible, using accurate and efficient methodology such as 3D modelling from histological sections. Molecular approaches such as sequence analyses may reveal further, cryptic species. This is the first case study on meiofaunal gastropods to test diversity estimations from traditional taxonomy against results from modern microanatomical methodology and molecular systematics.

Results

The examined meiofaunal Pseudunela specimens from several Indo-Pacific islands cannot be distinguished by external features. Their 3D microanatomy shows differences in the organ systems and allows for taxonomic separation in some cases. Additional molecular analyses based on partial mitochondrial cytochrome c oxidase subunit I (COI) and 16S rRNA markers revealed considerable genetic structure that is largely congruent with anatomical or geographical patterns. Two new species (Pseudunela viatoris and P. marteli spp. nov.) are formally described integrating morphological and genetic analyses. Phylogenetic analysis using partial 16S rRNA, COI and the nuclear 18S rRNA markers shows a clade of Pseudunelidae species as the sister group to limnic Acochlidiidae. Within Pseudunela, two subtypes of complex excretory systems occur. A complex kidney already evolved in the ancestor of Hedylopsacea. Several habitat shifts occurred during hedylopsacean evolution.

Conclusions

Cryptic species occur in tropical meiofaunal Pseudunela gastropods, and likely in other meiofaunal groups with poor dispersal abilities, boosting current diversity estimations. Only a combined 3D microanatomical and molecular approach revealed actual species diversity within Pseudunela reliably. Such integrative methods are recommended for all taxonomic approaches and biodiversity surveys on soft-bodied and small-sized invertebrates. With increasing taxon sampling and details studied, the evolution of acochlidian panpulmonates is even more complex than expected.     

The complete mitochondrial genomes of six heterodont bivalves (Tellinoidea and Solenoidea): variable gene arrangements and phylogenetic implications

Background

Taxonomy and phylogeny of subclass Heterodonta including Tellinoidea are long-debated issues and a complete agreement has not been reached yet. Mitochondrial (mt) genomes have been proved to be a powerful tool in resolving phylogenetic relationship. However, to date, only ten complete mitochondrial genomes of Heterodonta, which is by far the most diverse major group of Bivalvia, have been determined. In this paper, we newly sequenced the complete mt genomes of six species belonging to Heterodonta in order to resolve some problematical relationships among this subclass.

Principal Findings

The complete mt genomes of six species vary in size from 16,352 bp to 18,182. Hairpin-like secondary structures are found in the largest non-coding regions of six freshly sequenced mt genomes, five of which contain tandem repeats. It is noteworthy that two species belonging to the same genus show different gene arrangements with three translocations. The phylogenetic analysis of Heterodonta indicates that Sinonovacula constricta, distant from the Solecurtidae belonging to Tellinoidea, is as a sister group with Solen grandis of family Solenidae. Besides, all five species of Tellinoidea cluster together, while Sanguinolaria diphos has closer relationship with Solecurtus divaricatus, Moerella iridescens and Semele scaba rather than with Sanguinolaria olivacea.

Conclusions/Significance

By comparative study of gene order rearrangements and phylogenetic relationships of the five species belonging to Tellinoidea, our results support that comparisons of mt gene order rearrangements, to some extent, are a useful tool for phylogenetic studies. Based on phylogenetic analyses of multiple protein-coding genes, we prefer classifying the genus Sinonovacula within the superfamily Solenoidea and not the superfamily Tellinoidea. Besides, both gene order and sequence data agree that Sanguinolaria (Psammobiidae) is not monophyletic. Nevertheless, more studies based on more mt genomes via combination of gene order and phylogenetic analysis are needed to further understand the phylogenetic relationships in subclass Heterodonta.     

Comparing the usefulness of distance, monophyly and character-based DNA barcoding methods in species identification: a case study of neogastropoda

Background

DNA barcoding has recently been proposed as a promising tool for the rapid species identification in a wide range of animal taxa. Two broad methods (distance and monophyly-based methods) have been used. One method is based on degree of DNA sequence variation within and between species while another method requires the recovery of species as discrete clades (monophyly) on a phylogenetic tree. Nevertheless, some issues complicate the use of both methods. A recently applied new technique, the character-based DNA barcode method, however, characterizes species through a unique combination of diagnostic characters.

Methodology/Principal Findings

Here we analyzed 108 COI and 102 16S rDNA sequences of 40 species of Neogastropoda from a wide phylogenetic range to assess the performance of distance, monophyly and character-based methods of DNA barcoding. The distance-based method for both COI and 16S rDNA genes performed poorly in terms of species identification. Obvious overlap between intraspecific and interspecific divergences for both genes was found. The “10× rule” threshold resulted in lumping about half of distinct species for both genes. The neighbour-joining phylogenetic tree of COI could distinguish all species studied. However, the 16S rDNA tree could not distinguish some closely related species. In contrast, the character-based barcode method for both genes successfully identified 100% of the neogastropod species included, and performed well in discriminating neogastropod genera.

Conclusions/Significance

This present study demonstrates the effectiveness of the character-based barcoding method for species identification in different taxonomic levels, especially for discriminating the closely related species. While distance and monophyly-based methods commonly use COI as the ideal gene for barcoding, the character-based approach can perform well for species identification using relatively conserved gene markers (e.g., 16S rDNA in this study). Nevertheless, distance and monophyly-based methods, especially the monophyly-based method, can still be used to flag species.     

Genetic structure of the tiger mosquito, Aedes albopictus, in Cameroon (Central Africa)

Background

Aedes albopictus (Skuse, 1884) (Diptera: Culicidae), a mosquito native to Asia, has recently invaded all five continents. In Central Africa it was first reported in the early 2000s, and has since been implicated in the emergence of arboviruses such as dengue and chikungunya in this region. Recent genetic studies of invasive species have shown that multiple introductions are a key factor for successful expansion in new areas. As a result, phenotypic characters such as vector competence and insecticide susceptibility may vary within invasive pest species, potentially affecting vector efficiency and pest management. Here we assessed the genetic variability and population genetics of Ae. albopictus isolates in Cameroon (Central Africa), thereby deducing their likely geographic origin.

Methods and Results

Mosquitoes were sampled in 2007 in 12 localities in southern Cameroon and analyzed for polymorphism at six microsatellite loci and in two mitochondrial DNA regions (ND5 and COI). All the microsatellite markers were successfully amplified and were polymorphic, showing moderate genetic structureamong geographic populations (F_ST = 0.068, P<0.0001). Analysis of mtDNA sequences revealed four haplotypes each for the COI and ND5 genes, with a dominant haplotype shared by all Cameroonian samples. The weak genetic variation estimated from the mtDNA genes is consistent with the recent arrival of Ae. albopictus in Cameroon. Phylogeographic analysis based on COI polymorphism indicated that Ae. albopictus populations from Cameroon are related to tropical rather than temperate or subtropical outgroups.

Conclusion

The moderate genetic diversity observed among Cameroonian Ae. albopictus isolates is in keeping with recent introduction and spread in this country. The genetic structure of natural populations points to multiple introductions from tropical regions.     

Molecular Identification of Birds: Performance of Distance-Based DNA Barcoding in Three Genes to Delimit Parapatric Species

Background

DNA barcoding based on the mitochondrial cytochrome oxidase subunit I gene (cox1 or COI) has been successful in species identification across a wide array of taxa but in some cases failed to delimit the species boundaries of closely allied allopatric species or of hybridising sister species.

Methodology/Principal Findings

In this study we extend the sample size of prior studies in birds for cox1 (2776 sequences, 756 species) and target especially species that are known to occur parapatrically, and/or are known to hybridise, on a Holarctic scale. In order to obtain a larger set of taxa (altogether 2719 species), we include also DNA sequences of two other mitochondrial genes: cytochrome b (cob) (4614 sequences, 2087 species) and 16S (708 sequences, 498 species). Our results confirm the existence of a wide gap between intra- and interspecies divergences for both cox1 and cob, and indicate that distance-based DNA barcoding provides sufficient information to identify and delineate bird species in 98% of all possible pairwise comparisons. This DNA barcoding gap was not statistically influenced by the number of individuals sequenced per species. However, most of the hybridising parapatric species pairs have average divergences intermediate between intraspecific and interspecific distances for both cox1 and cob.

Conclusions/Significance

DNA barcoding, if used as a tool for species discovery, would thus fail to identify hybridising parapatric species pairs. However, most of them can probably still assigned to known species by character-based approaches, although development of complementary nuclear markers will be necessary to account for mitochondrial introgression in hybridising species.     

Phylogeny and Classification of the Trapdoor Spider Genus Myrmekiaphila: An Integrative Approach to Evaluating Taxonomic Hypotheses

Background

Revised by Bond and Platnick in 2007, the trapdoor spider genus Myrmekiaphila comprises 11 species. Species delimitation and placement within one of three species groups was based on modifications of the male copulatory device. Because a phylogeny of the group was not available these species groups might not represent monophyletic lineages; species definitions likewise were untested hypotheses. The purpose of this study is to reconstruct the phylogeny of Myrmekiaphila species using molecular data to formally test the delimitation of species and species-groups. We seek to refine a set of established systematic hypotheses by integrating across molecular and morphological data sets.

Methods and Findings

Phylogenetic analyses comprising Bayesian searches were conducted for a mtDNA matrix composed of contiguous 12S rRNA, tRNA-val, and 16S rRNA genes and a nuclear DNA matrix comprising the glutamyl and prolyl tRNA synthetase gene each consisting of 1348 and 481 bp, respectively. Separate analyses of the mitochondrial and nuclear genome data and a concatenated data set yield M. torreya and M. millerae paraphyletic with respect to M. coreyi and M. howelli and polyphyletic fluviatilis and foliata species groups.

Conclusions

Despite the perception that molecular data present a solution to a crisis in taxonomy, studies like this demonstrate the efficacy of an approach that considers data from multiple sources. A DNA barcoding approach during the species discovery process would fail to recognize at least two species (M. coreyi and M. howelli) whereas a combined approach more accurately assesses species diversity and illuminates speciation pattern and process. Concomitantly these data also demonstrate that morphological characters likewise fail in their ability to recover monophyletic species groups and result in an unnatural classification. Optimizations of these characters demonstrate a pattern of “Dollo evolution” wherein a complex character evolves only once but is lost multiple times throughout the group''s history.     

Phylogenetic relationships among the colobine monkeys revisited: new insights from analyses of complete mt genomes and 44 nuclear non-coding markers

Background

Phylogenetic relationships among Asian and African colobine genera have been disputed and are not yet well established. In the present study, we revisit the contentious relationships within the Asian and African Colobinae by analyzing 44 nuclear non-coding genes (>23 kb) and mitochondrial (mt) genome sequences from 14 colobine and 4 non-colobine primates.

Principal Findings

The combined nuclear gene and the mt genome as well as the combined nuclear and mt gene analyses yielded different phylogenetic relationships among colobine genera with the exception of a monophyletic ‘odd-nosed’ group consisting of Rhinopithecus, Pygathrix and Nasalis, and a monophyletic African group consisting of Colobus and Piliocolobus. The combined nuclear data analyses supported a sister-grouping between Semnopithecus and Trachypithecus, and between Presbytis and the odd-nosed monkey group, as well as a sister-taxon association of Pygathrix and Rhinopithecus within the odd-nosed monkey group. In contrast, mt genome data analyses revealed that Semnopithecus diverged earliest among the Asian colobines and that the odd-nosed monkey group is sister to a Presbytis and Trachypithecus clade, as well as a close association of Pygathrix with Nasalis. The relationships among these genera inferred from the analyses of combined nuclear and mt genes, however, varied with the tree-building methods used. Another remarkable finding of the present study is that all of our analyses rejected the recently proposed African colobine paraphyly and hybridization hypothesis and supported reciprocal monophyly of the African and Asian groups.

Significance

The phylogenetic utility of large-scale new non-coding genes was assessed using the Colobinae as a model, We found that these markers were useful for distinguishing nodes resulting from rapid radiation episodes such as the Asian colobine radiation. None of these markers here have previously been used for colobine phylogenetic reconstruction, increasing the spectrum of molecular markers available to mammalian systematics.     

Macrodasyida (Gastrotricha): a cladistic analysis of morphology

Abstract. A cladistic analysis based on 33 morphological characters was performed for the 31 genera currently assigned to the order Macrodasyida (Gastrotricha). Outgroup analysis indicated that the order is monophyletic and that it is defined by the structure of the pharynx and the complex distribution of duo-gland adhesive organs. Of the 6 currently recognized families in Macrodasyida, our analysis confirmed that 4 families are monophyletic: Dactylopodolidae, Macrodasyidae, Thaumastodermatidae and Turbanellidae. Dactylopodolidae was further confirmed as the most basal family within the order based on the retention of several plesiomorphies. The other three families have well-defined autapomorphies but will require further investigation to increase inter- and intrafamilial phylogenetic resolution. Planodasyidae appeared to be a paraphyletic taxon with no obvious autapomorphies; genera clustered among members of a polyphyletic family, Lepidodasyidae. We recommend that future research on macrodasyidan phylogeny focus on issues of comparative morphology and ultrastructure in lesser-known taxa such as the Dactylopodolidae, and on the taxa Lepidodasyidae and Planodasyidae.