An integrative approach to characterize cryptic species in the Thoracostoma trachygaster Hope, 1967 complex (Nematoda: Leptosomatidae)

Nematode diversity may seriously be underestimated when taking into account cryptic speciation. Thoracostoma trachygaster is commonly found in kelp holdfasts along the California coastline and was recently shown to consist of at least two distinct molecular clades (I and II). Here, we provide detailed morphological analysis of both clades, based on measurements taken from video vouchers of respectively eight and 16 individuals from the previous study, as well as 80 newly collected specimens from four Californian beaches. The latter were vouchered, measured, and then subjected to molecular analyses of the mitochondrial cytochrome oxidase c subunit I (COI) gene, and the ribosomal D2D3 and internal transcribed spacer (ITS) regions. This integrative approach shows that the three molecular clades are phylogenetically and morphologically distinct species, but a combination of morphological characters is needed to distinguish them. Two new species, Thoracostoma fatimae sp. nov. and Thoracostoma igniferum sp. nov. , are identified and described. The spicule length of T. fatimae sp. nov. is significantly shorter than that of T. trachygaster. Thoracostoma igniferum sp. nov. can be distinguished by the irregular posterior edge of the cephalic capsule and the two internal subdorsal tropis‐like projections in the wall of the cephalic capsule, which are lacking in T. fatimae sp. nov. and T. trachygaster. © 2012 The Linnean Society of London, Zoological Journal of the Linnean Society, 2012, 164 , 18–35.     

Diversity of the rotifer Brachionus plicatilis species complex (Rotifera: Monogononta) in Iran through integrative taxonomy

Faunistic survey using a DNA taxonomy approach may provide different results from morphological methods, especially for small and understudied animals. In this study, we report the results from morphometric analyses (linear measurements of the lorica) and DNA taxonomy (generalized mixed Yule coalescent model on the barcoding mtDNA locus cytochrome c oxidase subunit I) performed on 15 clonal lineages of the rotifer Brachionus plicatilis species complex from six Iranian inland saltwaters. The DNA taxonomy approach found more units of diversity (four) than the morphometric approach (two) in the studied rotifers. Three of the taxa identified in this study are already known as described valid species or as‐yet unnamed lineages, but a new, additional lineage is also identified from Iran. © 2014 The Linnean Society of London     

Starting to unravel the toxoglossan knot: molecular phylogeny of the "turrids" (Neogastropoda: Conoidea)

The superfamily Conoidea is one of the most speciose groups of marine mollusks, with estimates of about 340 recent valid genera and subgenera, and 4000 named living species. Previous classifications were based on shell and anatomical characters, and clades and phylogenetic relationships are far from well assessed. Based on a dataset of ca. 100 terminal taxa belonging to 57 genera, information provided by fragments of one mitochondrial (COI) and three nuclear (28S, 18S and H3) genes is used to infer the first molecular phylogeny of this group. Analyses are performed on each gene independently as well as for a data matrix where all genes are concatenated, using Maximum Likelihood, Maximum Parsimony and Bayesian approaches. Several well-supported clades are defined and are only partly identifiable to currently recognized families and subfamilies. The nested sampling used in our study allows a discussion of the classification at various taxonomical levels, and several genera, subfamilies and families are found polyphyletic.     

An integrative approach to species delineation incorporating different species concepts: a case study of Limnadopsis (Branchiopoda: Spinicaudata)

The unambiguous delineation and identification of species remain central problems in systematic and taxonomic studies. Species delineation depends on the data utilized and the species concept applied. In recent years, morphology‐based species delineation has been complemented by DNA sequence data, leading to an integrative taxonomy. Such integrative approaches, however, are hampered by the partial incongruence of the various data types with certain species concepts. In this study, we delineated Australian Limnadopsis species employing one mitochondrial (cytochrome c oxidase subunit I, COI) and one nuclear (elongation factor 1α, EF1α) marker and a morphological character apparently part of the specific mate recognition complex, and therefore potentially indicative of reproductive isolation. By integrating the data over various species concepts (e.g. the ‘biological’, ‘Hennigian’, ‘recognition’, ‘phylogenetic’ and ‘evolutionary’ species concepts), the delineation of most species becomes straightforward and unambiguous. Conflicts are particularly interesting as they reveal different aspects of speciation considering the various species concepts. Our study emphasizes the benefits of a truly integrative approach to taxonomy. By combining molecular data with morphological characters indicative of reproductive isolation, it is possible to delineate species integrating not only different data types, but also different underlying species concepts. Overall, 11 Limnadopsis species could be delineated, including all eight currently recognized species, and three so far undescribed species. Most species were congruently delineated under all species concepts. A strict application of the evolutionary species concept, however, would have further split L. parvispinus into two species on the basis of the COI data. In addition, Limnadopsis tatei is consistently split into two sympatrically occurring species under all applied species concepts. © 2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2011, 104 , 575–599.     

An integrative taxonomic approach to infer the systematic position of Chalepotaxis Ancey, 1887 (Gastropoda: Stylommatophora: Helicarionidae)

In the absence of knowledge about its anatomy, the systematic position of the genus Chalepotaxis, which inhabits a large part of South East Asia, has been historically dubious. The genitalia, sole of the foot and the caudal region of the type species, Chalepotaxis infantilis (Gredler, V. 1881. Zur Conchylien-Fauna von China. III. Stück. Jahrbücher der Deutschen Malakozoologischen Gesellschaft 8, 110–128), are described, which place this genus into the Helicarionidae family. DNA markers (cytochrome oxidase subunit I [COI] and 28S rRNA genes) corroborate this assignment and unequivocally remove the species from the Camaenidae (=Bradybaenidae) and Ariophantidae, where it had previously been placed. Radular morphology of the type species is also re-described and illustrated.     

Paramacrobiotus lachowskae,a new species of Tardigrada from Colombia (Eutardigrada: Parachela: Macrobiotidae)

In this article we describe a new Paramacrobiotus species of the areolatus group by means of integrative taxonomy. Together with the detailed morphological and morphometric data (obtained from light and scanning electron microscopy) we also provide DNA sequences of four universal molecular markers used in tardigrade taxonomy (three nDNA fragments: 18S rRNA, 28S rRNA, ITS-2, and one mtDNA fragment: COI). The animals of Paramacrobiotus lachowskae sp. nov. are similar to several species of the areolatus complex and the eggshell ornamentation is similar to that of two species of the richtersi group. Therefore, the new species can be easily distinguished from the areolatus group species by egg morphology (dome-like, wrinkled processes with long flexible spines/filaments covered by fine short hairs) and from species of the richtersi complex by the absence of the microplacoid. Paramacrobiotus lachowskae sp. nov. is the 43rd species reported from Colombia and fifth Paramacrobiotus species from this South American country.

http://zoobank.org/urn:lsid:zoobank.org:pub:FEDC11D3-DCCF-4699-A704-CE6DEA1CDED4     

An integrative approach to delimiting species in a rare but widespread mycoheterotrophic orchid

In the spirit of recent calls for species delimitation studies to become more pluralistic, incorporating multiple sources of evidence, we adopted an integrative, phylogeographic approach to delimiting species and evolutionarily significant units (ESUs) in the Corallorhiza striata species complex. This rare, North American, mycoheterotrophic orchid has been a taxonomic challenge regarding species boundaries, displaying complex patterns of variation and reduced vegetative morphology. We employed plastid DNA, nuclear DNA and morphometrics, treating the C. striata complex as a case study for integrative species delimitation. We found evidence for the differentiation of the endangered C. bentleyi (eastern USA) + C. striata var. involuta (Mexico) from the remaining C. striata (= C. striata s.s.; USA, Canada, Mexico). Corallorhiza striata involuta and C. bentleyi, disjunct by thousands of kilometres (Mexico‐Appalachia), were genetically identical but morphologically distinct. Evidence suggests the C. striata complex represents three species: C. bentleyi, C. involuta and a widespread C. striata s.s under operational criteria of diagnosability and common allele pools. In contrast, Bayesian coalescent estimation delimited four species, but more informative loci and a resultant species tree will be needed to place higher confidence in future analyses. Three distinct groupings were identified within C. striata s.s., corresponding to C. striata striata, C. striata vreelandii, and Californian accessions, but these were not delimited as species because of occupying a common allele pool. Each comprises an ESU, warranting conservation considerations. This study represents perhaps the most geographically comprehensive example of integrative species delimitation for any orchid and any mycoheterotroph.     

Phylogeny of the Braconinae (Hymenoptera: Braconidae): A new tribal order!

Generic relationships within the parasitoid wasp subfamily Braconinae are assessed based on a molecular phylogenetic analysis of four gene fragments: mitochondrial cytochrome c oxidase subunit I, 16S rDNA, nuclear 28S D2-D3 rDNA and elongation factor 1-alpha. Our results support the recognition of Aphrastobraconini, Braconini and Coeloidini plus three new tribes: Compsobraconini tribus nov ., Tropobraconini tribus nov . and Virgulibraconini tribus nov . The first of these new tribes is restricted to the New World; the second includes the Old World genera Tropobracon Cameron, Trispinaria Quicke and Grangerbracon Samartsev and Belokobylskij and possibly others, whereas the third comprises the Australian genus Virgulibracon Quicke, plus several other described and undescribed Australian genera. Consistent placement of Amyosoma Viereck with members of the Virgulibraconini tribus nov . is discussed, whereas Amyosoma is left currently unplaced. A preliminary key to tribes is presented, and the characters used to differentiate between Aphrastobraconini and Braconini are revised. Megacoeloides Quicke was never recovered with Coeloides, the type genus of Coeloidini, so it is treated as Braconinae incertae sedis. By combining molecular and morphological traits, nearly all valid genera are assigned to tribes, and the possible relationships of the remainder are discussed. Alienoclypeus Shenefelt, 1978 is synonymised with Atanycolus Förster, 1862 (Atanycolus insolitus (Shenefelt) comb. nov .). Several new genera have been revealed and will be described elsewhere.     

An integrative approach to species delimitation in Echinodorus (Alismataceae) and the description of two new species

Summary  Taxonomy of the genus Echinodorus is partially revisited in the light of current understanding of the phylogenetic relationships of the genus. As a result of new taxonomy, the species status of some previously synonymised taxa are restored, other names are synonymised, and some nomenclatural problems unnoticed by previous authors are resolved. Two new species, Echinodorus reptilis and E. emersus are described. The subgeneric divisions of the genus are not accepted, and all subspecific taxa are either rejected or established as species. As a result, 28 species based on a phylogenetic species concept are now recognised in Echinodorus and an identification key to these species is provided.     

Species delimitation in the Andean grasshopper genus Orotettix Ronderos & Carbonell (Orthoptera: Melanoplinae): an integrative approach combining morphological,molecular and biogeographical data

The reciprocal illumination nature of integrative taxonomy through hypothesis testing, corroboration and revision is a powerful tool for species delimitation as more than one source has to support the hypothesis of a new species. In this study, we applied an integrative taxonomy approach combining molecular and morphological data sets with distributional patterns to examine the level of differentiation between and within the grasshopper Orotettix species. Orotettix was described based on five valid species distributed in the Andes of Peru. In our study, initially a molecular‐based hypothesis was postulated and tested against morphological data and geographical patterns of distribution. Results from molecular and morphological analyses showed agreement among the species delimitation in Orotettix, and were also consistent with the geographical distribution. The analyses allowed us to delimit five new species for the genus ( O. lunatus sp. nov. , O . astreptos sp. nov. , O. colcaensis sp. nov. , O . paucartambensis sp. nov. and O . dichrous sp. nov. ) from the Eastern and Western Cordilleras of Peru. We also provide critical knowledge on the phylogenetic relationships and distribution of the genus and conduct a revision of Orotettix. © 2015 The Linnean Society of London     

Multiple species delimitation approaches with COI barcodes poorly fit each other and morphospecies – An integrative taxonomy case of Sri Lankan Sericini chafers (Coleoptera: Scarabaeidae)

DNA taxonomy including barcoding and metabarcoding is widely used to explore the diversity in biodiversity hotspots. In most of these hotspot areas, chafers are represented by a multitude of species, which are well defined by the complex shape of male genitalia. Here, we explore how well COI barcode data reflect morphological species entities and thus their usability for accelerated species inventorization. We conducted dedicated field surveys in Sri Lanka to collect the species‐rich and highly endemic Sericini chafers (Coleoptera: Scarabaeidae). Congruence among results of a series of protocols for de novo species delimitation and with morphology‐based species identifications was investigated. Different delimitation methods, such as the Poisson tree processes (PTP) model, Statistical Parsimony Analysis (TCS), Automatic Barcode Gap Discovery (ABGD), Assemble Species by Automatic Partitioning (ASAP), and Barcode Index Number (BIN) assignments, resulted in different numbers of molecular operational taxonomic units (MOTUs). All methods showed both over‐splitting and lumping of morphologically identified species. Only 18 of the observed 45 morphospecies perfectly matched MOTUs from all methods. The congruence of delimitation between MOTUs and morphospecies expressed by the match ratio was low, ranging from 0.57 to 0.67. TCS and multirate PTP (mPTP) showed the highest match ratio, while (BIN) assignment resulted in the lowest match ratio and most splitting events. mPTP lumped more species than any other method. Principal coordinate analysis (PCoA) on a match ratio‐based distance matrix revealed incongruent outcomes of multiple DNA delimitation methods, although applied to the same data. Our results confirm that COI barcode data alone are unlikely to correctly delimit all species, in particular, when using only a single delimitation approach. We encourage the integration of various approaches and data, particularly morphology, to validate species boundaries.     

Molecular phylogenetics of Triculine snails (Gastropoda: Pomatiopsidae) from southern China

Triculine (Gastropoda: Rissooidea: Pomatiopsidae) snails are involved in the transmission of schistosomiasis and paragonimiasis; their distributions are mainly across southeastern Asia and southern China. In the present investigation, partial sequences of COI, 16S, and 28S were examined to infer the phylogenetic relationships among the species rich and poorly understood gastropod. Samples were collected from 12 geographic locations in six provinces of southern China. Several methods such as maximum parsimony, maximum likelihood and distance analysis were used in phylogenetic analyses among these taxa. The resultant phylogenetic trees showed a similar topology irrespective of the phylogenetic methods used. The taxa fell into two clades, with those from Fujian, Guangxi, and Zhejiang Provinces in one clade and those from Hunan, Sichuan and Hubei in the other. Among the taxa in Hubei Province, five formed a monophyletic clade, but Tricula sp. H-SHY fell into a sister clade of Tricula hortensis of Sichuan, whilst Tricula hongshanensis formed a single clade. Sister taxa Tricula pingi and Tricula hsiangi formed well-supported clade within almost all the trees. These results, while preliminary, represent the first attempt to reconstruct a phylogeny for Triculinae across China.     

DNA barcoding of six Ceroplastes species (Hemiptera: Coccoidea: Coccidae) from China

Ceroplastes Gray (wax scales) is one of the genera of Coccidae, most species of which are considered to be serious economic pests. However, identification of Ceroplastes species is always difficult owing to the shortage of easily distinguishable morphological characters. Mitochondrial cytochrome c oxidase I (COI) sequences (or DNA barcodes) and the D2 expansion segments of the large subunit ribosomal RNA gene 28S were used for accurate identification of six Ceroplastes species (C. floridensis Comstock, C. japonicus Green, C. ceriferus (Fabricius), C. pseudoceriferus Green, C. rubens Maskell and C. kunmingensis Tang et Xie) from 20 different locations in China. For COI data, low G·C content was found in all species, averaging about 20.4%. Sequence divergences (K2P) between congeneric species averaged 12.19%, while intra‐specific divergences averaged 0.42%. All 112 samples fell into six reciprocally monophyletic clades in the COI neighbour‐joining (NJ) tree. The NJ tree inferred from 28S showed almost same results, but samples of two closely related species, C. ceriferus and C. pseudoceriferus, were clustered together. This research indicates that the standard barcode region of COI can efficiently identify similar Ceroplastes species. This study provides an example of the usefulness of barcoding for Ceroplastes identification.     

DNA-based species delimitation in algae

Given the problems of species delimitation in algae using morphology or sexual compatibility, molecular data are becoming the standard for delimiting species and testing their traditional boundaries. The idea that species are separately evolving metapopulation lineages, along with theoretical progress in phylogenetic and population genetic analyses, has led to the development of new methods of species delimitation. We review these recent developments in DNA-based species delimitation methods, and discuss how they have changed and continue to change our understanding of algal species boundaries. Although single-locus approaches have proven effective for a first rapid and large-scale assessment of species diversity, species delimitation based on single gene trees falls short due to gene tree–species tree incongruence, caused by confounding processes like incomplete lineage sorting, trans-species polymorphism, hybridization and introgression. Data from unlinked loci and multi-species coalescent methods, which combine principles from phylogenetics and population genetics, may now be able to account for these complicating factors. Several of these methods also provide statistical support regarding species boundaries, which is important because speciation is a process and therefore uncertainty about precise species boundaries is inevitable in recently diverged lineages.     

An integrative approach underscores the taxonomic status of Lamellidens exolescens,a freshwater mussel from the Oriental tropics (Bivalvia: Unionidae)

The Oriental Region harbours the second richest fauna of freshwater bivalves in the world, including many endangered endemic taxa. However, the Oriental fauna of the Unionidae have been very poorly studied using an integrative taxonomic approach, which may provide reasonable revisions of complicated (cryptic) taxa based on morphological, molecular, biogeographic and ecological evidence. Here, we present the first example of an integrative taxonomic revision concerning the status of Unio exolescens Gould (1843 Gould, A.A. (1843). Dr. Gould had examined the shells not long since announced as having been received from the Rev. Francis Mason, missionary at Tavoy, in British Burmah. Proceedings of the Boston Society of Natural History, 1, 139–141. [Google Scholar]), a nominal mussel taxon that was accepted as a valid species within the genus Trapezoideus Simpson (1900). Currently, Trapezoideus exolescens is considered the type of the genus as far as the originally designated type species, U. foliaceus Gould (1843 Gould, A.A. (1843). Dr. Gould had examined the shells not long since announced as having been received from the Rev. Francis Mason, missionary at Tavoy, in British Burmah. Proceedings of the Boston Society of Natural History, 1, 139–141. [Google Scholar]), was considered to be a synonym of T. exolescens. Using nucleotide sequences obtained from mitochondrial (COI and 16S rRNA) and nuclear (28S rDNA) genes, we found that the topotypes of Unio exolescens Gould (1843 Gould, A.A. (1843). Dr. Gould had examined the shells not long since announced as having been received from the Rev. Francis Mason, missionary at Tavoy, in British Burmah. Proceedings of the Boston Society of Natural History, 1, 139–141. [Google Scholar]) cluster together with representatives of another mussel genus, Lamellidens Simpson (1900). Based on these results and on morphological data, we transfer Unio exolescens Gould (1843 Gould, A.A. (1843). Dr. Gould had examined the shells not long since announced as having been received from the Rev. Francis Mason, missionary at Tavoy, in British Burmah. Proceedings of the Boston Society of Natural History, 1, 139–141. [Google Scholar]) from Trapezoideus to Lamellidens and propose Lamellidens exolescens (Gould, 1843 Gould, A.A. (1843). Dr. Gould had examined the shells not long since announced as having been received from the Rev. Francis Mason, missionary at Tavoy, in British Burmah. Proceedings of the Boston Society of Natural History, 1, 139–141. [Google Scholar]) comb. nov. In addition, we revisited the status of Unio foliaceus Gould (1843 Gould, A.A. (1843). Dr. Gould had examined the shells not long since announced as having been received from the Rev. Francis Mason, missionary at Tavoy, in British Burmah. Proceedings of the Boston Society of Natural History, 1, 139–141. [Google Scholar]) as a valid species and the type of the genus Trapezoideus based on the morphological study of the type specimen, although a question concerning the true position of this taxon is still open because its molecular sequences are not available. Our findings highlight that an integrative taxonomic approach is an important tool, particularly when dealing with such species-rich Unionidae fauna as those of the Oriental Realm.     

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.     

Morphological and molecular description of new species of squat lobster (Crustacea: Decapoda: Galatheidae) from the Solomon and Fiji Islands (South‐West Pacific)

The family Galatheidae is among the most diverse families of anomuran decapod crustaceans, and the South‐West Pacific is a biodiversity hot spot for these squat lobsters. Attempts to clarify the taxonomic and evolutionary relationships of the Galatheidae on the basis of morphological and molecular data have revealed the existence of several cryptic species, differentiated only by subtle morphological characters. Despite these efforts, however, relationships among genera are poorly understood, and the family is in need of a detailed systematic review. In this study, we assess material collected in different surveys conducted in the Solomon Islands, as well as comparative material from the Fiji Islands, by examining both the morphology of the specimens and two mitochondrial markers (cytochrome oxidase subunit I, COI, and 16S rRNA). These two sources of data revealed the existence of eight new species of squat lobster, four of which were ascribed to the genus Munida, two to the genus Paramunida, one to the genus Plesionida, and the last species was ascribed to the genus Agononida. These eight species are described along with phylogenetic relationships at the genus level. Our findings support the taxonomic status of the new species, yet the phylogenetic relationships are not yet fully resolved. Further molecular analysis of a larger data set of species, and more conserved genes, will help clarify the systematics of this group. © 2009 The Linnean Society of London, Zoological Journal of the Linnean Society, 2009, 156 , 465–493.     

A new species of Camaenidae (Gastropoda,Pulmonata) from Nusa Penida and Lombok Islands,Indonesia: novelty in a well-known fauna

A new species of camaenid land snail, Vulnus wallacei Páll-Gergely, Otani & Hosoda n. sp. is described from Nusa Penida and Lombok Islands, Indonesia. The new species is putatively classified into the genus Vulnus, which is only known so far from the Northern Maluku Islands. The genital anatomy, especially the presence of a well-developed penial sheath in V. wallacei, suggests a close relationship with Australian camaenids. However, the molecular phylogeny based on partial cytochrome c oxidase I, and 16S and 28S ribosomal RNA genes shows that the new species falls outside the cluster of all Australian Camaenidae.

http://www.zoobank.org/urn:lsid:zoobank.org:pub:D28554D0-3578-4E95-8849-09EAFB7D30F7     

Diversification process of stag beetles belonging to the genus Platycerus Geoffroy (Coleoptera: Lucanidae) in Japan based on nuclear and mitochondrial genes

Molecular phylogenies of the genus Platycerus in Japan were characterized based on the nuclear 28S ribosomal RNA and mitochondrial cytochrome oxidase subunit I (COI) genes. These analyses, combined with our previous morphological information, revealed a detailed diversification process of Platycerus in Japan, as well as estimates of their divergence times. Japanese species were monophyletic and were inferred to have diverged to the acuticollis species group and all other species ca 1.69 Mya. The acuticollis species group then appeared to split into P. viridicuprus and a group including P. takakuwai, P. albisomni and P. acuticollis ca 1.26 Mya. Other specific divergences have occurred primarily since ca 0.33 Mya. Comparing the molecular trees and the morphological tree, we also found introgression of the COI gene in some species. Genetic divergence of Platycerus has occurred intensely in southwestern Japan.