Taking the discovery approach in integrative taxonomy: decrypting a complex of narrow‐endemic Alpine harvestmen (Opiliones: Phalangiidae: Megabunus)

Species delimitation is fundamental for biological studies, yet precise delimitation is not an easy task, and every involved approach has an inherent failure rate. Integrative taxonomy, a method that merges multiple lines of evidence, can profoundly contribute to reliable alpha‐taxonomy and shed light on the processes behind speciation. In this study, we explored and validated species limits in a group of closely related Megabunus harvestmen (Eupnoi, Phalangiidae) endemic to the European Alps. Without a priori species hypotheses, we used multiple sources of inference, including mitochondrial and multilocus nuclear DNA, morphometrics and chemistry. The results of these discovery approaches revealed morphological crypsis and multiple new species within two of the five hitherto known species. Based on our analyses, we discussed the most plausible evolutionary scenarios, invoked the most reasonable species hypotheses and validated the new species limits. Building upon the achieved rigour, three new species, Megabunus cryptobergomas Muster and Wachter sp. nov., Megabunus coelodonta Muster and Steiner sp. nov., and Megabunus lentipes Muster and Komposch sp. nov., are formally described. In addition, we provide a dichotomous morphological key to the Megabunus species of the Alps. Our work demonstrates the suitability of integrative, discovery‐based approaches in combination with validation approaches to precisely characterize species and enabled us to implement nomenclatural consequences for this genus.     

Integrative Taxonomy and Species Delimitation in Harvestmen: A Revision of the Western North American Genus Sclerobunus (Opiliones: Laniatores: Travunioidea)

Alpha taxonomy, and specifically the delimitation of species, is becoming increasingly objective and integrative. The use of coalescent-based methods applied to genetic data is providing new tools for the discovery and delimitation of species. Here, we use an integrative approach via a combination of discovery-based multivariate morphological analyses to detect potential new species. These potential species are then used as a priori species in hypothesis-driven validation analyses with genetic data. This research focuses on the harvestmen genus Sclerobunus found throughout the mountainous regions of western North America. Based on our analyses, we conduct a revision of Sclerobunus resulting in synonymy of Cyptobunus with Sclerobunus including transfer of S. cavicolens comb. nov. and elevation of both subspecies of S. ungulatus: S. ungulatus comb. nov. and S. madhousensis comb. nov., stat. nov. The three subspecies of S. robustus are elevated, S. robustus, S. glorietus stat. nov., and S. idahoensis stat. nov. Additionally, five new species of Sclerobunus are described from New Mexico and Colorado, including S. jemez sp. nov., S. klomax sp. nov., S. skywalkeri sp. nov., S. speoventus sp. nov., and S. steinmanni sp. nov. Several of the newly described species are single-cave endemics, and our findings suggest that further exploration of western North American cave habitats will likely yield additional new species.     

Species delimitation methods reveal cryptic diversity in the Hypnea cornuta complex (Cystocloniaceae,Rhodophyta)

The simple and convergent morphologies of many red algae make these species difficult to identify using traditional morphological characters. Many cryptic species have been described in recent years based on molecular datasets, and this has led to the application of an integrative taxonomy approach in species delimitation. Here, we performed several species delimitation methods (mBGD, ABGD, SPN, PTP, GMYCs and GMYCm) based on two different loci (COI-5P and rbcL) in species of the Hypnea cornuta complex. These methods were combined with morphological and phylogenetic data, extensive sampling, analysis of topotype material, and historically relevant herbarium samples. Our findings demonstrate that the groups morphologically assigned to H. cornuta and H. stellulifera consist of five different cryptic species. H. cornuta is a polyphyletic taxon composed of three well-separated lineages, thus requiring sequencing of type or topotype specimens to determine which one is Hypnea cornuta sensu stricto. We have revealed that the distribution of H. stellulifera is limited to Asia, while the Brazilian specimens initially assigned to this species were clarified as a new endemic species: Hypnea cryptica sp. nov. Our results indicated that only an integrative approach combining several lines of evidence, including morphology, nomenclature history, molecular data, biogeography and ecology can correctly solve the taxonomic status of widely distributed cryptic species.     

Integrative taxonomy reveals the exceptional species diversity of Eudonia (Lepidoptera: Crambidae) in Tibet,China

The genus Eudonia is rarely recorded in Tibet despite having a wide occurrence in the plateau. In the taxonomy of the genus, the morphological characters are insufficient to delimit the closely related congeners due to the subtly interspecific differences and conspicuously intraspecific variability. In this study, the Tibetan species are studied with an integrative approach using molecular data and morphological characters. It is found that the number of Eudonia species in Tibet accounts for about 20% of the total species previously catalogued for the genus in China. Five species are described as new to science: Eudonia galonglaensis Li, sp. nov., E. varians Li, sp. nov., E. triangulata Li, sp. nov., E. angusta Li, sp. nov., and E. bomiensis Li, sp. nov. The potential distribution of Chinese Eudonia is predicted and illustrated using MaxEnt and DIVA-GIS. The results reveal the exceptionally high species diversity of Eudonia in Tibet for the first time, demonstrate that the integrative morphological and molecular approach is highly effective for resolving the difficult-to-distinguish morphologies, and predict the region of China to the south of 35°N and to the east of 95°E as having potentially high Eudonia species diversity.

http://www.zoobank.org/urn:lsid:zoobank.org:pub:EA3C5D21-9674-46E4-838D-48D5A098314B     

Systematic revision of the genus Everettia Godwin‐Austen, 1891 (Mollusca: Gastropoda: Dyakiidae) in Sabah,northern Borneo

The species of the snail genus Everettia in the Malaysian state of Sabah are superficially similar and difficult to distinguish by their shells. This paper presents new data on the taxonomy and distribution of Everettia in Sabah that have accumulated since the revision by Godwin‐Austen in 1891. By using morphological and molecular phylogenetic approaches, we reveal at least seventeen species of Everettia in Sabah, of which eleven are new to science, namely: Everettia layanglayang sp. nov. , Everettia lapidini sp. nov. , Everettia paulbasintali sp. nov. , Everettia occidentalis sp. nov. , Everettia jasilini sp. nov. , Everettia safriei sp. nov. , Everettia interior sp. nov. , Everettia jucundior sp. nov. , Everettia planispira sp. nov. , Everettia monticola sp. nov. , and Everettia dominiki sp. nov. , and one new subspecies, namely, Everettia corrugata williamsi ssp. nov. Phylogenetic analysis of mitochondrial COI and 16S, and nuclear ITS‐1 sequences demonstrates the monophyly of most of the morphologically well‐defined species. Our results show that certain aspects of classical morphology‐based taxonomy for Everettia species, especially with regard to the unique combination of shell surface sculptures, animal head colour, and mantle pigmentation, are solid. A dichotomous key to the Sabah species and subspecies of Everettia is provided. © 2009 The Linnean Society of London, Zoological Journal of the Linnean Society, 2009, 157 , 515–550.     

Integrative taxonomy of the stalk-eyed bug genus Chauliops (Heteroptera: Malcidae: Chauliopinae) reveals orogeny-driven speciation

Economically significant bean pests of the genus Chauliops are species rich in the areas surrounding the Qinghai–Tibet Plateau and provide an excellent system for speciation studies. Here, an integrative taxonomic approach, employing morphological analyses, population genetic methods, and multiple molecular species delimitation methods, was used to clarify the taxonomy of Chauliops in East and Southeast Asia. Four new species (Chauliops parahorizontalis Li & Bu, sp. nov., Chauliops albida Li & Bu, sp. nov., Chauliops bicoloripes Li & Bu, sp. nov., and Chauliops paraconica Li & Bu, sp. nov.) were described, which increases the number of Chauliops species in this area from six to 10; a key for Chauliops species is also provided. Phylogenetic analysis and divergence time estimation revealed that Chauliops was divided into four clades: Clade A (Chauliops bisontula + [Chauliops horizontalis + C. parahorizontalis sp. nov.]), Clade B (C. albida sp. nov. and C. bicoloripes sp. nov.), Clade C (Chauliops quaternaria and Chauliops zhengi), and Clade D (Chauliops fallax + [Chauliops conica + C. paraconica sp. nov.]). Two species diversification events of Chauliops estimated to have occurred 7–1 million years ago (Ma) and 25–13 Ma were detected. These speciation events were consistent with the two historical uplift events of the Qinghai–Tibet Plateau, suggesting that orogeny might have provided opportunities for the diversification of Chauliops species on the southeastern margin of the Qinghai–Tibet Plateau. Our findings show that population genetic analyses can be used to delimit related species and that orogeny is a key driver of species diversification on the southeastern margin of the Qinghai–Tibet Plateau.     

The systematics of Lobophora (Dictyotales,Phaeophyceae) in the western Atlantic and eastern Pacific oceans: eight new species

Lobophora is a common tropical to temperate genus of brown algae found in a plethora of habitats including shallow and deep‐water coral reefs, rocky shores, mangroves, seagrass beds, and rhodoliths beds. Recent molecular studies have revealed that Lobophora species diversity has been severely underestimated. Current estimates of the species numbers range from 100 to 140 species with a suggested center of diversity in the Central Indo‐Pacific. This study used three molecular markers (cox3, rbcL, psbA), different single‐marker species delimitation methods (GMYC, ABGD, PTP), and morphological evidence to evaluate Lobophora species diversity in the Western Atlantic and the Eastern Pacific oceans. Cox3 provided the greatest number of primary species hypotheses(PSH), followed by rbcL and then psbA. GMYC species delimitation analysis was the most conservative across all three markers, followed by PTP, and then ABGD. The most informative diagnostic morphological characters were thallus thickness and number of cell layers in both the medulla and the dorsal/ventral cortices. Following a consensus approach, 14 distinct Lobophora species were identified in the Western Atlantic and five in the Eastern Pacific. Eight new species from these two oceans were herein described: L. adpressa sp. nov., L. cocoensis sp. nov., L. colombiana sp. nov., L. crispata sp. nov., L. delicata sp. nov., L. dispersa sp. nov., L. panamensis sp. nov., and L. tortugensis sp. nov. This study showed that the best approach to confidently identify Lobophora species is to analyze DNA sequences (preferably cox3 and rbcL) followed by comparative morphological and geographical assessment.     

A new genus of schizomids (Arachnida: Schizomida: Hubbardiidae) from Mexico,with notes on its systematics

During the systematic revision of Mayazomus, the third most diverse genus of micro whip-scorpions in North America, we found three species with peculiar morphological variation, which are different from those diagnostic characters for Mayazomus. These species share with Mayazomus the large body size and the unusual development of the male pedipalps; however, these three species share the shape of the female spermathecae with Stenochrus rather than with Mayazomus. Therefore, their phylogenetic placements create a dilemma. Here we perform analyses with morphological evidence, using two different methodologies (Parsimony and Bayesian inference) in order to hypothesize the phylogenetic relationships of the three species mentioned with each other and with respect to other North American and/or Central American genera. The results confirm our initial hypothesis that the species belong to a different genus, Olmeca gen. nov., which is recovered as the sister group of all hubbardiine genera included in these analyses, except for the genus Hubbardia. The results also indicate that despite sharing some morphological similarities, it is distantly related with Mayazomus. Three species are described herein: Olmeca brujo sp. nov., Olmeca cruzlopezi sp. nov., and Olmeca santibanezi sp. nov. Additionally, we provide a hypothesis of the relationships and biogeography of Olmeca gen. nov. with other genera.

http://zoobank.org/urn:lsid:zoobank.org:pub:F64CE801-7D82-4827-A5B9-0C0B41F58043     

Integrative taxonomy uncovers hidden species diversity in the rheophilic genus Potamometra (Hemiptera: Gerridae)

The genus Potamometra Bianchi, 1896 represents big rheophilic semi-aquatic bugs that typically inhabit middle-altitude mountainous streams. Here, we integrated molecular and morphological data for delimiting species boundaries and understanding the evolutionary history of the genus Potamometra. Twenty-seven complete mitochondrial genomes of Potamometra were sequenced, with samples representing most of the known geographically distributed locations around the Sichuan Basin. The results of different species delimitation methods (ABGD, bPTP, GMYC and BPP) based on the monolocus or multilocus data strongly supported the existence of two cryptic new species (Potamometra anderseni Zheng, Ye & Bu, sp. nov. and Potamometra zhengi Zheng, Ye & Bu, sp. nov.) although more entities were found in the tree-based delimitation methods. The two new species were successfully validated using morphological characters within a detailed taxonomic framework. Phylogenetic analyses supported the reciprocal monophyly of the seven highly node-supported clades, which were matched with the five known species and two new taxa. A novel gene arrangement pattern that two trnF (trnF1 and trnF2) genes separated by an intergenic spacer (IGS) were found in all the species except the sister group of Potamometra berezowskii Bianchi, 1896 and Potamometra linnavuorii Chen, Nieser & Bu, 2016. This gene rearrangement event could be explained by the tandem duplication and random loss (TDRL) model. Our study emphasized that the combination of molecular sequence data, morphological characters and mitochondrial structural information could improve the accuracy of species delimitation.     

Type locality and species identity of Pareuchiloglanis sinensis (Hora & Silas) with a description of a new species of the genus from the upper Yangtze River basin in southern China

The genus Pareuchiloglanis, distributed in the Salween, Mekong, Red, Pearl and Yangtze River basins in China, Laos, Myanmar and Vietnam, consists of 20 valid species and 5 uncertain species. This study provided a taxonomic revision to Pareuchiloglanis occurring in the Yangtze River. According to the results of a morphological comparison, the type locality of Pareuchiloglanis sinensis was updated to the Nanpan-jiang (the upper Pearl River) basin; five species were identified in Pareuchiloglanis from the Yangtze River basin. Specimens formerly identified as Pareuchiloglanis sinensis from this basin were described as a new species, Pareuchiloglanis chui sp. nov. Moreover, Pareuchiloglanis tianquanensis was synonymized with Pareuchiloglanis sichuanensis. This study provided a key to these two species and three others (Pareuchiloglanis anteanalis, Pareuchiloglanis hupingshanensis and Pareuchiloglanis robusta) from the Yangtze River basin, including information about their geographical distribution. These findings provide an insight into the evolution, distribution and taxonomy of this genus for future studies.     

First reports of Grania (Clitellata: Enchytraeidae) from Africa and South America: molecular phylogeny and descriptions of nine new species

In this article, our knowledge of the geographic distribution of Grania species is expanded by describing seven new species, G rania bekkouchei sp. nov. , G rania brasiliensis sp. nov. , G rania capensis sp. nov. , G rania chilensis sp. nov. , G rania cryptica sp. nov. , G rania hinojosai sp. nov. , and G rania simonae sp. nov. , from poorly investigated regions of the Southern Hemisphere, plus two new species, G rania carolinensis sp. nov. and G rania unitheca sp. nov. , from off the east coast of the USA. An immature achaetous specimen that we call Grania cf. levis was also included. The newly generated data were combined with a previously published data set in order to update the hypothesis of phylogenetic relationships among Grania species. All new species except G . cryptica sp. nov. are supported by both morphological and molecular data. In addition, we find that G . chilensis sp. nov. is structured in at least four distinct populations along the Chilean coast. The species described from South Africa form a monophyletic clade where two are morphologically indistinguishable but diverging in both mitochondrial and nuclear ribosomal genes, and thus we describe them as different species, G . bekkouchei sp. nov. and G . cryptica sp. nov. Among the North Carolinian species, G . unitheca sp. nov. is indicated as a close relative of Grania monospermatheca Erséus & Lasserre, 1976, and G . carolinensis sp. nov. is indicated as a close relative of G. cf. levis. The updated phylogeny is strongly concordant with geographical species distributions, thus supporting a low level of dispersal within this genus, as has previously been hypothesized. © 2015 The Linnean Society of London     

A taxonomic revision and species delimitation of the genus Purpuraria Enderlein, 1929 (Orthoptera: Pamphagidae) using an integrative approach

Recent studies on the endemic Canarian genus Purpuraria have shown that the taxonomy of its only recognized species (P. erna) is probably erroneous. In this study, an integrative revision of the genus is performed, based on a large number of specimens and geographical sampling. As a result, (1) the diagnostic characters at the genus level are re‐described, (2) Purpuraria magna n. sp. based on morphological, morphometric and genetic data is described and (3) the taxonomic status of a formerly described subspecies is clarified. Intraspecific and interspecific morphometric differences have been found, indicating that the genus is undergoing a process of morphological diversification. Nevertheless, the possibility of interspecific mating between individuals of the two species is suggested, because no significant differences have been found between their respective calling songs. Genetic analyses using mitochondrial and nuclear DNA sequences suggest that P. erna and P. magna are recent species with evidences of secondary contact episodes in the past.     

Integrative taxonomy of the stunt nematodes of the genera Bitylenchus and Tylenchorhynchus (Nematoda,Telotylenchidae) with description of two new species and a molecular phylogeny

Stunt nematodes are characterized by phenotypic plasticity, with overlapping morphology and morphometry leading to potential misidentification. Consequently, the application of integrative taxonomic approaches is useful to species delimitation based on a combination of different perspectives, e.g. morphology and DNA sequences. We conducted nematode surveys in cultivated and natural environments in Spain and the USA, from which we identified 18 known species of the family Telotylenchidae and two new taxa within the studied samples. These species were morphologically, morphometrically, and molecularly characterized. The results of light and scanning electron microscopic observations, and molecular and phylogenetic analysis also allowed two new species to be distinguished, described herein as B itylenchus hispaniensis sp. nov. and T ylenchorhynchus mediterraneus sp. nov. The phylogenetic analysis was carried out using molecular data from nuclear ribosomal DNA genes [D2–D3 expansion segments of the large ribosomal subunit (28S), internal transcribed spacer (ITS), and partial small ribosomal subunit (18S)]. We also provide here a test of alternative hypotheses that confirms the monophyly of both Tylenchorhynchus and Bitylenchus sensu Siddiqi's classification but does not support Fortuner & Luc's conceptual view of Tylenchorhynchus as a large genus. Ancestral state reconstructions of several diagnostic morphological characters using a maximum parsimony approach showed congruence in morphological and molecular evolution for stylet knob inclination and tail tip annulation. Our analysis emphasizes some of the problems related to the taxonomy and phylogeny of nematodes of Telotylenchinae. © 2014 The Linnean Society of London     

Integration of molecular, ecological, morphological and endosymbiont data for species delimitation within the Pnigalio soemius complex (Hymenoptera: Eulophidae)

Integrative taxonomy is a recently developed approach that uses multiple lines of evidence such as molecular, morphological, ecological and geographical data to test species limits, and it stands as one of the most promising approaches to species delimitation in taxonomically difficult groups. The Pnigalio soemius complex (Hymenoptera: Eulophidae) represents an interesting taxonomical and ecological study case, as it is characterized by a lack of informative morphological characters, deep mitochondrial divergence, and is susceptible to infection by parthenogenesis‐inducing Rickettsia. We tested the effectiveness of an integrative taxonomy approach in delimiting species within the P. soemius complex. We analysed two molecular markers (COI and ITS2) using different methods, performed multivariate analysis on morphometric data and exploited ecological data such as host–plant system associations, geographical separation, and the prevalence, type and effects of endosymbiont infection. The challenge of resolving different levels of resolution in the data was met by setting up a formal procedure of data integration within and between conflicting independent lines of evidence. An iterative corroboration process of multiple sources of data eventually indicated the existence of several cryptic species that can be treated as stable taxonomic hypotheses. Furthermore, the integrative approach confirmed a trend towards host specificity within the presumed polyphagous P. soemius and suggested that Rickettsia could have played a major role in the reproductive isolation and genetic diversification of at least two species.     

Integrative taxonomy and molecular phylogeny of genus aplysina (demospongiae: verongida) from mexican pacific

Integrative taxonomy provides a major approximation to species delimitation based on integration of different perspectives (e.g. morphology, biochemistry and DNA sequences). The aim of this study was to assess the relationships and boundaries among Eastern Pacific Aplysina species using morphological, biochemical and molecular data. For this, a collection of sponges of the genus Aplysina from the Mexican Pacific was studied on the basis of their morphological, chemical (chitin composition), and molecular markers (mitochondrial COI and nuclear ribosomal rDNA: ITS1-5.8-ITS2). Three morphological species were identified, two of which are new to science. A. clathrata sp. nov. is a yellow to yellow-reddish or -brownish sponge, characterized by external clathrate-like morphology; A. revillagigedi sp. nov. is a lemon yellow to green, cushion-shaped sometimes lobate sponge, characterized by conspicuous oscules, which are slightly elevated and usually linearly distributed on rims; and A. gerardogreeni a known species distributed along the Mexican Pacific coast. Chitin was identified as the main structural component within skeletons of the three species using FTIR, confirming that it is shared among Verongida sponges. Morphological differences were confirmed by DNA sequences from nuclear ITS1-5.8-ITS2. Mitochondrial COI sequences showed extremely low but diagnostic variability for Aplysina revillagigedi sp. nov., thus our results corroborate that COI has limited power for DNA-barcoding of sponges and should be complemented with other markers (e.g. rDNA). Phylogenetic analyses of Aplysina sequences from the Eastern Pacific and Caribbean, resolved two allopatric and reciprocally monophyletic groups for each region. Eastern Pacific species were grouped in general accordance with the taxonomic hypothesis based on morphological characters. An identification key of Eastern Pacific Aplysina species is presented. Our results constitute one of the first approximations to integrative taxonomy, phylogeny and evolutionary biogeography of Eastern Pacific marine sponges; an approach that will significantly contribute to our better understanding of their diversity and evolutionary history.