Linking DNA sequences to morphology: cryptic diversity and population genetic structure in the marine nematode Thoracostoma trachygaster (Nematoda,Leptosomatidae)

Derycke, S., De Ley, P., De Ley, I.T., Holovachov, O., Rigaux, A. & Moens, T. (2010). Linking DNA sequences to morphology: cryptic diversity and population genetic structure in the marine nematode Thoracostoma trachygaster (Nematoda, Leptosomatidae).—Zoologica Scripta, 39, 276–289. Recent taxonomic and population genetic studies have revealed the presence of substantial cryptic diversity through sequence analysis of nematode morphospecies classified in different major clades. Correct interpretations of intra‐ and interspecific genetic variation require certainty about the conspecificity of the sequenced specimens, which in turn must depend on appropriate protocols with built‐in verifiability procedures. In this study, we performed a population genetic study in the free‐living marine nematode Thoracostoma trachygaster, a member of one of the earliest major clades to diverge in nematode phylogeny. We collected 367 nematodes from 11 populations located in the Californian Bight, all of which were video captured before DNA extraction to document and verify their individual morphology. Sequences for the cytochrome c oxidase subunit 1 (COI), D2D3 and 18S genes showed eight deeply divergent clades, and using a reverse taxonomy approach, six of these clades proved to be other morphospecies than T. trachygaster. Phylogenetic analyses of COI, internal transcribed spacer and D2D3 showed evidence for two sympatrically distributed cryptic species within the morphospecies T. trachygaster. Population genetic analyses of the most widespread cryptic species showed a moderate genetic structuring (Φ_ST = 0.28), and 18% of this genetic variation was caused by differences between populations north and south of Point Conception. Within the southern Californian Bight, some genetic differentiation could be attributed to differences between populations north and south of Malibu, supporting the idea of a barrier to gene flow near Los Angeles region. The results for T. trachygaster support the contention that species diversity within free‐living nematodes is underestimated, and that dispersal of marine nematodes from tidal environments associated with kelp holdfasts is substantial at scales of a few 100 km.     

Three new species of deep‐sea Gromia (Protista,Rhizaria) from the bathyal and abyssal Weddell Sea,Antarctica

We describe three new species of the genus Gromia from bathyal and abyssal depths in the Weddell Sea. The new species are characterized by a combination of morphological and molecular criteria. All three species possess a distinct oral capsule and a layer of ‘honeycomb membranes’, which form the inner part of the organic test wall. Both these features are typical of gromiids. Their identification as gromiids is confirmed by analyses of partial small subunit ribosomal DNA (SSU rDNA) gene sequences. Gromia marmorea sp. nov. is a rounded species with a prominent oral capsule and a characteristically mottled appearance. In Gromia melinus sp. nov. , the test surface exhibits a polygonal pattern of ridges, with a layer of clay particles coating the surface between the ridges. Gromia winnetoui sp. nov. represents an elongate morphotype in which the organic test is enclosed within an agglutinated case, a feature previously unknown in gromiids. Phylogenetic analysis using the maximum‐likelihood method revealed that all three species form distinct clades, reflecting the morphological differences among Weddell Sea species, as well as between deep‐water Southern Ocean Gromia and previously described gromiids. © 2009 The Linnean Society of London, Zoological Journal of the Linnean Society, 2009, 157 , 451–469.     

Revision and cladistic analysis of the jumping spider genus Onomastus (Araneae: Salticidae)

The jumping spider genus Onomastus Simon, 1900 is revised. Four new species: Onomastus indra sp. nov. , Onomastus kaharian sp. nov. , Onomastus pethiyagodai sp. nov. , and Onomastus rattotensis sp. nov. are described. Parsimony analysis of 26 morphological characters supported the monophyly of Onomastus. Lyssomanes is sister to Onomastus. Onomastus separates into two clades: the widespread South‐East Asia clade and the South Asia clade. The South Asia clade is restricted to the Sri Lanka–Western Ghats biodiversity hotspot. Species of the South Asia clade appear to be spot endemics, highly in danger of extinction because of habitat loss and climate change. Male palps are complex and species‐specific, suggesting rapid divergent evolution. © 2010 The Linnean Society of London, Zoological Journal of the Linnean Society, 2010, 159 , 711–745.     

How many species of Hipposideros have occurred on Madagascar since the Late Pleistocene?

Populations of the Malagasy Hipposideros commersoni (family Hipposideridae) are threatened by deforestation and hunting. Maximum likelihood and Bayesian analysis of 148 cytochrome b sequences found this species to be paraphyletic and composed of three well‐supported monophyletic clades. Clades B and C form a monophyletic lineage that can be referred to H. commersoni; these two clades are separated by 6% sequence variation. Clade A represents a distinct evolutionary lineage separate (9–11% average sequence divergence) from H. commersoni (clades B and C) and is named herein as a new species, H ipposideros cryptovalorona sp. nov. In the phylogeny presented herein, this species is strongly associated with the outgroup taxa Hipposideros gigas and Hipposideros vittatus, both restricted to Africa. External, cranial and dental measurements taken from the same individuals used in the molecular study indicate no clear distinction in morphology amongst these three clades; this includes noseleaf structure and craniodental characteristics. Principal component analyses showed limited separation of the three clades. Comparison to a Quaternary fossil species from north‐west Madagascar, Hipposideros besaoka, found little morphological overlap between any of the three clades and this extinct species. Hence, at least three species of Hipposideros have occurred on Madagascar since the Late Pleistocene, two extant (H. commersoni s.s. and H. cryptovalorona sp. nov.) and one extinct (H. besaoka). © 2015 The Linnean Society of London     

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     

Intraspecific or interspecific variation: delimitation of species boundaries within the genus Gammarus (Crustacea,Amphipoda, Gammaridae), with description of four new species

The delineation of Gammarus species is controversial because of extensive intraspecific morphological variation. The current study examined DNA sequences from the mitochondrial cytochrome c oxidase subunit I and the nuclear 28S genes as well as morphological and ecological data to determine the species boundaries of Gammarus species from China. The results of molecular analyses showed that Gammarus sp1, G. sp2, G. sp3, and G. sp4 are monophyletic and deeply divergent from sister groups. Detailed morphological and ecological comparisons with closely related species were consistent with molecular analyses. Gammarus sp1, G. sp2, G. sp3, and G. sp4 were described as four new species: Gammarus illustris sp. nov. , Gammarus clarus sp. nov. , Gammarus hypolithicus sp. nov. , and Gammarus parvioculus sp. nov. We recommend that molecular detected species should be formally named and described for future research. © 2010 The Linnean Society of London, Zoological Journal of the Linnean Society, 2010, 160 , 215–253.     

DNA sequences corroborate Soesiladeepakius as a non‐salticoid genus of jumping spiders: placement with lapsiines,phylogeny, and description of six new species (Araneae,Salticidae)

Species of the Amazonian jumping spider genus Soesiladeepakius Makhan are confirmed as non‐salticoids. Sequences of nuclear (28S, Actin) and mitochondrial (16S through NADH dehydrogenase subunit I, ‘16S‐ND1’) gene regions, analysed under parsimony and maximum likelihood, placed the genus within the lapsiines, closely related to Galianora Maddison. Additionally, six new species of this genus are herein described, namely Soesiladeepakius lyra sp. nov. , Soesiladeepakius retroversus sp. nov. , Soesiladeepakius arthrostylus sp. nov. , Soesiladeepakius gasnieri sp. nov. , Soesiladeepakius biarmatus sp. nov. , and Soesiladeepakius uncinatus sp. nov. , all from the Amazon region in Brazil. To test the monophyly of Soesiladeepakius within lapsiines, a cladistic analysis was carried out using a data matrix comprising 24 morphological characters scored for 12 taxa. The analysis resulted in two equally parsimonious trees of 29 steps. One of these trees is used to discuss the relationships among the species of Soesiladeepakius and character evolution. © 2012 The Linnean Society of London, Zoological Journal of the Linnean Society, 2012, 165 , 274–295.     

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     

Phylogeny of the spider genus Ixchela Huber, 2000 (Araneae: Pholcidae) based on morphological and molecular evidence (CO1 and 16S), with a hypothesized diversification in the Pleistocene

The genus Ixchela Huber is composed of 20 species distributed from north‐eastern Mexico to Central America, including the five new species described here from Mexico: I xchela azteca sp. nov. , I xchela jalisco sp. nov. , I xchela mendozai sp. nov. , I xchela purepecha sp. nov. and I xchela tlayuda sp. nov. We test the monophyly and investigate the phylogenetic relationships among species of the genus Ixchela using morphological and molecular data. Parsimony (PA) analysis of 24 taxa and 40 morphological characters with equal and implied weights supported the monophyly of Ixchela with eight morphological synapomorphies. The PA analyses with equal and implied weights, and separate Bayesian inference (BI) analyses for the CO1 gene (506 characters), concatenated gene fragments CO1 + 16S (885 characters), morphology + CO1 (546 characters) and the combined evidence data set (morphology + CO1 + 16S) (925 characters) support the monophyly of Ixchela. Our preferred topology shows two large clades; clade 1 has a natural distribution in the Mesoamerican biotic component, whereas clade 2 predominates in the Mexican Montane biotic component. The genus Ixchela diverged in the late Miocene, and the divergence between the internal clades in the genus occurred in the late Pliocene; by contrast, most of the speciation events seem to have occurred mainly during the Pleistocene, where climatic changes brought on by repeated glaciations played an important role in the diversification of the genus. © 2015 The Linnean Society of London     

Neglected refugia of biodiversity: mountainous regions in Mozambique and Malawi yield two novel freshwater crab species (Potamonautidae: Potamonautes)

Phylogenetic relationships amongst the southern African freshwater crab fauna are reinvestigated following the recent collection of morphologically distinct Potamonautes specimens from remote mountainous regions in Malawi and Mozambique. Specimens were subjected to DNA sequencing of three mtDNA loci, cytochrome c oxidase subunit I (COI), 12S rRNA, and 16S rRNA and compared to the 14 described species from the region. Phylogenetic analysis using maximum parsimony and Bayesian inference revealed the presence of two novel evolutionary lineages. The phylogeny demonstrates that Potamonautes obesus (A. Milne‐Edwards, 1868) is sister to a morphologically distinct novel species from Mount Namuli in Mozambique. Two sympatric and genetically distinct species from Mount Mulanje, in Malawi (forms A and B) were recognized. Form B is sister to the large‐bodied South African riverine freshwater crabs and represents a novel lineage whereas the remaining species (form A) from Mulanje, in Malawi was sister to samples from Mounts Inago and Mabu, and in Mozambique was identified as Potamonautes choloensis (Chace, 1953). The two novel evolutionary lineages were genetically distinct and morphologically different from the described species in each of the respective regions. Two new freshwater crab species Potamonautes namuliensis sp. nov. and Potamonautes mulanjeensis sp. nov. , are described in the present study. The samples from Mount Mulanje in Malawi, and Mounts Mabu and Inago in Mozambique represent new distribution records for Potamonautes choloensis. © 2012 The Linnean Society of London, Zoological Journal of the Linnean Society, 2012, 164 , 498–509.     

Molecular phylogeny of the nematode genus Longidorus (Nematoda: Longidoridae) with description of three new species

The phylum Nematoda includes the genus Longidorus, a remarkable group of invertebrates that are polyphagous root‐ectoparasites of many plants including various agricultural crops and trees. Damage is caused by direct feeding on root cells as well as by transmitting nepoviruses. Species discrimination in Longidorus is complicated by phenotypic plasticity (intraspecific variability and minor interspecific differences) leading to potential misidentification. We conducted nematode surveys in cultivated and natural environments in southern Spain that detected 11 species of Longidorus. We developed a comparative study amongst these related species by considering morphological and morphometric features together with molecular data from nuclear ribosomal RNA genes [D2‐D3 expansion segments of large ribosomal subunit (28S), internal transcribed spacer 1 (ITS1), and partial small ribosomal subunit (18S)]. The results of our molecular and phylogenetic analyses confirmed the morphological hypotheses and allowed the delimitation and discrimination of three new species of the genus, described herein as Longidorus baeticus sp. nov. , Longidorus oleae sp. nov. , and Longidorus andalusicus sp. nov. , and eight known species (Longidorus alvegus, Longidorus crataegi, Longidorus fasciatus, Longidorus intermedius, Longidorus iuglandis, Longidorus magnus, Longidorus rubi, and Longidorus vineacola). Phylogenetic analyses of Longidorus spp. based on the three molecular markers resulted in a general consensus of these species grouping, as lineages were maintained for the majority of species (i.e. species with a conoid‐rounded lip region, amphidial fovea asymmetrically bilobed, female tail bluntly rounded), but not in some others (i.e. positions of L. crataegi, L. intermedius, and L. rubi were quite variable). To date, this is the most complete phylogenetic analysis for Longidorus and Paralongidorus species, with the highest number of species included. No correspondence between phylogenetic trees and morphological characters was found for ribosomal markers, with the exception of amphidial shape. Thus, polyphasic identification, based on integration of molecular analysis with morphology, is a tool beyond doubt in Longidorus identification. © 2013 The Linnean Society of London     

Unmasking cryptic species: morphometric and phylogenetic analyses of the Ibero‐North African Linaria incarnata complex

Linaria incarnata has been treated as a widely distributed Ibero‐North African species in the latest taxonomic reviews. Morphological and phylogenetic disparity between populations of this species has been previously reported. Here we present new morphological and phylogenetic evidence for the disintegration of L. incarnata into three distinct species: L. incarnata from the western Iberian Peninsula; L . mamorensis sp. nov. from north‐western Morocco; and L. onubensis from south‐western Spain. The relatively poor morphological differentiation between these taxa (which can be regarded as cryptic species) and their distinct phylogenetic positions indicate that characters of the L. incarnata morphotype have been acquired multiple times in the evolution of Linaria section Versicolores. © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 00, 000–000.     

Sayrevilleinae Legalov,a newly recognised subfamily of fossil weevils (Coleoptera,Curculionoidea, Attelabidae) and the use of synchrotron microtomography to examine inclusions in amber

Two genera of extinct weevils, Sayrevilleus Gratshev & Zherikhin from Cretaceous New Jersey amber and Baltocar Kuschel from Eocene Baltic amber, are recognized as close relatives based on similarities revealed by the use of synchrotron tomography and the availability of new amber inclusions. The subfamily Sayrevilleinae Legalov stat. nov. is characterized by possessing mandibles with an external cutting edge and an inner blunt edge. The subfamily is placed in the family Attelabidae (s.l.), although some characters also suggest a possible relationship with the ‘higher weevils’ comprising Caridae, Brentidae, and Curculionidae. Sayrevilleus is transferred from the tribe Auletini of Rhynchitinae to Sayrevilleinae, and Sayrevilleus grimaldii Gratshev & Zherikhin is redescribed. Baltocar Kuschel is transferred from Caridae to Sayrevilleinae and revised, its type species, Baltocar succinicus (Voss), is redescribed and three new species, Baltocar groehni Riedel sp. nov. , Baltocar hoffeinsorum Riedel sp. nov. , and Baltocar subnudus Riedel sp. nov. are described based on eight well‐preserved inclusions. The genera Orapauletes Legalov and Zherichiniletes Legalov previously assigned to Sayrevilleini are regarded as Curculionoidea incertae sedis. The Sayrevilleinae were distributed over areas of North America and Europe at least since the Late Cretaceous (c. 90 Mya) and were probably relatively diverse until the Eocene (c. 44 Mya). It is speculated that they became extinct through competition with Curculionidae, which used a similar oviposition strategy. © 2012 The Linnean Society of London, Zoological Journal of the Linnean Society, 2012, 165 , 773–794.     

Phylogenetic systematics of Erythronium (Liliaceae): morphological and molecular analyses

For a nearly complete set of species of Erythronium (Liliaceae), we examined two plastid loci (the rps16 intron and the 5′ trnK intron, excluding the matK exon), one nuclear locus (nrITS) and morphology to evaluate species relationships and that of Erythronium to Amana, the putatively most closely allied genus. A matrix of morphological characters was developed through observation of around 900 living and herbarium specimens; evolution of these was examined using character optimization on the combined (total‐evidence) tree. Parsimony methods were used to examine the morphological and molecular data sets produced, both separately and in combination, with Bayesian methods also used on the molecular data sets. These established that the genus is probably sister to Amana (although most analyses placed Amana inside Erythronium) and that Tulipa is sister to the pair of Erythronium and Amana. Within Erythronium, there are three strongly supported geographically distinct clades: (1) Eurasian and (2) eastern and (3) western North American. Separation of species in these three clades is less clear, particularly among the western North American taxa. © 2012 The Linnean Society of London, Botanical Journal of the Linnean Society, 2012, ●●, ●●–●●.     

Revision and phylogeny of the European species of the Eurytoma morio species group (Hymenoptera: Eurytomidae), parasitoids of bark and wood boring beetles

A revision of Eurytoma (Hymenoptera, Eurytomidae) species belonging to the morio group is proposed. Species discrimination is based on morphological and, partly, on molecular data, including the barcoding fragment of the mitochondrial cytochrome c oxidase subunit I (COI) gene and the D2 expansion region of the 28S ribosomal gene. Morphological and molecular phylogenetic analyses largely support the morphological evidence. E urytoma cristata Delvare sp. nov. , E urytoma saliphila Delvare sp. nov. , and E urytoma sylviae Delvare sp. nov. are described from France, E urytoma ithma Delvare sp. nov. is described from France and Italy, and E urytoma gatesi Delvare sp. nov. is described from North America and France. Decatomidea polygraphi Ashmead, 1894 and Ipideurytoma spessivtsevi Bou?ek & Novicky, 1954 are synonymized with Eurytoma afra Boheman, 1836. Eurytoma auricoma Mayr, 1878 is removed from synonymy with Eurytoma arctica Thomson, 1875 and is synonymized with Eurytoma maura Boheman, 1836. Eurytoma eccoptogastri Ratzeburg, 1844, Eurytoma flavoscapularis Ratzeburg, 1844, Eurytoma flavovaria Ratzeburg, 1844, and Eurytoma masii var. flavonigra Russo, 1938 are synonymized with Eurytoma morio Boheman, 1836. Eurytoma masii Russo, 1925 and Eurytoma kemalpasensis Narendran, Tezcan & Civelek, 1995 are synonymized with Eurytoma striolata Ratzeburg, 1848. Eurytoma melanoneura Walker, 1871 and E. masii are removed from synonymy with E. morio. Lectotypes are designated for E. afra, E. auricoma, E. masii, Decatoma aloisifilippoi Russo, 1938, and E. masii var. flavonigra. © 2014 The Linnean Society of London     

Taxonomy and molecular phylogeny of the Asian Paraleucophenga Hendel (Diptera,Drosophilidae)

This paper reports on nine Asian species of the genus Paraleucophenga, of which four are new to science: Paraleucophenga brevipenis sp. nov. , Paraleucophenga hirtipenis sp. nov. , Paraleucophenga longiseta sp. nov. , and Paraleucophenga tanydactylia sp. nov. We also report on a new synonym, Paraleucophenga shanyinensis Chen & Toda, 1994 syn. nov. A key to all of the species examined, based on morphological data, is provided, together with a ‘molecular’ key to seven Paraleudophenga species based on DNA sequence data of the mitochondrial NADH dehydrogenase subunit 2 (ND2) gene. The phylogenetic relationships among seven Paraleucophenga species are reconstructed based on DNA sequences of the mitochondrial ND2 gene, using two Leucophenga species as outgroups. © 2009 The Linnean Society of London, Zoological Journal of the Linnean Society, 2009, 155 , 615–629.     

Morphological and molecular taxonomy of a new Daptonema (Nematoda,Xyalidae) with comments on the systematics of some related taxa

A new species of Daptonema is described based upon morphological characters and 18S rRNA sequence. Daptonema matrona sp. nov. was collected in Pina Basin (north‐eastern Brazil). It differs from all other species of the genus by the presence of reduced cephalic setae and straight spicules. These features require an adaptation of the generic diagnosis. Moreover, the females are characterized by intra‐uterine development of the offspring, considered herein as their major autapomorphic feature. Molecular systematic analyses supported Daptonema matrona sp. nov. as a distinct genetic and evolutionary lineage. The data also indicate hypotheses of taxonomic synonymies amongst some related taxa from Xyalidae as well as the paraphyly of Daptonema. © 2010 The Linnean Society of London, Zoological Journal of the Linnean Society, 2010, 158 , 1–15.     

Tilletiopsis derxii, Tilletiopsis oryzicola and Tilletiopsis penniseti, three new species of the ustilagionomycetous anamorphic genus Tilletiopsis isolated from leaves in Thailand

Four strains of ballistoconidium-forming yeast-like fungi (K-95, K-125, K-132 and K139), isolated from plants collected in Bangkok, Thailand, were assigned to the genus Tilletiopsis based on morphological and chemotaxonomical characteristics. On the basis of sequence data of 18S rDNA and the D1/D2 region of 26S rDNA, strains K-95, K-125 and K-132 were close to T. flava and T. fulvescens, and strain K-139 each formed related to T. minor. DNA-DNA reassociation experiments with related species revealed that strains K-125, K-132 and K-139 each formed a new and distinct species whereas strain K-95 was identified as T. flava. Tilletiopsis derxii Takashima et Nakase sp. nov. (JCM 10217^T; K-125), Tilletiopsis oryzicola Takashima et Nakase sp. nov. (JCM 10218^T; K-132), and Tilletiopsis penniseti Takashima et Nakase sp. nov. (JCM 10216^T; K-139) are the names proposed for the new taxa.