A polyphasic approach for the characterization of endophytic Alternaria strains isolated from grapevines

A polyphasic approach was set up and applied to characterize 20 fungal endophytes belonging to the genus Alternaria, recovered from grapevine in different Italian regions.Morphological, microscopical, molecular and chemical investigations were performed and the obtained results were combined in a pooled cluster analysis. Following morphological analyses, all strains were grouped according to their three-dimensional sporulation pattern on PCA and to the colony characteristics on different substrates. After DNA extraction, all strains were analyzed by RAPD-PCR and the resulting profiles were subjected to cluster analysis. The metabolites extracted from the 20 Alternaria endophytes were analyzed by a HPLC and the resulting metabolite profiles were subjected to multivariate statistic analyses. In comparison with reference ‘small-spored’ Alternaria species, the 20 strains were segregated into two morphological groups: one belonging to the A. arborescens species-group and a second to the A. tenuissima species-group. RAPD analysis also showed that grapevine endophytes belonged to either the A. arborescens or the A. tenuissima species-group and that they were molecularly distinct from strains belonging to A. alternata. Chemotaxonomy gave the same grouping: the grapevine endophytic strains belong to A. arborescens or A. tenuissima species-groups producing known metabolites typical of these species-groups. Interestingly, the 20 grapevine endophytes were able to produce also a number of unknown metabolites, whose characterization could be useful for a more precise segregation of the two species-groups.The results show how complementary morphological, molecular and chemical data can clarify relationships among endophyte species-groups of low morphological divergence.     

Staphylorchis cymatodes (Gorgoderidae: Anaporrhutinae) from carcharhiniform, orectolobiform and myliobatiform elasmobranchs of Australasia: Low host specificity, wide distribution and morphological plasticity

Anaporrhutine gorgoderids (Digenea: Gorgoderidae: Anaporrhutinae) found in the body cavity of six species of elasmobranchs from the orders Carcharhiniformes, Myliobatiformes and Orectolobiformes from Australian waters were found to belong to the genus Staphylorchis. Although these specimens were morphologically variable, sequences of ITS2 and 28S ribosomal DNA from specimens from three host families and two host orders were identical. Based on morphological and molecular data these specimens were identified as the type-species of the genus, Staphylorchis cymatodes. New measurements are provided for S. cymatodes, and for the first time genetic data are presented for this species. In addition to providing new morphological and molecular data for S. cymatodes, the previously described species S. gigas, S. parisi and S. scoliodonii, are here synonymised with S. cymatodes. This implies that S. cymatodes, as conceived here, has remarkably low host-specificity, being recorded from eight elasmobranch species from four families and three orders, has a wide geographical distribution in the Indo-west Pacific from off India, in the Bay of Bengal, to Moreton Bay in the Coral Sea, and is morphologically plastic, with body size, size of specific organs and body shape differing dramatically between specimens from different host species. The genus Staphylorchis now contains only two valid species, S. cymatodes and S. pacifica.     

Diversity and Systematics of Schizomavella Species (Bryozoa: Bitectiporidae) from the Bathyal NE Atlantic

Eight NE Atlantic and Mediterranean species, which were originally assigned to the genus Schizoporella (Family Schizoporellidae) when introduced, are redescribed and stabilized by typification. Seven of these species are transferred to the bitectiporid genus Schizomavella: S. fischeri, S. glebula, S. neptuni, S. obsoleta, S. richardi, S. triaviculata, and S. triaviculata var. paucimandibulata, which is here raised to species rank. The eighth species, Schizoporella fayalensis, is transferred to the lanceoporid genus Stephanotheca. Schizomavella obsoleta and S. glebula are considered junior subjective synonyms of S. fischeri and S. richardi, respectively. Two new species are described: Schizomavella rectangularis n. sp. from the Strait of Gibraltar, and Schizomavella phterocopa n. sp. from the Great Meteor Bank. A new subgenus, Calvetomavella n. subgen. is established as a result of a phylogenetic analysis based on morphological characters; it includes S. neptuni, S. triaviculata, S. paucimandibulata and S. phterocopa n. sp., together with Schizomavella discoidea and Schizomavella noronhai. The rest of the species remain in the nominotypical subgenus Schizomavella.     

Characterization of Alternaria isolates from the infectoria species-group and a new taxon from Arrhenatherum,Pseudoalternaria arrhenatheria sp. nov.

The infectoria species-group within the genus Alternaria was originally conceived by Simmons in 1993 and was based upon common morphological characteristics that included the development of conidial chains with primary, secondary, and tertiary branching resulting in substantial three-dimensional complexity. These characters can overlap to varying degrees with numerous taxa in another Alternaria group, the alternata species-group, making species-group differentiation difficult. However, members of the infectoria species-group are also distinguished from other small-spored Alternaria species based upon colony characteristics that typically include white or nearly white floccose colonies on DRYES medium and clumps of sporulation islands on low sugar media such as V8 agar, PCA, and weak PDA. In addition, the infectoria species-group contains representatives that are known to produce teleomorphs (Lewia), whereas the members of the alternata species-group and other Alternaria species-groups are strictly asexual. In this study, an assemblage of isolates recovered from varied hosts from the west coast of the United States were examined based upon morphological characters and compared to previously described members of the infectoria species-group. These isolates and members of the infectoria species-group typically produce arachnoid vegetative hyphae with multiple primary conidiophores, whereas other small-spored Alternaria species produce primary conidiophores predominately directly from the agar surface. Additionally, molecular phylogenetic analyses resolved these isolates and members of the infectoria species-group as distinctly nested amongst other sexual taxa in Allewia (Embellisia anamorph) and Macrospora (Nimbya anamorph) and phylogenetically distant to asexual lineages of Alternaria. One taxon among these isolates was novel and clustered with the asexual A. rosae in a distinct clade basal to all other members of the infectoria species-group. A new genus is proposed, Pseudoalternaria gen. nov. and a new taxon is described, Pseudoalternaria arrhenatheria sp. nov.. Moreover, a second taxon is reclassified, Pseudoalternaria rosae comb. nov.     

Aspergillus yunnanensis,a new and rare species in the Aspergillus section Jani

Aspergillus is a monophyletic genus comprising the subgenera Aspergillus, Circumdati, Cremei, Fumigati, Nidulantes and Polypaecilum. The subgenus Circumdati contains many economically important species and mycotoxin producers. Section Jani was recently introduced with morphological and molecular support. In the present study, two strains isolated from farmland soil were assigned in section Jani based on multi-locus phylogenetic analyses but showed low similarity with existing species. Further morphological observation found they had wider vesicles and conidia connections which were different from the known species. Based on phylogenetic and morphological data, Aspergillus yunnanensis was introduced as the third species in section Jani. Members in section Jani are rarely distributed, this is the first report of this section in China.     

Phylogenetic relationships of Microdontinae (Diptera: Syrphidae) based on molecular and morphological characters

The intrasubfamilial classification of Microdontinae Rondani (Diptera: Syrphidae) has been a challenge: until recently more than 300 out of more than 400 valid species names were classified in Microdon Meigen. We present phylogenetic analyses of molecular and morphological characters (both separate and combined) of Microdontinae. The morphological dataset contains 174 characters, scored for 189 taxa (9 outgroup), representing all 43 presently recognized genera and several subgenera and species groups. The molecular dataset, representing 90 ingroup species of 28 genera, comprises sequences of five partitions in total from the mitochondrial gene COI and the nuclear ribosomal genes 18S and 28S. We test the sister‐group relationship of Spheginobaccha with the other Microdontinae, attempt to elucidate phylogenetic relationships within the Microdontinae and discuss uncertainties in the classification of Microdontinae. Trees based on molecular characters alone are poorly resolved, but combined data are better resolved. Support for many deeper nodes is low, and placement of such nodes differs between parsimony and Bayesian analyses. However, Spheginobaccha is recovered as highly supported sister group in both. Both analyses agree on the early branching of Mixogaster, Schizoceratomyia, Afromicrodon and Paramicrodon. The taxonomical rank in relation to the other Syrphidae is discussed briefly. An additional analysis based on morphological characters only, including all 189 taxa, used implied weighting. A range of weighting strengths (k‐values) is applied, chosen such that values of character fit of the resulting trees are divided into regular intervals. Results of this analysis are used for discussing the phylogenetic relationships of genera unrepresented in the molecular dataset.     

DNA barcoding of genus Metapenaeopsis (Decapoda: Penaeidae) and molecular phylogeny inferred from mitochondrial and nuclear DNA sequences

Metapenaeopsis Bouvier, 1905 is the most diverse genus within Penaeidae. Metapenaeopsis shrimps exhibited subtle morphological differences, which make identification a difficult task based on taxonomic keys alone. In this study, we carried out DNA barcoding and phylogenetic analyses to examine taxonomy and phylogeny of genus Metapenaeopsis based on mitochondrial (COI) and nuclear (PEPCK and NaK) genes. Despite limited performance of DNA barcoding in delineating Metapenaeopsis shrimps, it questioned the taxonomic status of the two subspecies, Metapenaeopsis mogiensis intermedia and Metapenaeopsis mogiensis mogiensis, as well as three separate species: Metapenaeopsis provocatoria longirostris, Metapenaeopsis quinquedentata and Metapenaeopsis velutina. The major pattern of relationships between all studied taxa of Metapenaeopsis was similar across all analytical methods in which species with one-valved petasma were genetically distinct from those with two-valved petasma. As expected from morphology, the remaining species with stridulating organ constituted a strongly supported clade. In contrast, a paraphyletic clade was resolved for species without stridulating organ which contradicts Crosnier's morphological classification scheme for Metapenaeopsis. Overall, the present molecular data indicated that the shape of petasma and stridulating organ were both phylogenetically significant morphological characters for this genus, adding further evidence for the Crosnier's proposal.     

Polyphyly of Xylosandrus Reitter inferred from nuclear and mitochondrial genes (Coleoptera: Curculionidae: Scolytinae)

The Xyleborina ambrosia beetle genus Xylosandrus contains 54 species, several of which are of economic importance. The monophyly of the genus was tested using a data set comprised of multiple gene loci: 28S rDNA; the mitochondrial gene cytochrome oxidase I (COI); and the nuclear genes arginine kinase (ArgK), rudimentary (CAD), and Elongation Factor 1α (EF-1α). The nuclear protein-coding genes CAD and ArgK were used for the first time in phylogenetics of Scolytinae. Analyses were performed using Parsimony and Bayesian optimality criteria. Our analyses included 43 specimens representing 15 Xylosandrus species and 20 species from Amasa, Anisandrus, Cnestus, Euwallacea and Xyleborus, and two species from the outgroup genus Coccotrypes. All analyses recovered a polyphyletic Xylosandrus. Several species of Xylosandrus were consistently placed in clades with the genera Anisandrus and Cnestus with high support values (100% bootstrap support). Among these, was the economically important invasive species X. mutilatus, which was consistently recovered as part of the “Cnestus” clade. In our analyses, both CAD and ArgK demonstrated phylogenetic utility across varying nodal depths. Despite the selection of genes with signals at complementary phylogenetic depths, the data set used herein did not resolve the phylogeny of Xylosandrus and related genera. Since the taxon sample available for molecular work represents only a fraction of Xylosandrus species, a complete revision that combines molecular and morphological data in a total evidence approach is recommended for the genus.     

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     

Phylogenetic Molecular Species Delimitations Unravel Potential New Species in the Pest Genus Spodoptera Guenée, 1852 (Lepidoptera,Noctuidae)

Nowadays molecular species delimitation methods promote the identification of species boundaries within complex taxonomic groups by adopting innovative species concepts and theories (e.g. branching patterns, coalescence). As some of them can efficiently deal with large single-locus datasets, they could speed up the process of species discovery compared to more time consuming molecular methods, and benefit from the existence of large public datasets; these methods can also particularly favour scientific research and actions dealing with threatened or economically important taxa. In this study we aim to investigate and clarify the status of economically important moths species belonging to the genus Spodoptera (Lepidoptera, Noctuidae), a complex group in which previous phylogenetic analyses and integrative approaches already suggested the possible occurrence of cryptic species and taxonomic ambiguities. In this work, the effectiveness of innovative (and faster) species delimitation approaches to infer putative species boundaries has been successfully tested in Spodoptera, by processing the most comprehensive dataset (in terms of number of species and specimens) ever achieved; results are congruent and reliable, irrespective of the set of parameters and phylogenetic models applied. Our analyses confirm the existence of three potential new species clusters (for S. exigua (Hübner, 1808), S. frugiperda (J.E. Smith, 1797) and S. mauritia (Boisduval, 1833)) and support the synonymy of S. marima (Schaus, 1904) with S. ornithogalli (Guenée, 1852). They also highlight the ambiguity of the status of S. cosmiodes (Walker, 1858) and S. descoinsi Lalanne-Cassou & Silvain, 1994. This case study highlights the interest of molecular species delimitation methods as valuable tools for species discovery and to emphasize taxonomic ambiguities.     

Discopersicus n. gen., a New Member of the Family Tylenchidae ?rley, 1880 with Detailed SEM Study on Two Known Species of the Genus Discotylenchus Siddiqi, 1980 (Nematoda; Tylenchidae) from Iran

Discopersicus iranicus n. gen., n. comb., previously described from Iran as a new species under the genus Discotylenchus, is illustrated using light microscope and scanning electron microscope (SEM) observations and further studied using molecular characters. SEM studies revealed the newly proposed genus has oblique amphidial apertures on the lateral sides of the lip region. SEM images are also provided for two species of Discotylenchus, namely D. discretus and D. brevicaudatus, as the first SEM study of the genus. These results confirmed longitudinal amphidial aperture type on lateral sides of the lip region in genus Discotylenchus, as noted by Siddiqi while erecting the genus with D. discretus as the type species. Molecular phylogenetic analyses using partial small subunit (SSU) and large subunit (LSU) rDNA sequences revealed the affinity of the genus Discopersicus n. gen. with members of the subfamily Boleodorinae, as supported by morphological characters (mainly, the oblique amphidial opening).     

Phylogenetic relationships and biogeography of Pseudoxiphophorus (Teleostei: Poeciliidae) based on mitochondrial and nuclear genes

Phylogenetic relationships of species of genus Pseudoxiphophorus have been only tackled in detail based on morphology so far. However, phylogenetic evidence based on molecular data is still lacking. In this study, we have used five molecular markers (mitochondrial cytb, 16S, atp6-8, and nuclear actB and S7) to reconstruct a robust, inclusive phylogeny of Pseudoxiphophorus. Our phylogenetic results strongly disagree with the main morphological hypothesis, and indicate different phylogenetic relationships among the recognized species of Pseudoxiphophorus. Pseudoxiphophorus jonesii is recovered as the sister group of all other Pseudoxiphophorus lineages, and this initial splitting may be associated to the extension of the Mexican Neovolcanic Plateau at the Punta del Morro site (event used to calibrate our dating analysis). The branch leading to all other Pseudoxiphophorus separated subsequently into two major groups, one comprising those lineages occurring in southern Mexico and Guatemala–Belize, and another with those lineages that extended further southwards to Honduras and Nicaragua. This event took place during the Pliocene, and is likely associated with periods of inundation of the Polochic–Motagua fault area. The Isthmus of Tehuantepec also appears to have been a strong biogeographic barrier triggering cladogenesis in Pseudoxiphophorus. Heterandria formosa (traditionally placed as sister to Pseudoxiphophorus) is not sharing the most recent common ancestor with Pseudoxiphophorus, and is recovered as more distantly related to them. Furthermore, Pseudoxiphophorus bimaculatus (the most cosmopolitan species) is also recovered as a polyphyletic assemblage that appears to comprise those Pseudoxiphophorus that have not been assigned to the other eight, more localized species. All this suggests that Pseudoxiphophorus needs a major taxonomic revision as a whole in order to incorporate all existing diversity.     

Taxonomic Boundaries and Craniometric Variation in the Treeshrews (Scandentia,Tupaiidae) from the Palawan Faunal Region

The taxonomy of treeshrews (Order Scandentia) has long been complicated by ambiguous morphological species boundaries, and the treeshrews of the Palawan faunal region of the Philippines are no exception. Four named forms in the genus Tupaia Raffles, 1821, have been described from four island groups based on subtle qualitative morphological characters, and as many as three distinct species have been recognized. A recent molecular phylogenetic study of relationships among Tupaia species suggests that the two currently-recognized treeshrew species from the Palawan faunal region diverged very recently relative to other sister-species divergences within the genus and may not represent species-level taxonomic entities. Here we review the taxonomic and biogeographic histories of the Tupaia taxa from this region. We also collected craniodental data from 133 skulls of all four named forms, representing five island populations, and conducted univariate and multivariate analyses on these data. Our morphometric results are consistent with molecular results, further suggesting that there is insufficient evidence to recognize T. moellendorffi Matschie, 1898, as a separate species from T. palawanensis Thomas, 1894. Our analyses also revealed a craniodentally divergent population from the island of Balabac, which has never been considered a distinct subspecies (or species) from the population on Palawan. These results have conservation implications for the island populations in our analyses, but additional surveys and molecular evidence will be required to fully assess conservation priorities for the treeshrews of the Palawan faunal region.     

Unravelling the Biodiversity and Molecular Phylogeny of Needle Nematodes of the Genus Longidorus (Nematoda: Longidoridae) in Olive and a Description of Six New Species

The genus Longidorus includes a remarkable group of invertebrate animals of the phylum Nematoda comprising polyphagous root-ectoparasites of numerous plants including several agricultural crops and trees. Damage is caused by direct feeding on root cells as well as by transmitting nepoviruses that cause disease on those crops. Thus, correct identification of Longidorus species is essential to establish appropriate control measures. We provide the first detailed information on the diversity and distribution of Longidorus species infesting wild and cultivated olive soils in a wide-region in southern Spain that included 159 locations from which 449 sampling sites were analyzed. The present study doubles the known biodiversity of Longidorus species identified in olives by including six new species (Longidorus indalus sp. nov., Longidorus macrodorus sp. nov., Longidorus onubensis sp. nov., Longidorus silvestris sp. nov., Longidorus vallensis sp. nov., and Longidorus wicuolea sp. nov.), two new records for wild and cultivate olives (L. alvegus and L. vineacola), and two additional new records for wild olive (L. intermedius and L. lusitanicus). We also found evidence of some geographic species associations to western (viz. L. alvegus, L. intermedius, L. lusitanicus, L. onubensis sp. nov., L. vineacola, L. vinearum, L. wicuolea sp. nov.) and eastern distributions (viz. L. indalus sp. nov.), while only L. magnus was detected in both areas. We developed a comparative study by considering morphological and morphometrical features together with molecular data from nuclear ribosomal RNA genes (D2–D3 expansion segments of 28S, ITS1, and partial 18S). Results of molecular and phylogenetic analyses confirmed the morphological hypotheses and allowed the delimitation and discrimination of six new species of the genus described herein and four known species. Phylogenetic analyses of Longidorus spp. based on three molecular markers resulted in a general consensus of these species groups, since lineages were maintained for the majority of species. This study represents the most complete phylogenetic analysis for Longidorus species to date.     

End of an enigma: <Emphasis Type="Italic">Aenigmopteris</Emphasis> belongs in <Emphasis Type="Italic">Tectaria</Emphasis> (Tectariaceae: Polypodiopsida)

The phylogenetic affinities of the fern genus Aenigmopteris have been the subject of considerable disagreement, but until now, no molecular data were available from the genus. Based on the analysis of three chloroplast DNA regions (rbcL, rps16-matK, and trnL-F) we demonstrate that Aenigmopteris dubia (the type species of the genus) and A. elegans are closely related and deeply imbedded in Tectaria. The other three species of genus are morphologically very similar; we therefore transfer all five known species into Tectaria. Detailed morphological comparison further shows that previously proposed diagnostic characters of Aenigmopteris fall within the range of variation of a broadly circumscribed Tectaria.     

Molecular validation of Sarcodon quercinofibulatus, a species of the S. imbricatus complex associated with Fagaceae, and notes on Sarcodon

Morphological and molecular phylogenetic analyses revealed that Italian and Mexican collections of an unknown Sarcodon species of the S. imbricatus complex associated with Fagaceae (Castanea and Quercus), were assignable to Sarcodon quercinofibulatus, a species recently described from Spain. The species, characterized by a light brown-hazelnut coloured pileus surface eventually breaking into large and coarse scales, was recognized as independent from Sarcodon imbricatus and S. squamosus. S. aspratus, usually synonymized with S. imbricatus, is a different species. S. squamosus collections from montane and Mediterranean pine woodlands were shown to be conspecific. Four sections of Sarcodon (Sarcodon, Violacei, Squamiceps and Scabrosi) established by Maas Geesteranus (Verh K ned Akad Wet III, 65: 1–127, 1975) only on morphological basis, are here confirmed as monophyletic.     

A phylogenetic study of the squid family Onychoteuthidae (Cephalopoda: Oegopsida)

The oegopsid squid family Onychoteuthidae presently comprises six genera (Moroteuthis, Onychoteuthis, Ancistroteuthis, Kondakovia, Onykia and Chaunoteuthis) but the status of some of these is still uncertain. An interdisciplinary study was undertaken to clarify the phylogenetic relationships, which included a morphological approach (morphometric analysis), and a molecular study of a mitochondrial gene portion (l-rRNA gene, 16S). The morphometric analysis identified two groups, one including some Onychoteuthis and some Moroteuthis knipovitchi, and another including the remaining genera or species. At the intrageneric level, M. knipovitchi appears to be separated from the other species of the genus; M. robusta is closely related to M. ingens and M. pacifica. Morphometric analysis confirmed that Kondakovia longimana is different from M. ingens. Likewise, Onychoteuthis was clearly separated from the other genera, but there is neither geographical grouping nor morphometric differentiation of the Onychoteuthis species with the parameters measured. Some specimens were apparently intermediate between the Onychoteuthis and Ancistroteuthis groups. On a molecular basis, the Onychoteuthidae appeared to be monophyletic. Monophyly of the genus Moroteuthis (four species studied) was not strongly supported: M. knipovitchi was distinct from the others. The molecular analysis showed three Hawaiian species, Onychoteuthis compacta, O. sp. B and O. sp. C, to be closely related. The gene sequence for the newly created species of Onykia is clearly different from all the others, indicating that it is a true species.