DNA barcoding from the hooked squids (Cephalopods: Onychoteuthidae) of the Sargasso Sea

Squids of the family Onychoteuthidae are ecologically important in pelagic food webs and have been reported from every ocean except the Arctic. Although they are abundant and caught frequently as bycatch in fisheries, the biogeography of many species remains poorly understood. Species identification within the Atlantic Ocean is usually restricted to two species: Onychoteuthis banksii and Onykia carriboea. Here, we report the occurrence of four species of the family Onychoteuthidae (Onychoteuthis cf. banksii, Onykia carriboea, Walvisteuthis jeremiahi, and Onychoteuthis sp. AL 2) from the Sargasso Sea in the western Atlantic, identified using DNA barcoding (cytochrome c oxidase subunit I) and morphology. Our results have doubled the known onychoteuthid biodiversity in the Sargasso Sea, which has implications for the ecology of this oceanic region.     

Capsalids (Platyhelminthes: Monogenea) from marine fishes off Okinawa in Japan with the proposal of two new genera

Molecular studies of the Capsalidae suggested that the genus Benedenia is polyphyletic, but a taxonomic organization of the genus that reflects molecular data has not yet been proposed. As a result of molecular analysis (28S rDNA, ITS1–5.8S-ITS2, and cox1 data) including specimens of Benedeniinae newly obtained from Okinawa-jima Island in Japan, two new genera and the revival of Tareenia independent to the genus Benedenia are proposed. Gracilobenedenia n. gen. is distinguished from the other genera of Benedeniinae based on morphological characteristics. This new genus comprises 6 species: G. lutjani n. comb. (type species), G. anticavaginata n. comb., G. rohdei n. comb., G. beverleyburtonae n. comb., G. kuremibai n. gen., n. sp., and G. hichi n. gen., n. sp. Armatobenedenia n. gen. is a monotypic genus for A. armata n. comb. The present molecular phylogenetic analysis showed the independence of Tareenia, and it can be morphologically separated from the other benedeniines. Four species including two new species obtained from Okinawa-jima Island are reported: G. kuremibai n. sp., G. hichi n. sp., G. lutjani n. comb., and Metabenedeniella parva. Furthermore, in the species identification and phylogenetic analysis of capsalids, the usefulness of not only the 28S rDNA but also ITS and the cox1 regions was suggested. These genes were evaluated the efficacy of those regions in DNA barcoding, and the ITS and cox1 regions shown to be useful for DNA barcoding in capsalids compared to the 28S rDNA sequence.     

Characterization of the Cystoid Nematode Meloidoderita kirjanovae (Nemata: Sphaeronematidae) from Southern Italy

A population of the cystoid nematode Meloidoderita kirjanovae was detected parasitizing water mint (Mentha aquatica) in southern Italy. The morphological identification of this species was confirmed by molecular analysis using the internal transcribed spacer 1 (ITS1) and 5.8S gene sequences of nuclear ribosomal DNA (rDNA), which clearly separated it from the closely related species Meloidoderita polygoni. A phylogenetic analysis of M. kirjanovae with species of related genera was conducted using sequences of the D2-D3 expansion segments of the 28S nuclear ribosomal RNA gene. The resulting phylogenetic tree was congruent with trees from an extended dataset for Criconematina and Tylenchida. The basal position of the genus Meloidoderita together with Sphaeronema within the Criconematina clade in this tree may indicate their close relationships. The anatomical changes induced by M. kirjanovae population from Italy in water mint were similar to those reported for a nematode population infecting roots of M. longifolia in Israel. Nematode feeding caused the formation of a stellar syncytium that disorganized the pericycle and vascular root tissues.     

Diversimorbus metrosiderotis gen. et sp. nov. and three new species of Holocryphia (Cryphonectriaceae) associated with cankers on native Metrosideros angustifolia trees in South Africa

The Cryphonectriaceae includes important tree pathogens, especially on the Myrtales. During a routine disease survey in the Western Cape Province of South Africa, a fungus resembling the Eucalyptus pathogen Holocryphia eucalypti was observed on native Metrosideros angustifolia (Myrtales). The aims of this study were to identify the fungus and to expand surveys for fungi in the Cryphonectriaceae on M. angustifolia. Fungi were identified based on DNA sequence comparisons and morphological features, and their pathogenicity was tested on M. angustifolia under field conditions. Based on morphology and multigene phylogenetic analyses of DNA sequence data from six gene regions, we describe a new genus including a single species and three new species of Holocryphia (Cryphonectriaceae) from M. angustifolia. These fungi are provided with the names Diversimorbus metrosiderotis gen. et sp. nov., Holocryphia capensis sp. nov., Holocryphia gleniana sp. nov., and Holocryphia mzansi sp. nov. We also revise H. eucalypti, the type of the genus, to include only isolates from Eucalyptus in South Africa. Research results indicated that H. mzansi may undergo host shifts between different tree genera in the Myrtaceae. Inoculation tests showed that isolates of all the newly described species can cause lesions on the branches of M. angustifolia, indicating that they are all pathogens of this tree.     

Taxonomic revision of European Apium L. s.l.: Helosciadium W.D.J.Koch restored

European representatives of Apium sensu lato (Apiaceae), and Apium prostratum and Naufraga balearica, were studied with morphological, fruit anatomical, and palynological methods. Morphometric data were compared with phylogenetic results from previous molecular studies. This confirms that most of the European Apium species belong to a separate group corresponding to the previously named genus Helosciadium. All these species had previously been formally named as Helosciadium species, except for the new combination Helosciadium bermejoi, which is formally described here. Molecular studies place Apium prostratum and Naufraga balearica close to Apium graveolens, the type species of Apium. Our morphometric results show similarities of Naufraga with H. bermejoi, but fruit anatomy distinguishes it both from Helosciadium and from A. graveolens/prostratum. The placement of Cyclospermum leptophyllum in a separate genus is confirmed. Diagnostic keys to the genera and Helosciadium species, and an annotated checklist are given.     

A comparative study on the phylogenetic diversity of culturable actinobacteria isolated from five marine sponge species

A cultivation-based approach was employed to compare the culturable actinobacterial diversity associated with five marine sponge species (Craniella australiensis, Halichondria rugosa, Reniochalina sp., Sponge sp., and Stelletta tenuis). The phylogenetic affiliation of the actinobacterial isolates was assessed by 16S rDNA-RFLP analysis. A total of 181 actinobacterial strains were isolated using five different culture media (denoted as M1–M5). The type of medium exhibited significant effects on the number of actinobacteria recovered, with the highest number of isolates on M3 (63 isolates) and the lowest on M1 (12 isolates). The genera isolated were also different, with the recovery of three genera on M2 and M3, and only a single genus on M1. The number of actinobacteria isolated from the five sponge species was significantly different, with a count of 83, 36, 30, 17, and 15 isolates from S. tenuis, H. rugosa, Sponge sp., Reniochalina sp., and C. australiensis, respectively. M3 was the best isolation medium for recovery of actinobacteria from S. tenuis, H. rugosa, and Sponge sp., while no specific medium preference was observed for the recovery of actinobacteria from Reniochalina sp., and C. australiensis. The RFLP fingerprinting of 16S rDNA genes digested with HhaI revealed six different patterns, in which 16 representative 16S rDNAs were fully sequenced. Phylogenetic analysis indicated that 12 strains belong to the group Streptomyces, three strains belong to Pseudonocardia, and one strain belongs to Nocardia. Two strains C14 (from C. australiensis) and N13 (from Sponge sp.) have only 96.26% and 96.27% similarity to earlier published sequences, and are therefore potential candidates for new species. The highest diversity of three actinobacteria genera was obtained from Sponge sp., though the number of isolates was low. Two genera of actinobacteria, Streptomyces, and Pseudonocardia, were isolated from both S. tenuis and C. australiensis. Only the genus of Streptomyces was isolated from H. rugosa and Reniochalina sp. Sponge species have been demonstrated here to vary as sources of culturable actinobacterial diversity, and the methods for sampling such diversity presented may be useful for improved sampling of such diversity.     

Aliarcobacter vitoriensis sp. nov., isolated from carrot and urban wastewater

Two isolates, one recovered from a carrot and another one from urban wastewater, were characterized using a polyphasic approach. Phylogenetic analysis based on 16S rRNA gene sequences revealed that both isolates clustered together, and were most closely related to Aliarcobacter lanthieri. Multilocus phylogenetic analysis (MLPA) using the concatenated sequences of five housekeeping genes (atpA, gyrA, gyrB, hsp60 and rpoB) suggested that these isolates formed a distinct phylogenetic lineage among the genera derived from the former genus Arcobacter. Whole-genome sequence, in silico DNA-DNA hybridization (isDDH) and the average nucleotide identity (ANI) value between the genome of strain F199^T and those of related species confirmed that these isolates represent a novel species. These strains can be differentiated from its phylogenetically closest species A. lanthieri by its inability to growth on 1% glycine and by their enzyme activity of esterase lipase (C8) and acid phosphatase. Our results, by the application of a polyphasic analysis, confirmed that these two isolates represent a novel species of the genus Aliarcobacter, for which the name Aliarcobacter vitoriensis sp. nov. is proposed. The type strain is F199^T (=CECT 9230^T=LMG 30050^T).     

Ultrastructure and Development of Pasteuria sp. (S-1 strain), an Obligate Endoparasite of Belonolaimus longicaudatus (Nemata: Tylenchida)

Pasteuria sp., strain S-1, is a gram-positive, obligate endoparasitic bacterium that uses the phytoparasitic sting nematode, Belonolaimus longicaudatus, as its host in Florida. The host attachment of S-1 appears to be specific to the genus Belonolaimus with development occurring only in juveniles and adults of B. longicaudatus. This bacterium is characterized from other described species of Pasteuria using ultrastructure of the mature endospore. Penetration, development, and sporogenesis were elucidated with TEM, LTSEM, and SEM and are similar to other nematode-specific Pasteuria. Recent analysis of 16S rDNA sequence homology confirms its congeneric ranking with other Pasteuria species and strains from nematodes and cladocerans, and corroborates ultrastructural, morphological, morphometric, and host-range evidence suggesting separate species status.     

Description of a new species of Moniliformis (Acanthocephala: Moniliformidae) from Peromyscus hylocetes (Rodentia: Cricetidae) in Mexico

Moniliformis ibunami n. sp., is described from the intestine of the transvolcanic deermouse Peromyscus hylocetes Merriam 1898 (Cricetidae) from Parque Nacional Nevado de Colima “El Floripondio”, Jalisco, Mexico. The new species can be distinguished morphologically from the other 18 congeneric species of Moniliformis by a combination of morphological and molecular characters including the number of hooks on the proboscis (12 longitudinal rows, each one with six to eight transversally arranged unrooted hooks), the proboscis length (230–270 μm), the female trunk length (159–186 mm) and egg size (40–70 × 20–40). For molecular distinction, nearly complete sequences of the small subunit (SSU) and large subunit (LSU) of the nuclear ribosomal DNA and cytochrome oxidase subunit 1 (cox 1) of the mitochondrial DNA of the new species were obtained and compared with available sequences downloaded from GenBank. Phylogenetic analyses inferred with the three molecular markers consistently showed that Moniliformis ibunami n. sp. is sister to other congeneric species of Moniliformis. The genetic distance with cox 1 gene among Moniliformis ibunami n. sp., M. saudi, M. cryptosaudi, M. kalahariensis, M. necromysi and M. moniliformis ranged from 20 to 27%. Morphological evidence and high genetic distance, plus the phylogenetic analyses, indicate that acanthocephalans collected from the intestines of transvolcanic deer mice represent a new species which constitutes the seventh species of the genus Moniliformis in the Americas.     

Fukuyoa paulensis gen. et sp. nov., a New Genus for the Globular Species of the Dinoflagellate Gambierdiscus (Dinophyceae)

The marine epiphytic dinoflagellate Gambierdiscus is a toxicologically important genus responsible for ciguatera fish poisoning, the principal cause of non-bacterial illness associated with fish consumption. The genus currently contains species exhibiting either globular or anterior-posteriorly compressed morphologies with marked differences in cell shape and plate arrangement. Here we report a third globular, epiphytic and tychoplanktonic species from the coasts of Ubatuba, Brazil. The new species can be distinguished from G. yasumotoi and G. ruetzleri by its broader first apical plate that occupies a larger portion of the epitheca. Accordingly, phylogenetic trees from small subunit (SSU) and large subunit (LSU) ribosomal DNA sequences also showed strongly supported separation of the new species from the G. yasumotoi / G. ruetzleri group albeit with short distance. The molecular phylogenies, which included new sequences of the planktonic species Goniodoma polyedricum, further indicated that the globular species of Gambierdiscus formed a tight clade, clearly separated (with strong bootstrap support) from the clade of lenticular species including the type for Gambierdiscus. The morphological and molecular data in concert support the split of Gambierdiscus sensu lato into two genera. Gambierdiscus sensu stricto should be reserved for the species with lenticular shapes, highly compressed anterioposteriorly, with short-shank fishhook apical pore plate, large 2'' plate, low and ascending cingular displacement, and pouch-like sulcal morphology. The new genus name Fukuyoa gen. nov. should be applied to the globular species, slightly laterally compressed, with long-shank fishhook apical pore plate, large 1'' plate, greater and descending cingular displacement, and not pouch-like vertically-oriented sulcal morphology. Fukuyoa contains the new species Fukuyoa paulensis gen. et sp. nov., and F. yasumotoi comb. nov. and F. ruetzleri comb. nov.     

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.     

Allocreadium khankaiensis sp.nov. and Allocreadium hemibarbi Roitman, 1963 (Trematoda: Allocreadiidae) from the Russian Far East: Morphological,molecular, and phylogenetic studies

Three species of the genus Allocreadium were collected from cyprinid fish in the territory of the Russian Far East: Allocreadium khankaiensis sp.nov; Allocreadium hemibarbi Roitman, 1963, and Allocreadium sp. 1. It is established that, according to morphometric values, the species A. khankaiensis sp.nov. has high morphological similarity with Allocreadium elongatum Achmerov, 1960; Allocreadium erythroculteris Achmerov, 1960; and Allocreadium aburahaya Shimazu, 2003. However, A. khankaiensis sp.nov. differs from A. elongatum and A. erythroculteris by vitellaria extension and size of cirrus pouch. In addition, A. khankaiensis sp.nov. differs from A. erythroculteris by the smaller size of the body and ventral sucker. A. khankaiensis sp.nov. and A. aburahaya have similar metrical values but A. khankaiensis sp.nov. differs by the shape of the testes: rounded vs. lobed. Species affiliation of another Allocreadium species is based on the morphometric identity with the species A. hemibarbi. Phylogenetic tree reconstruction using partial sequences of the 28S rRNA gene revealed a clear resolution of all Allocreadium species, excluding Allocreadium lobatum and Allocreadium neotenicum. The genetic analysis detected Allocreadium sp. 1 as a presumably independent species from the Razdolnaya River basin.     

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.     

A synopsis of gall midges associated with conifers,with description of a new species of the genus <Emphasis Type="Italic">Kaltenbachiola</Emphasis> Hedicke (Diptera,Cecidomyiidae: Lasiopterinae) from Yakutia,damaging cones of the Siberian spruce (<Emphasis Type="Italic">Picea obovata</Emphasis>)

The world fauna comprises 134 species from 52 genera of gall midges associated with plants of the order Pinales (3 families, 16 genera and 74 species); 14 genera are specific to Pinales. The distribution of genera and species of gall midges over host plant taxa is described. The Holarctic genus Kaltenbachiola comprises four species all developing in the spruce (Picea spp.) cones. Earlier, K. strobi (Winnertz), widely distributed in Europe, was the only species known in the Palaearctic. A new species Kaltenbachiola anastasiae sp. n. is described, which damages cones of Picea obovata in Central Yakutia. The phylogenetic relationships of Kaltenbachiola with close genera specific to Pinales are characterized, and an updated diagnosis of the genus with additional morphometric parameters and keys to the pine-specific genera of the tribe Dasineurini and to species of the genus Kaltenbachiola are given. The host associations and specific traits of biology and distribution of Kaltenbachiola species are considered.     

Metulocladosporiella gen. nov. for the causal organism of Cladosporium speckle disease of banana

Cladosporium musae, a widespread leaf-spotting hyphomycete on Musa spp., is genetically and morphologically distinct from Cladosporium s. str. (Davidiella anamorphs, Mycosphaerellaceae, Dothideales). DNA sequence data derived from the ITS and LSU gene regions of C. musae isolates show that this species is part of a large group of hyphomycetes in the Chaetothyriales with dematiaceous blastoconidia in acropetal chains. Cladosporium adianticola, a foliicolous hyphomycete known from leaf litter in Cuba is also a member of this clade and is closely related to C. musae. A comparison with other genera in the Cladosporium complex revealed that C. musae belongs to a lineage for which no generic name is currently available, and for which the genus Metulocladosporiella gen. nov. is proposed. Two species of Metulocladosporiella are currently known, namely M. musae, which is widely distributed, and M. musicola sp. nov., which is currently known from Africa.     

Molecular and morphometric identification of Trypanosoma (Megatrypanum) minasense in blood samples of marmosets (Callithrix: Callithrichidae) from the city of Rio de Janeiro,Brazil.

Callithrix jacchus and C. penicillata marmosets are invasive to the state of Rio de Janeiro, Brazil, threatening the native and vulnerable C. aurita. Both invasive species can be hosts of Trypanosoma cruzi, T. minasense, T. rangeli and T. devei. We aim to investigate the occurrence of trypanosomatids in Callithrix sp. from Jardim Botânico do Rio de Janeiro, located in a central and populous area of the city. Fifteen marmosets were captured. Blood samples were collected for light microscopy and molecular genetics analysis. Parasites morphometric values were evaluated for species identification. DNA was extracted from blood samples by phenol-chloroform method, for partial amplification of the 18S rRNA gene. PCR products were sequenced and aligned using BLAST®. A maximum likelihood phylogenetic tree was constructed to analyze the proximity between the observed sequences. By light microscopy, trypomastigotes were detected in five of the fifteen marmosets. Morphometric measurements and size polymorphism corresponded to those previously described for T. minasense. The DNA sequences of approximately 600 base pairs of the 18S rRNA gene were obtained for three samples with 99% identity with T. minasense sequence, forming a cluster in the phylogenetic tree and corroborating morphometric analysis. Trypanosoma minasense is a highly specific parasite to non-human primates considered as non-pathogenic. There is no evidence of infection in humans and these parasite findings from invasive marmosets do not support additional risks for the native species.     

Fungal pathogen (mis-) identifications: A case study with DNA barcodes on Melampsora rusts of aspen and white poplar

Wide variation and overlap in morphological characters have led to confusion in species identification within the fungal rust genus Melampsora. The Melampsora species with uredinial–telial stages on white poplar and aspens are especially prone to misidentification. This group includes the Melampsora populnea species complex and the highly destructive pine twisting rust, Melampsora pinitorqua, which alternates between hosts in Populus section Populus and Pinus. Our objective was to compare morphologically based identification to genetic material extracted from Melampsora species pathogenic to aspen and white poplar. We compared morphometric traits and DNA barcodes obtained from internal transcribed spacer (ITS), large ribosomal RNA subunit (28S), and mitochondrial cytochrome oxidase 1 (CO1) sequences to delimit within this taxonomically difficult group. Eight different Melampsora species were initially defined based on host specificity and morphometric data. DNA barcodes were then overlaid on these initial species definitions. The DNA barcodes, specifically those defined on ITS and 28S sequences, provided a highly accurate means of identifying and resolving Melampsora taxa. We highlighted species misidentification in specimens from Canadian herbaria related to either Melampsora medusae f. sp. tremuloidae or Melampsora aecidioides. Finally, we evidenced that the north-American species found on Populus alba, M. aecidioides is closely related but distinct from the four species of the M. populnea complex (Melampsora larici-tremulae, Melampsora magnusiana, Melampsora pinitorqua, and Melampsora rostrupii) found in Eurasia.     

Morphogenesis and evolution of Kutorgina Billings, 1861 (Brachiopoda, Kutorginida)

A comparative morphological analysis of all known members of the Lower-Middle Cambrian genus Kutorgina, one of the brachiopod genera with a carbonate shell that are oldest and richest in species. The evolution of the genus is discussed, and the borders between the Kutorgina species are emended. A new species, Kutorgina elanica sp. nov., is described.