Nuclear Ribosomal Gene Research Articles

Exploring Environmental DNA (eDNA) to Assess Biodiversity of Hard Substratum Faunal Communities on the Lucky Strike Vent Field (Mid-Atlantic Ridge) and Investigate Recolonization Dynamics After an Induced Disturbance

Deep hydrothermal vent ecosystems face physical disturbances from naturally occurring volcanic and tectonic activities and are at increasing risk of mineral resource exploitation, raising concerns about the resilience of biological communities. Following destructive events, efficient and rapidly applicable surveys of organisms are required to monitor the state, evolution and recovery of these ecosystems to their baseline status. In this study, we explored the eDNA approach as a tool to assess biodiversity of communities associated with deep-sea hard substrata and tracked the recolonization dynamics of benthic communities living on the Montsegur edifice within the Lucky Strike vent field (Mid-Atlantic Ridge), after an induced disturbance. Hard substratum samples were collected prior to and one year after the disturbance, for environmental DNA (eDNA) metabarcoding using one marker of the mitochondrial Cytochrome Oxidase I (COI) gene and three markers of the nuclear 18S ribosomal RNA (18S) gene. We also generated a DNA barcoding inventory that consisted of taxa physically collected and morphologically identified. This inventory contained amplified barcodes from COI, 18S and the nuclear ribosomal RNA 28S gene. The resulting sequence information from the COI and 18S were used for eDNA taxonomic assignment. The eDNA datasets uncovered a high diversity of metazoan OTUs. Baseline data collected at the start of the experiment identified higher OTU richness at sites peripheral to the active edifice, as well as inactive sites. One year following the initial disturbance, analysis of recolonization data found no statistical difference in presence/absence from baseline communities. The eDNA protocols provide a reproducible strategy to quickly assess biodiversity associated with deep sea hard substratum, enabling comparisons across various habitats. To follow recolonization dynamics at small spatial scales however, we recommend an approach that uses both molecular and morphological-based traditional methods. Finally, we present original data on the “unseen” diversity of the fauna inhabiting the poorly studied inactive sites, locations that are targeted by commercial mining. Continued monitoring of these sites is currently ongoing and will bring new insight on recovery potential over time, with the ultimate goal of informing conservation and management decisions in relation to the protection of hydrothermal vent ecosystems.

A morphological and combined phylogenetic analysis of pirate spiders (Araneae, Mimetidae): evolutionary relationships, taxonomy and character evolution

Mimetidae is one of the three families within Araneoidea whose members do not spin foraging webs, but are unique in displaying a complex prey-capture behaviour known as aggressive mimicry. Mimetids are distributed worldwide and are most diverse in the tropics of Central and South America. Here we provide a comprehensive phylogeny of pirate spiders (Mimetidae) based on analyses that combine morphological and multigene nucleotide sequence data. We scored 147 morphological characters for 55 mimetids and 16 outgroup taxa and combined it in a total-evidence approach with the sequence data of Benavides et al. (2017) which included two nuclear ribosomal genes, 18S rRNA and 28S rRNA, two mitochondrial ribosomal genes, 12S rRNA and 16S rRNA, the nuclear protein-encoding gene histone H3 and the mitochondrial protein-encoding gene cytochromec oxidase subunitI. We analysed the combined dataset using parsimony, maximum-likelihood and Bayesian inference methods. Our results support the monophyly of Mimetidae and of the genera Gelanor, Ero, Anansi and Australomimetus. Mimetidae is sister to Arkyidae + Tetragnathidae. Mimetus as currently circumscribed is not monophyletic under any analytical approach used, although several lineages within the genus are consistently found in our analyses. We describe, illustrate and discuss the morphological synapomorphies that support the main clades of Mimetidae. The following nomenclatural changes are proposed: Ermetus koreanus (Paik, 1967), the sole species of the genus, is transferred to Ero C.L. Koch, 1836 and thus Ermetus Ponomarev, 2008 is a junior synonym of Ero C.L. Koch, 1836 (new synonymy) and Ero koreana Paik, 1967 becomes a revalidated combination. Phobetinus sagittifer Simon, 1895, the type species of the genus, is transferred to Mimetus Hentz, 1832 and thus Phobetinus Simon, 1895 is a junior synonym of Mimetus Hentz, 1832 (new synonymy), which results in two changes: Mimetus sagittifer (Simon, 1895), new combination and Mimetus investus (Simon, 1909), new combination. Reo latro Brignoli, 1979, the type species of the genus, is transferred to Mimetus and thus Reo Brignoli, 1979 is a junior synonym of Mimetus (new synonymy), which results in the following two changes: Mimetus latro Brignoli, 1979, new combination and Mimetus eutypus Chamberlin & Ivie, 1935, revalidated combination. Arochoides integrans Mello-Leitão, 1935 is transferred to Tetragnathidae (new family placement). The type specimen of Arochoides integrans, the only species in this genus, is a subadult male of Azilia (Tetragnathidae), most likely Azilia histrio Simon, 1895. Arochoides is a junior synonym of Azilia (new synonymy). http://zoobank.org/urn:lsid:zoobank.org:pub:90F6B3DA-232B-428C-BF38-AEA8953D7685

Taenia asiatica: Historical overview of taeniasis and cysticercosis with molecular characterization.

Asian Taenia is a human-infecting Taenia tapeworm known as Taenia asiatica following morphological examination of adult and larval stages of the tapeworm by Eom and Rim (1993). The life cycle of T. asiatica differs from that of T. saginata in its intermediate host (pigs versus cattle) as well as in the infected organs (liver versus muscle). T. asiatica can be differentiated from T. solium and T. saginata by examination of morphological characteristics such as the scolex, mature and gravid proglottids in the adult stage, and the scolex and bladder surface in the larval stage. T. asiatica has been identified in Korea, Taiwan, the Philippines, China, Thailand, Indonesia, Vietnam, Japan, Lao PDR, Nepal and India. The molecular tools employed for T. asiatica identification have been developed to differentiate T. asiatica from other human-infecting Taenia tapeworms based on genetic information such as nucleotide sequence of mitochondrial genes, nuclear ribosomal genes and nuclear genes that lead to development of the subsequent molecular techniques, such as PCR-RFLP, PCR-RAPD, BESST-base, LAMP and qPCR. Investigation of the phylogenetic relationships among human Taenia species revealed that T. asiatica is a sister species with T. saginata, which is genetically more similar than other Taenia species in terms of the nucleotide sequences of cox1, nad1 and 28S rDNA. The mitochondrial genomes of human Taenia tapeworms comprise 13,703bp (T. asiatica), 13,670bp (T. saginata) and 13,709bp (T. solium), and contain 36 genes including 12 protein-coding genes, 2 ribosomal RNAs (rRNAs, a small and a large subunit), and 22 transfer RNAs (tRNAs). Sequence differences in the full genome of T. asiatica and T. saginata mitochondria is 4.6%, while T. solium differs by 11%. Hox gene orthology in T. asiatica was established by comparative analysis with Platyhelminthes Hox genes. T. asiatica Hox revealed six Hox orthologs including two lab/Hox1, two Hox3, one Dfd/Hox4 and one Lox/Lox4. Hybridization between T. asiatica and T. saginata was definitely observed in these species which are sympatrically endemic in the regions of Korea, Thailand, China and Lao PDR. Comparative analyses of T. asiatica, T. saginata and T. solium genomes were also reported with genome features. Taenia asiaticus nomen novum was proposed for T. asiaticaEom and Rim, 1993 which is a homonym of T. asiatica Linstow, 1901 (Davaineidae).

<p><strong>Additions to <em>Ochroconis</em> from India</strong></p>

Ochroconis helicteris, a novel species of the oligotrophic genus Ochroconis (Sympoventuriaceae, Venturiales), has been described and illustrated. Morphologically, this species has characters similar to allied species in the genus Ochroconis, but differs in conidial dimensions and shape. Phylogenetic analyses using nuclear ribosomal DNA gene ITS and protein coding gene BT2 revealed it clustering as monophyletic clade which was separated from known Ochroconis species. New species was isolated from leaf surface of medicinal plant Helicteris isora L.

Phylogenetic Position of Codonocephalus Diesing, 1850 (Digenea, Diplostomoidea), an Unusual Diplostomid with Progenetic Metacercariae

Codonocephalus is a monotypic genus of diplostomid digeneans and is the only genus in the sub-family Codonocephalinae. The type-species Codonocephalus urniger has an unusual progenetic metacercaria that uses frogs as intermediate hosts and can use snakes as paratenic hosts. Adult C. urniger parasitize ardeid wading birds in the Palearctic. Despite the broad distribution of Codonocephalus, no DNA sequence data are currently available for the genus. In this study, we generated sequence data for nuclear ribosomal and mitochondrial DNA from progenetic metacercaria of the type-species C. urniger from marsh frog, Pelophylax ridibundus, collected in Ukraine. We used partial sequences of the nuclear ribosomal 28S gene to examine for the first time the phylogenetic position of Codonocephalus among the Diplostomoidea.

Two new species of Rohdendorfia (Diptera: Syrphidae) from Central Asia

Abstract Two new species of the flower fly genus Rohdendorfia Smirnov, 1924 (Diptera: Syrphidae) are described, Rohdendorfia bella Mengual, sp. nov. and Rohdendorfia khakimovi Barkalov, sp. nov. A full description and images of both new species are given, as well as an identification key for the species of this genus. Molecular sequences from mitochondrial cytochrome c oxidase I (COI) gene and nuclear ribosomal 28S and 18S rRNA genes are also provided for the new species. The studied specimens of Rohdendorfia bella Mengual, sp. nov. are the first record of the genus Rohdendorfia from India and represent the first individuals collected in the state of Jammu and Kashmir.

Integrative species delimitation in Nearctic ambush bugs (Heteroptera: Reduviidae: Phymatinae): insights from molecules, geometric morphometrics and ecological associations

AbstractAmbush bugs (Hemiptera: Reduviidae: Phymatinae) are sit‐and‐wait predators of flower‐visiting insects including pollinators. Broad species distribution ranges, intraspecific polymorphism, sexual dimorphism and subtle interspecific differences all contribute to making species delimitation especially difficult in this group, which is used as a model in the study of interactions between sexual dimorphism and sexual selection. Species boundaries among Nearctic ambush bugs in the common and frequently collected erosa species group (11 species, nine subspecies) have therefore remained unclear, resulting in a complex and poorly justified taxonomy. Recent molecular phylogenetic research suggested that several widespread Nearctic species are para‐ or polyphyletic. We here build on this research, integrating geometric morphometrics, molecular species delimitation approaches and host plant association data to provide a comprehensive dataset with respect to both taxon and character sampling with the goal of teasing apart evolutionary lineages of Nearctic Phymata Latreille. Although molecular‐based species delimitation analyses suggested a variety of species hypotheses, probably as a result of striking discordance between mitochondrial and nuclear ribosomal genes, the combination of these with geometric morphometric data enabled us to confidently delimit several of these problematic taxa. In addition, geometric morphometric analysis of pronotal shape revealed undocumented morphological patterns that appear to be useful in the diagnosis of many of the surveyed taxa. The results from this study provide an objective foundation for the much‐needed taxonomic revision of the most ubiquitous ambush bugs in North America.

Morphology, Ultrastructure, and Mitochondrial Genome of the Marine Non-Photosynthetic Bicosoecid Cafileria marina Gen. et sp. nov.

In this paper, we describe a novel bacteriophagous biflagellate, Cafileria marina with two smooth flagellae, isolated from material collected from a rock surface in the Kvernesfjorden (Norway). This flagellate was characterized by scanning and transmission electron microscopy, fluorescence, and light microscopy. The sequence of the small subunit ribosomal RNA gene (18S) was used as a molecular marker for determining the phylogenetic position of this organism. Apart from the nuclear ribosomal gene, the whole mitochondrial genome was sequenced, assembled, and annotated. Morphological observations show that the newly described flagellate shares key ultrastructural characters with representatives of the family Bicosoecida (Heterokonta). Intriguingly, mitochondria of C. marina frequently associate with its nucleus through an electron-dense disc at the boundary of the two compartments. The function of this association remains unclear. Phylogenetic analyses corroborate the morphological data and place C. marina with other sequence data of representatives from the family Bicosoecida. We describe C. marina as a new species from a new genus in this family.

Impatiens maculifera sp. nov. (Balsaminaceae) Yunnan, China

Impatiens (Balsaminaceae) is a notoriously difficult genus to study for reasons that include small, remote and endemic areas of occurrence, and difficulty in obtaining well‐dried herbarium specimens and separating and reconstructing floral parts from available dried specimens. Impatiens has also been notoriously difficult when it comes to phylogenetic resolution at the infrageneric level, but a new system with two subgenera (Impatiens and Clavicarpa) was recently proposed by Yu et al. and it has made it easier to classify newly discovered species.Impatiens maculifera, a new species of Balsaminaceae from Malipo County, Yunnan, China described here, is similar to I. parvisepala S.X. Yu & Y.T. Hou in having racemose inflorescences, four lateral sepals, clavate capsules and ellipsoid seeds, but differs in having leaves narrowly oval or with elliptic–lanceolate blades (versus obovate or obovate–lanceolate), obvious petioles (versus leaves subsessile), racemose inflorescence with 8–12 flowers (versus 6–8 flowers), flowers greenish yellow (versus yellow), lateral united petals and lower sepal with dense red spots (versus red spots absent), and dorsal petal with apparent stalk at base (versus unapparent stalk). Morphological characters and molecular phylogenetic analyses of DNA sequences from both nuclear ribosomal and plastid genes show that the new species differs distinctively from I. parvisepala. Furthermore, I. maculifera is distinguished from other Impatiens species based on morphological, micromorphological and palynological evidence, and molecular data.

A study on the non-monophyletic genera Australothrix and Holostichides based on multigene and morphological analyses with a reexamination of type materials (Protozoa: Ciliophora)

In a study of ciliate diversity, we collected two new species of the genera Australothrix and Holostichides. Based on nuclear ribosomal genes, our study shows that these two genera are genetically non-monophyletic. To clarify the issues of the non-monophyly, we reexamined type material of H. heterotypicus, H. terrae, and Birojimia soyaensis. Based on multigene and morphometric analyses, Australothrix and Holostichides are clearly non-monophyletic even in mitochondrial CO1 gene trees. The multigene analyses show a clade composed of A. lineae sp. nov., H. terrae, and B. soyaensis, suggesting that a cytopharynx with argyrophilic structures might be their synapomorphy. A list of species with this type of cytopharynx is provided for further studies to either accept or reject this hypothesis. Based on the reexamination, we discriminate the morphologically nearly identical (cryptic/sibling) species H. obliquocirratus sp. nov. from H. heterotypicus and they show significant genetic dissimilarities in the multigene trees. They showed only few morphological (non-quantitative) differences and thus distinguishing them morphologically needs careful investigation.

Fusarium incarnatum-equiseti complex from China.

The Fusarium incarnatum-equiseti species complex (FIESC) is shown to encompass 33 phylogenetic species, across a wide range of habitats/hosts around the world. Here, 77 pathogenic and endophytic FIESC strains collected from China were studied to investigate the phylogenetic relationships within FIESC, based on a polyphasic approach combining morphological characters, multi-locus phylogeny and distribution patterns. The importance of standardised cultural methods to the identification and classification of taxa in the FIESC is highlighted. Morphological features of macroconidia, including the shape, size and septum number, were considered as diagnostic characters within the FIESC. A multi-locus dataset encompassing the 5.8S nuclear ribosomal gene with the two flanking internal transcribed spacers (ITS), translation elongation factor (EF-1α), calmodulin (CAM), partial RNA polymerase largest subunit (RPB1) and partial RNA polymerase second largest subunit (RPB2), was generated to distinguish species within the FIESC. Nine novel species were identified and described. The RPB2 locus is demonstrated to be a primary barcode with high success rate in amplification, and to have the best species delimitation compared to the other four tested loci.

MOLECULAR-GENETIC IDENTIFICATION OF CAUSAL AGENTS OF FUNGAL DISEASES ON POTATO AND TOMATO COLLECTED UNDER PRODUCTION STORAGE

The expansion of exports and imports of potato and tomato crops between countries contributes to the rapid spread of the most harmful species and strains of phytopathogenic fungi around the world. Due to it, the identification of phytopathogenic fungi is important for agriculture. Currently, molecular-genetic methods are considered as one of the modern and promising methods for the diagnosis and identification of plant diseases, which allow identifying microorganisms without any knowledge of its biological features. One of the recommended methods for identification and classification of taxa is the determination of the nucleotide sequence of nuclear ribosomal gene regions.
 276 infected potato tubers and tomato fruits were used to identify phytopathogenic fungi isolates, which were collected from production storages. Molecular-genetic identification of microorganisms based on the analysis of the nucleotide sequences of the ITS region made it possible to taxonomically identify 102 isolates of phytopathogenic fungi that belong to 7 genera. It was revealed that the proportion of fungi, which belohg to the genera Fusarium (60%), Penicillium (15%) and Clonostachys (12%), Alternaria (9%) were dominated in the generic structure.
 The isolation frequency (Fr) and relative density (RD) of 7 generaphytopathogenic fungi were calculated according to the results of molecular-genetic identification.Isolation frequency (Fr)of fungi genera ranged from 0.3% to 22.1%. Relative density of fungi species ranged varied 0.9% to 59.8%.
 Thereby, molecular-genetic identification based on the analysis of the nucleotide sequence of the ITS region made it possible to perform identification of pathogens on potato and tomato under production storage.Also, fungi Clonostachys and Acrostalagmus genera were isolated, which are not pathogens of potato and tomato diseases.

First Report of Black Root Disease Caused by Chaetomium globosum on Rubber Tree Seedlings in Yunnan, China

In September 2016, a new disease of the root tips of rubber tree seedlings (Hevea brasiliensis Mull Arg.) was found in Jinghong City, Yunnan, China. Diseased seedlings had stunted, blackened, but not rotten root tips. The disease incidence at the seedbeds could reach as high as 98%. Some stems of the seedlings developed lesions or cankers, but their leaves were free of any disease symptoms. Samples of infected root tips were washed with tap water, dried, and cut into 0.2- to 0.3-cm pieces and then surface sterilized with 0.1% mercuric chloride solution for 30 s followed by three 1-min rinses with sterilized water. The pieces near the edge of the symptomatic tissues were placed on potato dextrose agar (PDA) medium and incubated at 25°C for 5 days. Such infected tissues consistently yielded a filamentous fungus with similar cultural and morphological characteristics on PDA. A representative pure culture designated as strain XJ160930 was finally obtained after several isolation transfers to PDA using a single ascospore. Strain XJ160930 had the following culture characteristics: A colony plug on 7-day PDA culture at 25°C exhibited an entire edge, with sparse white to olivaceous buff aerial hyphae when young, then without aerial hyphae and becoming citrine greenish olivaceous to gray olivaceous with fulvous, apricot to sienna exudates, or olivaceous to olivaceous gray exudates diffusing into the medium. Ascomata superficial, ostiolate, greenish olivaceous or slightly dark olivaceous buff to gray or dull green in reflected light owing to ascomatal hairs, subglobose, ovate or obovate, 110 to 240 μm diameter, and 160 to 410 µm high. Ascomatal wall brown, textura intricata in surface view. Terminal hairs finely warty, brown, erect or undulate to loosely coiled with erect or flexuous lower part, tapering and fading toward the tips, 2 to 5 μm diameter near the base. Lateral hairs brown, flexuous, fading and tapering toward the tips. Ascospores brown when mature, limoniform, usually biapiculate, bilaterally flattened, 7 to 8.5 × 5.5 to 7 μm, with an apical germ pore. Based on these morphological characteristics, the fungus was identified as Chaetomium globosum (Wang et al. 2016). To determine the pathogenicity, isolate XJ160930 was inoculated onto GT1 rubber tree seeds. Thirty seeds each were sowed in three 30-cm pots of clean sand, and 500 ml of spore suspension (1.0 × 10⁶ conidia/ml) prepared from 10-day-old culture on PDA was sprayed onto these pots. Water was used as a control treatment. All the pots were placed in a greenhouse at approximately 25 to 28°C and sprayed with sterilized water once every 3 days. After 20 days of incubation, the seeds began to germinate and grow. When these seedlings were pulled out then, all those from the fungus-inoculated pots showed root-tip stunts and darkening, like the symptoms observed in the field-infected seedlings. The pathogen was successfully isolated from the infected root tip tissues and confirmed as C. globosum by microscopic observation. No symptom was observed on the control seedlings. The experiment was repeated three times, and the same results were obtained. For molecular characterization, DNA was extracted from a pure culture of strain XJ160930 with a UNlQ-10 Column Fungal Genomic DNA Isolation Kit (Shanghai Sangon Biotech, China). A 555-bp segment of 5.8S nuclear ribosomal gene with two flanking internal transcribed spacers (ITS), a 676-bp segment of DNA dependent RNA polymerase subunit II gene region (RPB2), and a 733-bp segment of β-tubulin (TUB2) were amplified and then sequenced, using the primers ITS5/ITS4, RPB2AM-1bf/RPB2AM-7R (Miller and Huhndorf 2005), and T1 (O’Donnell and Cigelnik 1997) and TUB4Rd (Groenewald et al. 2013). The sequences, deposited in GenBank as accession numbers KY355130 (ITS), KY355133 (RPB2), and KY355135 (TUB2), showed 100, 99, and 100% sequence identity to C. globosum (GenBank HQ529775.1, KM655411.1, and KX976958.1, respectively). Therefore, both molecular and morphological analyses indicated that the pathogenic fungus was C. globosum. C. globosum has been reported to cause leaf blight disease of Chrysanthemum morifolium in Zhejiang, China (Ma et al. 2015), and decay the stem tissues of the H. brasiliensis (Wong 1988). Here, C. globosum caused failed development of the main roots in rubber seedlings, resulting in susceptibility to wind and proneness to toppling later in their lives. As far as we know, this is the first report of this pathogen involved in disease in H. brasiliensis in China occurring in the early phases of the host life.

Invasion genetics of the silver carp Hypophthalmichthys molitrix across North America: Differentiation of fronts, introgression, and eDNA metabarcode detection.

In the 1970s, the introduced silver carp Hypophthalmichthys molitrix (which is indigenous to eastern Asia) escaped from southern U.S. aquaculture to spread throughout the Mississippi River basin, and since has steadily moved northward. This large, prolific filter-feeder reduces food availability for other fishes. It now has reached the threshold of the Laurentian Great Lakes, where it likely will significantly impact food chains and fisheries. Our study evaluates population genetic variability and differentiation of the silver carp using 10 nuclear DNA microsatellite loci, and sequences of two mitochondrial genes–cytochrome b and cytochrome c oxidase subunit 1, along with the nuclear ribosomal protein S7 gene intron 1. We analyze population samples from: two primary Great Lakes’ invasion fronts (at the Illinois River outside of Chicago, IL in Lake Michigan and in the Wabash River, which leads into the Maumee River and western Lake Erie), the original establishment “core” in the Lower Mississippi River, and expansion areas in the Upper Mississippi and Missouri rivers. We analyze and compare our results with bighead and other invasive carps, and cyprinid relatives. Results reveal that the silver carp invasion possesses moderate levels of genetic diversity, with more mtDNA haplotypes and unique microsatellite alleles in the “core” Lower Mississippi River population, which also diverges the most. The two invasion fronts also significantly genetically differ. About 3% of individuals (including all populations except the Illinois River) contain a unique and very divergent mtDNA haplotype, which likely stems from historic introgression in Asia with female largescale silver carp H. harmandi. The nuclear microsatellites and S7 sequences of the introgressed individuals do not differ from silver carp and are very distant from bighead carp. These sequence variation data are employed to design and evaluate a targeted high-throughput metabarcoding sequence assay that identifies and distinguishes among species of invasive carps (i.e., silver, bighead, grass, black, and common carps, along with goldfish), as well as native cyprinids, using cytochrome b. Our assay further differentiates among selected silver carp haplotypes (including between H. molitrix and H. harmandi), for use in population genetics and future analyses of spread pathways. We test and evaluate this assay on environmental (e)DNA water samples from 48 bait shops in the Great Lakes’ region (along the Lake Erie, Lake St. Clair, and Wabash River watersheds), using positive and negative controls and custom bioinformatic processing. Test results discern silver carp eDNA in four of the shops–three in Lake Erie and one in the Wabash River watershed–and bighead carp from one of the same Lake Erie venues, suggesting that retailers (who often source from established southerly populations) comprise another introduction vector. Our overall findings thus provide key population genetic and phylogenetic data for understanding and tracing introductions, vectors, and spread pathways for silver carp, their variants, and their relatives.

Cost‐efficient high throughput capture of museum arthropod specimen DNA using PCR‐generated baits

Abstract Gathering genetic data for rare species is one of the biggest remaining obstacles in modern phylogenetics, particularly for megadiverse groups such as arthropods. Next generation sequencing techniques allow for sequencing of short DNA fragments contained in preserved specimens >20 years old, but approaches such as whole‐genome sequencing are often too expensive for projects including many taxa. Several methods of reduced representation sequencing have been proposed that lower the cost of sequencing per specimen, but many remain costly because they involve synthesizing nucleotide probes and target hundreds of loci. These datasets are also frequently unique for each project and thus generally incompatible with other similar datasets. Here, we explore utilization of in‐house generated DNA baits to capture commonly utilized mitochondrial and ribosomal DNA loci from insect museum specimens of various ages and preservation types without the a priori need to know the sequence of the target loci. Both within species and cross‐species capture are explored, on preserved specimens ranging in age from one to 54 years old. We found most samples produced sufficient amounts of data to assemble the nuclear ribosomal rRNA genes and near complete mitochondrial genomes and produce well‐resolved phylogenies in line with expected results. The dataset obtained can be straightforwardly combined with the large cache of existing Sanger‐sequencing‐generated data built up over the past 30 years and targeted loci can be easily modified to those commonly used in different taxa. Furthermore, the protocol we describe allows for inexpensive data generation (as low as ~$35/sample), of at least 20 kilobases per specimen, for specimens at least as old as ~1965, and can be easily conducted in most laboratories. If widely applied, this technique will accelerate the accurate resolution of the Tree of Life especially on non‐model organisms with limited existing genomic resources.

Septonema lohmanii G. Delgado & O. Koukol, sp. nov., a New Species in Mytilinidiales (Dothideomycetes) and the Phylogenetic Position of S. fasciculare (Corda) S. Hughes

During independent surveys of microfungi associated with Pinus spp. in the United States and the Czech Republic, a distinct fungus matching the generic concept of Septonema Corda was collected. It is characterized by distinctly ornamented conidiophores, branches, conidia and hyphae, ranging from verruculose to strongly verrucose with prominent rounded warts, yellowish brown to brown or reddish brown in color and forming densely floccose, dark brown or dark reddish brown colonies on pine wood and bark. Conidia are cylindrical or subcylindrical and produced in short, simple or branched acropetal chains. Multigene phylogenetic analyses including nuclear ribosomal (LSU) and protein coding gene (EF1-α) sequence data suggest that both collections are conspecific and belong to the order Mytilinidiales (Dothideomycetes, Ascomycota) where they group distant from other mytilinidiaceous fungi with known septonema-like anamorphs. To provide a proper name based on phylogenetic placement and to possibly circumscribe Septonema sensu stricto, a non-sporulating, putative strain belonging to S. secedens Corda, the generic type, was included in the analyses. DNA sequence data placed this strain within the family Venturiaceae (Venturiales, Dothideomycetes) but morphological examination of the corresponding herbarium specimen revealed that it belongs instead to S. fasciculare (Corda) S. Hughes. Because of the polyphyletic nature of the genus and the unknown phylogenetic position of its type species, our fungus is accommodated in Septonema as a new species named S. lohmanii G. Delgado & Koukol, sp. nov.

Towards a phylogeny ofCyclops(Copepoda): (in)congruences among morphology, molecules and zoogeography

AbstractThe phylogeny ofCyclops(~30 spp.), a predominantly Palearctic cold‐adapted genus, was reconstructed based on morphological and molecular characters. The morphological analysis used extensive taxon sampling from the entire Holarctic range of the genus and included 53 morphological characters. Polymorphic traits were coded by the “unordered,” “unscaled” and “scaled” methods; maximum parsimony criterion was applied in tree building. Molecular phylogenetic reconstructions utilized partial nuclear 18S and 28S ribosomal genes, mitochondrial cytochrome oxidase I and complete internal transcribed spacer regions I and II, albeit with limited taxon sampling. Bayesian inference and maximum likelihood were used in these tree reconstructions. The molecular characters were used both in combination with morphology and as an independent test of the basal relationships inferred from morphology. Monophyly of the genus received strong support in both the morphological and molecular phylogenies; the basal relationships remain unresolved. The morphology‐based phylogenies, along with the geographic distribution patterns and ecological traits, supported monophyly of theankyrae−ladakanusclade,scutifer‐clade (C. scutifer,C. jashnovi,C. columbianus),kolensis‐clade (C. kolensis,C. kikuchii,C. vicinus,C. furcifer,C. insignis,C. alaskaensis),abyssorum‐clade (C. abyssorums. str.,C. abyssorum larianus,C. ricae,C. sevani) anddivergens‐clade (South Carpathian “Cyclopssp. Y,”C. mauritaniae,C. divergens,C. bohater,C. lacustris). Relationships among European and North American populations ofC. scutiferandC. columbianusbased on partial sequences of the 12S mitochondrial gene showC. scutiferto be paraphyletic, suggesting two independent invasions into North America via the Bering Land Bridge from Siberia to Alaska.

Two new species of Verruconis from Hainan, China.

Two new species of the genus Verruconis, V.hainanensis and V.pseudotricladiata, were described using combined morphological and DNA sequence data. The DNA sequences of respective strains including nuclear ribosomal DNA genes (nuSSU, ITS, nuLSU) and fragments of three protein-coding genes (ACT1, BT2, TEF1) were sequenced and compared with those from closely-related species to genera Ochroconis and Verruconis (Family Sympoventuriaceae, Order Venturiales). Morphologically, both species showed typical ampulliform conidiophores and conidiogenous cells, features not seen in other species of Verruconis. The conidia of V.hainanensis are fusiform and those of V.pseudotricladiata are Y or T shaped, similar to old members of a closely-related genus Scolecobasidium. The addition of these two new species provides a new perspective on the heterogeneity of Scolecobasidium.

Planktotrophic Brachiopod Larvae from the Pacific and Caribbean of Panama

Lingulids and discinids are the only brachiopods that exhibit life histories that include a feeding planktonic stage usually referred to as a “larva”. We collected planktotrophic brachiopod larvae from the Pacific and Caribbean coasts of Panama and took a DNA barcoding approach with mitochondrial cytochrome c oxidase subunit I (COI), mitochondrial ribosomal 16S, and nuclear ribosomal 18S genes to identify those larvae and to estimate their diversity in the region. We observed specimens from both coasts with distinct morphologies typical of lingulid and discinid larvae. COI and 16S were sequenced successfully for the lingulid larvae but failed consistently for all discinid larvae. 18S was sequenced successfully for larvae from both families. Sequence data from each gene revealed one lingulid operational taxonomic unit (OTU) from Bocas del Toro on the Caribbean coast, and one lingulid OTU from the Bay of Panama on the Pacific coast. These OTUs differed by >20% for COI, >10% for 16S and ~0.5% for 18S. Both OTUs clustered with GenBank sequences of Glottidia species, the only genus of lingulids in the Americas, but were distinct from G. pyramidata the only species reported for the Caribbean. Analysis of 18S sequence data for discinid larvae recovered 2 OTUs, one exclusively from the Pacific and one with a mixture of Pacific and Caribbean larvae. The 18S marker does not provide enough resolution to distinguish between species, and comparisons with GenBank sequences suggest that one OTU includes Pelagodiscus species, while the other may include Discradisca species. When compared with other marine invertebrates, our surveys of brachiopod larvae through DNA barcoding show relatively low levels of diversity for Panama.

Identification and characterization of the causative triatomine bugs of anaphylactic shock in Zhanjiang, China

BackgroundTwo health concerns primarily related to triatomine bugs are transmission of Trypanosoma cruzi through infective feces, and allergic reactions induced by triatomine bites. In the Southwestern United States, reduviid bugs bites commonly cause insect allergy. In South China, four cases of anaphylactic shock have been reported after this bite exposure. To further classify the species of these bugs and confirm the sensitization of the triatomine saliva, we caught triatomine bugs from the region where the bites occurred and performed phylogenetic and immunohistochemical (IHC) analysis.MethodsTriatomine bugs were collected in Donghai Island of Zhanjiang City in South China. The genomic DNA was extracted from three legs of the bugs. The fragments of mitochondrial 16S rRNA, cytochrome c oxidase subunit I (COI) gene and nuclear ribosomal 18S and 28S rRNA genes were obtained by PCR and sequenced. A phylogenetic tree was constructed based on the sequence of 16S rRNA gene using a maximum likelihood method with MEGA 7.0 software. Trypanosomal specific fragments and vertebrate COI genes were amplified from the fecal DNA to detect the infection of trypanosomes and analyze the blood feeding patterns, respectively. Paraffin-embedded sections were then prepared from adult triatomines and sent for IHC staining.ResultsWe collected two adult triatomine bugs in Donghai Island. Morphological and molecular analyses indicated that the triatomines were Triatoma rubrofasciata. No fragments of T. cruzi or other trypanosomes were detected from the fecal DNA. Mitochondrial gene segments of Homo sapiens and Mus musculus were successfully amplified. The allergens which induced specific IgE antibodies in human serum were localized in the triatomine saliva by IHC assay.ConclusionsThe two triatomine bugs from Donghai Island were T. rubrofasciata. They had bitten humans and mice. Their saliva should contain the allergens related to the allergic symptoms and even anaphylactic shock of exposed residents. Great consideration should be given to this triatomine bugs due to their considerable distribution and potential threat to public health in South China.