COI Fragments Research Articles

Just a Fragment of Undescribed Diversity: Twenty New Oriental and Palearctic Species of Sciaroidea (Diptera), including DNA Sequence Data and Two New Fossil Genera.

Simple SummaryTwo-winged flies (Diptera) are one of the megadiverse insect orders, in terms of biodiversity. The suborder Nematocera (=lower Diptera) includes several particularly species-rich groups, such as fungus gnats (Mycetophilidae and related families of the superfamily Sciaroidea) and gall midges (Cecidomyiidae), the latter presumably even being the most diverse family of Diptera of all. Although every year dozens of new species from these families are being described, either from extant fauna or from the fossil record, an amazing number of species still remain undescribed, especially outside Europe or North America. In this paper, another portion of new taxa of fungus gnats is described, enabling to better understand the fascinating and still little-known world of insects on Earth.The following 17 extant new species of Sciaroidea (Diptera: Bibionomorpha) are described: Bolitophila nikolae Ševčík sp. nov. (Bolitophilidae, Taiwan), Catocha jingfui sp. nov. (Cecidomyiidae, Taiwan), Catocha manmiaoe sp. nov. (Cecidomyiidae, Taiwan), Catocha shengfengi sp. nov. (Cecidomyiidae, Taiwan), Planetella taiwanensis sp. nov. (Cecidomyiidae, Taiwan), Diadocidia pseudospinusola sp. nov. (Diadocidiidae, Taiwan), Asioditomyia bruneicola sp. nov. (Ditomyiidae, Brunei), Asioditomyia lacii sp. nov. (Ditomyiidae, Taiwan), Ditomyia asiatica sp. nov. (Ditomyiidae, Thailand), Chetoneura davidi sp. nov. (Keroplatidae, Brunei), Euceroplatus mantici sp. nov. (Keroplatidae, Thailand), Setostylus fangshuoi sp. nov. (Keroplatidae, Taiwan), Platyceridion yunfui sp. nov. (Keroplatidae, Hainan), Terocelion adami sp. nov. (Keroplatidae, Taiwan), Hadroneura martini sp. nov. (Mycetophilidae, Taiwan), Paratinia furcata sp. nov. (Mycetophilidae, Czech Republic, Slovakia), and Nepaletricha sikorai sp. nov. (Sciaroidea incertae sedis, Thailand). Two new genera are described from the mid-Cretaceous Burmese amber, Burmasymmerus gen. nov. (Ditomyiidae, type species Burmasymmerus korneliae sp. nov., including also B. wieslawi sp. nov.), representing the first record of the family Ditomyiidae from the Mesozoic, and Burmatricha gen. nov. (Sciaroidea incertae sedis, type species Burmatricha mesozoica sp. nov.). Molecular phylogeny of Ditomyiidae, based on two DNA markers (28S, COI), as well as that of Catocha Haliday, 1833, based on the mitochondrial COI and 16S fragments, are also presented.

Genetic diversity, gene flow and hybridization in fan-shaped sponges (Phakellia spp.) in the North-East Atlantic deep sea

Deep-sea North Atlantic sponge grounds are crucial components of the marine fauna providing a key role in ecosystem functioning. To properly develop effective conservation and management plans, it is crucial to understand the genetic diversity, molecular connectivity patterns and turnover at the population level of the species involved. Here we present the study of two congeneric sponges, Phakellia robusta and Phakellia hirondellei, using multiple sources of evidence. Our phylogenetic study using a fragment of COI placed these two species as sister. Haplotype network analysis using COI revealed no genetic structure for P. hirondellei in samples from the Cantabrian Sea (<100 km). Contrastingly, P. robusta showed a clear genetic structure separating deep-water samples from the Cantabrian Sea and the Hatton-Rockall Basin, from samples from shallower waters from Kerry Head Reefs, NW of Orkney, and Norway. ddRADseq-derived SNPs for P. robusta also segregated samples by bathymetry rather than by geographical distances, and detected a predominant northwards migration for shallow-water specimens connecting sites separated ca. 2,000 km, probably thanks to prevalent oceanographic currents. Importantly, our analysis using SNPs combining the datasets of the two species revealed the presence of potential hybrids, which was corroborated by morphological (spicule) and microbial (16S amplicon sequencing) analyses. Our data suggest that hybridization between these two species occurred at least two times in the past. We discuss the importance of using next-generation techniques to unveil hybridization and the implications of our results for conservation.

Molecular phylogenetics and taxonomic reassessment of the widespread agamid lizard Calotes versicolor (Daudin, 1802) (Squamata, Agamidae) across South Asia

Abstract The genus Calotes Cuvier, 1817 (Agamidae: Draconinae) is highly diverse, with species occurring in South and Southeast Asia, and Oceania. Most species of the subfamily except C. versicolor have narrow geographic distributions. Calotes versicolor is distributed from western Iran in the west to south China and Indonesia in the east and has been introduced to parts of Africa and North America. The species has had a complicated taxonomic history; multiple species and subspecies related to C. versicolor were described from India and adjoining regions, which were synonymized in subsequent revisions. However, a study of Burmese C. versicolor yielded two new species, C. htunwini and C. irawadi, indicating that C. versicolor is a species complex. Such integrative taxonomic studies have not been carried out in India, the supposed type locality of C. versicolor. Hence, we studied C. versicolor sensu lato from the Indian subcontinent and generated sequences of mitochondrial 16S and COI fragments from tissues sampled from multiple localities in the region, including the type localities of its synonyms. Phylogenetic analyses revealed four well-supported, deeply-divergent lineages, supported by morphological data. These lineages represent (i) C. versicolor sensu stricto, from South India and parts of the east coast, (ii) C. irawadi sensu lato from northeast India and Southeast Asia, (iii) a synonym from the eastern Indo-Gangetic Plains which we resurrect here, and (iv) a subspecies from Pakistan which we elevate to species level. We provide re-descriptions for the resurrected or elevated species, and a diagnostic key to the species of the C. versicolor complex. The study shows that C. versicolor sensu stricto is endemic to parts of southern and eastern India, and not widely distributed, though it may have been introduced to other parts of the world.

Coming of age for COI metabarcoding of whole organism community DNA: Towards bioinformatic harmonisation

Metabarcoding of DNA extracted from community samples of whole organisms (whole organism community DNA, wocDNA) is increasingly being applied to terrestrial, marine and freshwater metazoan communities to provide rapid, accurate and high resolution data for novel molecular ecology research. The growth of this field has been accompanied by considerable development that builds on microbial metabarcoding methods to develop appropriate and efficient sampling and laboratory protocols for whole organism metazoan communities. However, considerably less attention has focused on ensuring bioinformatic methods are adapted and applied comprehensively in wocDNA metabarcoding. In this study we examined over 600 papers and identified 111 studies that performed COI metabarcoding of wocDNA. We then systematically reviewed the bioinformatic methods employed by these papers to identify the state‐of‐the‐art. Our results show that the increasing use of wocDNA COI metabarcoding for metazoan diversity is characterised by a clear absence of bioinformatic harmonisation, and the temporal trends show little change in this situation. The reviewed literature showed (i) high heterogeneity across pipelines, tasks and tools used, (ii) limited or no adaptation of bioinformatic procedures to the nature of the COI fragment, and (iii) a worrying underreporting of tasks, software and parameters. Based upon these findings we propose a set of recommendations that we think the metabarcoding community should consider to ensure that bioinformatic methods are appropriate, comprehensive and comparable. We believe that adhering to these recommendations will improve the long‐term integrative potential of wocDNA COI metabarcoding for biodiversity science.

Congruence between morphology-based species and Barcode Index Numbers (BINs) in Neotropical Eumaeini (Lycaenidae).

BackgroundWith about 1,000 species in the Neotropics, the Eumaeini (Theclinae) are one of the most diverse butterfly tribes. Correct morphology-based identifications are challenging in many genera due to relatively little interspecific differences in wing patterns. Geographic infraspecific variation is sometimes more substantial than variation between species. In this paper we present a large DNA barcode dataset of South American Lycaenidae. We analyze how well DNA barcode BINs match morphologically delimited species.MethodsWe compare morphology-based species identifications with the clustering of molecular operational taxonomic units (MOTUs) delimitated by the RESL algorithm in BOLD, which assigns Barcode Index Numbers (BINs). We examine intra- and interspecific divergences for genera represented by at least four morphospecies. We discuss the existence of local barcode gaps in a genus by genus analysis. We also note differences in the percentage of species with barcode gaps in groups of lowland and high mountain genera.ResultsWe identified 2,213 specimens and obtained 1,839 sequences of 512 species in 90 genera. Overall, the mean intraspecific divergence value of CO1 sequences was 1.20%, while the mean interspecific divergence between nearest congeneric neighbors was 4.89%, demonstrating the presence of a barcode gap. However, the gap seemed to disappear from the entire set when comparing the maximum intraspecific distance (8.40%) with the minimum interspecific distance (0.40%). Clear barcode gaps are present in many genera but absent in others. From the set of specimens that yielded COI fragment lengths of at least 650 bp, 75% of the a priori morphology-based identifications were unambiguously assigned to a single Barcode Index Number (BIN). However, after a taxonomic a posteriori review, the percentage of matched identifications rose to 85%. BIN splitting was observed for 17% of the species and BIN sharing for 9%. We found that genera that contain primarily lowland species show higher percentages of local barcode gaps and congruence between BINs and morphology than genera that contain exclusively high montane species. The divergence values to the nearest neighbors were significantly lower in high Andean species while the intra-specific divergence values were significantly lower in the lowland species. These results raise questions regarding the causes of observed low inter and high intraspecific genetic variation. We discuss incomplete lineage sorting and hybridization as most likely causes of this phenomenon, as the montane species concerned are relatively young and hybridization is probable. The release of our data set represents an essential baseline for a reference library for biological assessment studies of butterflies in mega diverse countries using modern high-throughput technologies an highlights the necessity of taxonomic revisions for various genera combining both molecular and morphological data.

Hidden diversity of Ctenophora revealed by new mitochondrial COI primers and sequences.

The mitochondrial gene cytochrome‐c‐oxidase subunit 1 (COI) is useful in many taxa for phylogenetics, population genetics, metabarcoding, and rapid species identifications. However, the phylum Ctenophora (comb jellies) has historically been difficult to study due to divergent mitochondrial sequences and the corresponding inability to amplify COI with degenerate and standard COI “barcoding” primers. As a result, there are very few COI sequences available for ctenophores, despite over 200 described species in the phylum. Here, we designed new primers and amplified the COI fragment from members of all major groups of ctenophores, including many undescribed species. Phylogenetic analyses of the resulting COI sequences revealed high diversity within many groups that was not evident from more conserved 18S rDNA sequences, in particular among the Lobata (Ctenophora; Tentaculata; Lobata). The COI phylogenetic results also revealed unexpected community structure within the genus Bolinopsis, suggested new species within the genus Bathocyroe, and supported the ecological and morphological differences of some species such as Lampocteis cruentiventer and similar undescribed lobates (Lampocteis sp. “V” stratified by depth, and “A” differentiated by colour). The newly designed primers reported herein provide important tools to enable researchers to illuminate the diversity of ctenophores worldwide via quick molecular identifications, improve the ability to analyse environmental DNA by improving reference libraries and amplifications, and enable a new breadth of population genetic studies.

Report of a new genus Arboramima belonging to tribe Diestramimini, with a new species A. cattusis from Guangxi, China (Orthoptera: Rhaphidophoridae: Aemodogryllinae).

A new genus (Arboramima Zong, Qin He gen. nov.) with a new species (Arboramima cattusis Zong, Qin He sp. nov.) is reported from Guangxi, China. The morphology of the new genus and species are provided. Samples of Aemodogryllinae species were selected for the reconstruction of phylogenetic tree based on mitochondrial COI fragment. The results supported the validation of the new genus. The type specimens are deposited in Museum of Biology, East China Normal University (ECNU).

Assessment of current taxonomic assignment strategies for metabarcoding eukaryotes.

The effective use of metabarcoding in biodiversity science has brought important analytical challenges due to the need to generate accurate taxonomic assignments. The assignment of sequences to genus or species level is critical for biodiversity surveys and biomonitoring, but it is particularly challenging as researchers must select the approach that best recovers information on species composition. This study evaluates the performance and accuracy of seven methods in recovering the species composition of mock communities by using COI barcode fragments. The mock communities varied in species number and specimen abundance, while upstream molecular and bioinformatic variables were held constant, and using a set of COI fragments. We evaluated the impact of parameter optimization on the quality of the predictions. Our results indicate that BLAST top hit competes well with more complex approaches if optimized for the mock community under study. For example, the two machine learning methods that were benchmarked proved more sensitive to reference database heterogeneity and completeness than methods based on sequence similarity. The accuracy of assignments was impacted by both species and specimen counts (query compositional heterogeneity) which ultimately influence the selection of appropriate software. We urge researchers to: (i) use realistic mock communities to allow optimization of parameters, regardless of the taxonomic assignment method employed; (ii) carefully choose and curate the reference databases including completeness; and (iii) use QIIME, BLAST or LCA methods, in conjunction with parameter tuning to better assign taxonomy to diverse communities, especially when information on species diversity is lacking for the area under study.

Integrated Taxonomic Revision of AfrotropicalXyleborinus(Curculionidae: Scolytinae) Reveals High Diversity After Recent Colonization of Madagascar

AbstractThe ambrosia beetle genus Xyleborinus Reitter, 1913 is particularly species rich in Madagascar where the genus exhibits extraordinary morphological variation not seen elsewhere. This study provides the first detailed molecular phylogeny of the genus based on COI, 28S, and CAD gene fragments. Biogeographical and taxonomic hypotheses were tested for the Afrotropical fauna with a particular focus on the Malagasy radiation. Analyses revealed a single colonization of Madagascar no earlier than 8.5–11.0 Ma, indicating an extraordinary recent radiation on the island which has given rise to at least 32 species. Two recolonization events of the African mainland were strongly supported by the molecular data, with several other intraspecific dispersals to the mainland inferred from species distributions. A taxonomic re-evaluation of all Afrotropical Xyleborinus resulted in several taxonomic changes. We found that morphological differences associated with COI divergence higher than 7% indicated different species. Twelve new species are described: Xyleborinus castriformis Eliassen &amp; Jordal, sp. nov., Xyleborinus clivus Eliassen &amp; Jordal, sp. nov., Xyleborinus concavus Eliassen &amp; Jordal, sp. nov., Xyleborinus coronatus Eliassen &amp; Jordal, sp. nov., Xyleborinus diadematus Eliassen &amp; Jordal, sp. nov., Xyleborinus laevipennis Eliassen &amp; Jordal, sp. nov., Xyleborinus magnispinosus Eliassen &amp; Jordal, sp. nov., Xyleborinus margo Eliassen &amp; Jordal, sp. nov., Xyleborinus ntsoui Eliassen &amp; Jordal, sp. nov., Xyleborinus singularis Eliassen &amp; Jordal, sp. nov., Xyleborinus tuberculatus Eliassen &amp; Jordal, sp. nov., and Xyleborinus turritus Eliassen &amp; Jordal, sp. nov., all from Madagascar. New synonyms are proposed for Xyleborinus aemulus (Wollaston, 1869) [=Xyleborinus spinifer (Eggers, 1920)], Xyleborinus andrewesi (Blandford, 1896) [=Xyleborinus mimosae (Schedl, 1957)], Xyleborinus dentellus (Schedl, 1953) [=Xyleborinus forcipatus (Schedl, 1957)], Xyleborinus octospinosus (Eggers, 1920) [=Xyleborinus mitosomipennis (Schedl, 1953)], and Xyleborinus similans (Eggers, 1940) [=Xyleborinus sclerocaryae (Schedl, 1962)]. Two species were given new status: Xyleborinus profundus (Schedl, 1961) is elevated from subspecies of Xyleborinus aduncus (Schedl, 1961), and Xyleborinus mitosomus (Schedl, 1965) is reinstated from its previous synonymy with Xyleborinus spinosus (Schaufuss, 1891). Xyleborus gracilipennis Schedl 1957 is reverted to its original genus, and a similar status is confirmed for Xyleborus collarti Eggers 1932. The number of taxonomically valid Xyleborinus species in the Afrotropical region is now 47, which includes 3 adventive species. Revised diagnoses for all species and a key for species identification are provided.

Morphotype and haplotype identification of Varroa destructor (Acari: Varroidae), and its importance for apiculture in Nicaragua

The ectoparasite Varroa (Acari: Varroidae) is considered to be the main pest of honey bees (Apis mellifera L.) in Nicaragua. The aim of this study was to determine morphotypes and mitochondrial haplotypes of the Varroa mites, related to infestation levels in A. mellifera hives in Nicaragua in a cross-sectional study (2013-2016). Samples were taken from 34 sentinel apiaries in five geographical zones; from 713 Varroa females collected during the study, 153 were selected for measurement of various morphometric characters for further classification into morphotypes. The mitochondrial haplotype was assigned to one of the two (Korean or Japanese), using the restriction by SacI of the PCR product of a fragment of the gene CO-I. Morphometric measurement and sequencing revealed the affiliation to the species Varroa destructor with a mean (± SD) body width of 1699.1 ± 60.2µm and a body length of 1161.1 ± 34.9µm. Body characters were significantly different among the 34 apiaries. Varroa destructor samples were classified into four morphotypes, with no significant differences in the geographical zones. As GAGCTC SacI enzyme cleavage sequences were not observed, all PCR products were identified as V. destructor Korean haplotype. The CO-I gene nucleotide sequences from two samples V. destructor showed both 100% similarity with the Korean haplotype and 99.8% similarity to the Japanese haplotype. Although the V. destructor mite was identified as a Korean haplotype, host-parasite association in 2 decades has led into a balance without entering into severe losses in the Nicaraguan apiculture.

Systematics, Phylogeny, and Evolution of &lt;i&gt;Phrynocephalus&lt;/i&gt; (Superspecies &lt;i&gt;przewalskii&lt;/i&gt;) (Reptilia: Agamidae)

The superspecies przewalskii group of Phrynocephalus includes several taxa with unclear taxonomic status. We analyze a fragment of the mitochondrial DNA gene COI and the body patterns of 275 specimens including type specimens. The results resolve the taxonomy and phylogeny of the group and we provide a diagnostic key for the species.

Development of a set of oligonucleotides for the identification of the Sakhalin sturgeon Acipenser mikadoi Hilgendorf, 1892 by PCR

Sturgeons (family Acipenseridae) are valuable commercial fish and aquaculture resources. The Acipenser genus includes about 20 species, 12 of which are recorded for the Russian territory. One of the rarest is the Sakhalin sturgeon A. mikadoi Hilgendorf, 1892. At present, its population size is significantly decreased and the species is close to extinction. Natural populations of the species have survived in the Tumnin River in Khabarovsk Region and the Viakhtu River in Sakhalin Region. Vihtu River in Sakhalin Region. Due to the small numbers of rare and endangered fish species, noninvasive approaches, including the use of DNA from the environment, appear to be the most suitable for monitoring their species and genetic diversity. The method is well established in this area and has been successfully tested for the monitoring of several species of rare and endangered sturgeons. This paper presents the results of the development of primers specific for the mitochondrial DNA of the species A. mikadoi. A matrix of 15 sequences of complete mitochondrial genomes from 3 sturgeon species whose ranges may overlap - A. mikadoi (KX276658), A. dauricus (KJ402277) and A. schrenckii (MH973728- MH973734, KX276660, KX276659, KF150287, KC905169, KC820796) - was generated. A. medirostris (NC_028405), which is genetically closest to the Sakhalin sturgeon, was also included in the analysis. A sliding window algorithm was applied to the sequence matrix. A graph showing the distribution of divergence values along the mitochondrial genome of sturgeon was plotted based on the results of the analysis Fig. 1. Based on matrices of individual genome fragments (16S rRNA, COI, ND1, and D-loop), a search for species-specific primers was performed in the DECIPHER program. A Primer-BLAST algorithm was run based on each pair of primers. Taxa Actinopterygii and Acipenseridae were used as references separately in different runs. In addition, primers were checked using local reference sequences of the complete mitochondrial genome of representatives of the genus Acipenser. The results of primer evaluation are summarized in the table Table 1. As a result, a combination of primer pairs developed on the basis of D-loop and COI fragments seems to be the most successful for error-free identification of Sakhalin sturgeon.

The first step towards a DNA barcode reference database for mayflies (Ephemeroptera) of Slovakia

Mayflies (Ephemeroptera) represent a small but diverse order of amphibiotic insects, whose larvae contribute to several essential processes in freshwater habitats, such as bioturbation and bioirrigation, decomposition, nutrient cycling, and also serve as a primary source of nutrients for numerous organisms. Due to their cosmopolitan distribution and high-quality water requirements, they are also important indicators of ecosystem health and an integral part of biomonitoring protocols. Although the Slovak mayfly fauna is well researched, studies on genetic diversity, including DNA barcoding, are still lacking. The absence of the comprehensive DNA barcode reference libraries from various biogeographical regions and the presence of so-called cryptic lineages may prevent further efficient use and application of new approaches to aquatic ecosystem biomonitoring (Biomonitoring 2.0) based on eDNA analyses. Therefore, in the initial stage of our research, we bring the first insight into the genetic diversity of mayflies (based on mtDNA COI-5P barcoding fragment) from 47 localities of Slovakia mostly situated in the biogeographically significant Western Carpathians' territory. A total of 403 sequences of 42 morphologically determined species were added to the BOLD (Barcode of Life Data System) database, representing more than 1/3 of the mayfly fauna of Slovakia and covering 10 of 16 families. Sequences of these species were finally assigned to 62 BINs (Barcode Index Numbers) in BOLD (Fig. 1), whereby sequences of 12 species were divided into more than one BIN, indicating the presence of cryptic lineages. The largest number of BINs was represented by widely distributed species such as Baetis rhodani Pictet, 1843-1845 (6 BINs), Habroleptoides confusa Sartori &amp;amp; Jacob, 1986 (4 BINs) and Ecdyonurus venosus (Fabricius, 1775) (3 BINs). The sequences of the remaining nine species were split into two BINs. Maximum intraspecific variability (calculated by K2P) of some representatives was surprisingly high [e.g., E. venosus – 27.1 %; Baetis muticus (Linneaus, 1758) – 23.6 %; Caenis luctuosa (Burmeister, 1839) – 23.34 %, Baetis rhodani – 18.66 % and B. vernus Curtis, 1834 – 15.25 %] and far exceeded the level of intraspecific variability of the COI fragment based on the BOLD standards. The sequences of 23 individuals determined as Habroleptoides confusa, Baetis rhodani, B. buceratus Eaton, 1870, Caenis beskidensis Sowa, 1973 and Torleya major (Klapálek, 1905) created seven unique BINs, which represent the distant phylogenetic lineages of already existing BINs, that are currently unique to Slovakia. The coexistence of Baetis rhodani individuals of two different BINs was confirmed at five localities. Our study indicates clear importance of more detailed sampling and DNA barcoding due to the presence of unexpected intraspecific genetic diversity of mayflies captured in a relatively small area of the Western Carpathians.

Optimization and Application of direct PCR in community metabarcoding

Direct PCR allows the amplification of DNA from animal or plant tissue samples without the need for DNA extraction and purification steps. For this procedure, dry tissue is homogenized, dissolved in water and subsequently amplified, thus, its successful application largely depends on the absence of PCR inhibitors. Although this method has been successfully applied in barcoding approaches of invertebrates, it has not yet been attempted in metabarcoding approaches. We used nonbiting midges (Diptera: Chironomidae) to test if amplicons produced by direct PCR could be used for next-generation sequencing. To access whether direct PCR is applicable for a variety of chironomid species, we tested 236 adult specimens randomly selected from emergence traps of an artificial pond mesocosm. We used ground tissue, corresponding to 0.1% of the specimens’ biomass, and a direct PCR protocol following Wong et al. (2014) for amplification. In total, 98 % of the samples were successfully amplified and we found a diverse community comprised of 20 different genera. In order to compare direct PCR and ´traditional´ DNA isolation-PCR, we created mock communities (14 species) and used both approaches for the amplification of a 421 bp COI fragment. After a second PCR for indexing and adapter ligation, samples were sequenced on an Illumina sequencer. We found only slightly lower recovery rates for mock communities with the direct PCR approach compared to traditional protocols. These recovery rates were further improved for both methods when an equal biomass (ca. 0.006 mg) of chironomid specimens was used. With our approach, it was possible to detect species which constituted only 1% of the entire biomass of a sample. Generally, direct PCR did not have a large effect on sequence read abundance. However, read abundance varied strongly between species. We are currently investigating whether this was caused by primer bias or an artifact of differently degraded tissue. This study is a proof of principle that the amplicons produced by direct PCR can be used for next-generation sequencing, with possible applications for future biomonitoring projects and portable laboratory technologies. We are currently using this technique to monitor a large-scale chironomid community experiment (artificial pond mesocosm facility) covering weekly samples taken over two summer half-years.

An elongated COI fragment to discriminate botryllid species and as an improved ascidian DNA barcode

Botryllids are colonial ascidians widely studied for their potential invasiveness and as model organisms, however the morphological description and discrimination of these species is very problematic, leading to frequent specimen misidentifications. To facilitate species discrimination and detection of cryptic/new species, we developed new barcoding primers for the amplification of a COI fragment of about 860 bp (860-COI), which is an extension of the common Folmer's barcode region. Our 860-COI was successfully amplified in 177 worldwide-sampled botryllid colonies. Combined with morphological analyses, 860-COI allowed not only discriminating known species, but also identifying undescribed and cryptic species, resurrecting old species currently in synonymy, and proposing the assignment of clade D of the model organism Botryllus schlosseri to Botryllus renierii. Importantly, within clade A of B. schlosseri, 860-COI recognized at least two candidate species against only one recognized by the Folmer's fragment, underlining the need of further genetic investigations on this clade. This result also suggests that the 860-COI could have a greater ability to diagnose cryptic/new species than the Folmer's fragment at very short evolutionary distances, such as those observed within clade A. Finally, our new primers simplify the amplification of 860-COI even in non-botryllid ascidians, suggesting their wider usefulness in ascidians.

Multidisciplinary approach detects speciation within the kissing bug Panstrongylus rufotuberculatus populations (Hemiptera, Heteroptera, Reduviidae).

BACKGROUND Panstrongylus rufotuberculatus (Hemiptera-Reduviidae) is a triatomine species with a wide geographic distribution and a broad phenotypic variability. In some countries, this species is found infesting and colonising domiciliary ecotopes representing an epidemiological risk factor as a vector of Trypanosoma cruzi, etiological agent of Chagas disease. In spite of this, little is known about P. rufotuberculatus genetic diversity.METHODS Cytogenetic studies and DNA sequence analyses of one nuclear (ITS-2) and two mitochondrial DNA sequences (cyt b and coI) were carried out in P. rufotuberculatus individuals collected in Bolivia, Colombia, Ecuador and Mexico. Moreover, a geometric morphometrics study was applied to Bolivian, Colombian, Ecuadorian and French Guiana samples.OBJECTIVES To explore the genetic and phenetic diversity of P. rufotuberculatus from different countries, combining chromosomal studies, DNA sequence analyses and geometric morphometric comparisons.FINDINGS We found two chromosomal groups differentiated by the number of X chromosomes and the chromosomal position of the ribosomal DNA clusters. In concordance, two main morphometric profiles were detected, clearly separating the Bolivian sample from the other ones. Phylogenetic DNA analyses showed that both chromosomal groups were closely related to each other and clearly separated from the remaining Panstrongylus species. High nucleotide divergence of cyt b and coI fragments were observed among P. rufotuberculatus samples from Bolivia, Colombia, Ecuador and Mexico (Kimura 2-parameter distances higher than 9%).MAIN CONCLUSIONS Chromosomal and molecular analyses supported that the two chromosomal groups could represent different closely related species. We propose that Bolivian individuals constitute a new Panstrongylus species, being necessary a detailed morphological study for its formal description. The clear morphometric discrimination based on the wing venation pattern suggests such morphological description might be conclusive.

A molecular perspective on the systematics of the spider crab genus

Libinia, a genus of spider crabs, encompasses 10 valid species, three from the eastern Pacific and seven from the western Atlantic. Previous phylogenetic hypotheses based on molecular and larval morphological data suggested an uncertain position of the genus Libinia within the family Epialtidae. Therefore, we investigated the evolutionary relationships among the species of Libinia, and also suggested a position for this genus within the family. Phylogenetic trees were obtained from concatenated mtDNA sequences of 16S and COI fragments. Molecular dating methods were used to estimate the relative timing of origin and diversification within Libinia. Eighteen new primers were designed, and PCR conditions were standardised to amplify the COI fragments of older or poorly preserved pinned specimens, generating ~550 base pair sequences. Phylogenetic hypotheses supported the monophyly of the genus. Species that occur in the eastern Pacific and the ones found in the western Atlantic do not form monophyletic clades related to this geographical separation. In the eastern Pacific, there is low phylogenetic proximity between Libinia mexicana and L. setosa; and since we did not sample L. peruana, we inferred that it should cluster with L. setosa or L. mexicana due to morphological similarity. Libinia spinosa is separate from the other species, L. mexicana was a sister group of L. cavirostris, and L. ferreirae was closely related to L. dubia and L. rhomboidea, which occur in the Gulf of Mexico and in the Caribbean, respectively. In the Gulf of Mexico, L. dubia and L. emarginata are closely related. Although L. erinacea was not sampled, we suggest, from previous studies, that it should group with L. dubia due to morphological similarities. The phylogenetic hypothesis suggests the monophyly of Libinia, with its origins at the late Eocene. Finally, the recovered clades based on two molecular markers agreed with a parallel, linked morphological study that is still in progress.

Variation of cytochrome oxidase-I gene and venom proteins of Rhopalurus junceus populations in the Moanicum sector: Pharmacological implications

Context: In Cuba, the venom of the endemic scorpion Rhopalurus junceus has been used in traditional medicine for cancer treatment. The genetic variability in populations from distinct origin and differences in the composition of venom –a little explored topic- could influence the pharmacological effects of products used in medical practice. Aims: To characterize the populations of R. junceus from six different districts of the Moanicum sector in northeastern Cuba, taking into account the variations in the sequences of cytochrome oxidase subunit I gene (COI) and peptide masses of venom. Methods: For the genetic characterization, a 658 bp gene fragment of COI was amplified by PCR. The genetic structure of populations was analyzed hierarchically, using an analysis of molecular variance (AMOVA). In proteomic analysis, molecular masses of venom constituents were arranged in increasing order, and different components were considered when masses differed by 2 Da. Results: R. junceus species in the six districts studied in the Moanicum sector belong to different populations. The sequences of COI gene showed high levels of genetic variability, recovering 47 haplotypes (87% unique). The composition of the venom was also significantly different between the districts. The most abundant components presented masses of 3-6 kDa (K+-channel specific peptides) and 6,1-10 kDa (Na+-channel specific peptides). Conclusions: Populations of R. junceus showed high genetic differentiation and a broad intraspecific variation in the venom peptides (3-10 kDa) in six districts of Moanicum sector of Cuba. This richness in scorpion varieties with different toxins profile should be considered for pharmacological applications.

Tracing the Invasion and Expansion Characteristics of the Flatid Planthopper, Metcalfa pruinosa (Hemiptera: Flatidae), in Korea Using Mitochondrial DNA Sequences.

Simple SummaryAfter the invasion of Korea in 2005, the first lines of studies on Metcalfa pruinosa inferred the origin of the species using a fragment of mitochondrial COI sequences. However, the low variability of the sequences limited further scrutinized inference on the invasion dynamics. In this study, we sequenced a fragment of the COI gene from 536 individuals of the species and combined the sequence data with the available GenBank data, totaling 830 individuals. These data indicated that the North-West region is a point of entry in addition to the South-East region, the presumed sole point of entry to Korea. Furthermore, it suggested that North-West entry involves the M. pruinosa originating from the USA. In an effort to find further variable regions in the mitochondrial genome, one region provided substantially increased variability compared to the fragment of the COI. The concatenated sequences of COI and the newly obtained variable region, which were used to infer the expansion pattern in Korea, indicated that the main highway, running obliquely between the North-West and South-East regions, appears to be responsible for the current population genetic structure of M. pruinosa in Korea, facilitating gene flow through this highway traffic.The flatid planthopper, Metcalfa pruinosa (Hemiptera: Flatidae), which is an invasive species, is widespread in Korea. We sequenced a fragment of the COI from 536 individuals collected mainly in Korea and the European countries and combined these sequence data with the public data, totaling 830 individuals worldwide. The identification of one shared haplotype only between Korea and the USA, the presence of this haplotype only in the North-West region of Korea, and the highest haplotype diversity in this region suggested that the North-West region is another point of entry in addition to the South-East region, which is the presumed sole point of entry to Korea. Furthermore, it suggested that North-West entry involves the M. pruinosa originating from the USA. In an effort to find further variable regions in the mitochondrial genome, one region provided substantially increased variability compared to that of the fragment of COI. FST estimation, PCoA, and BAPS analysis, using the concatenated sequences of COI and the newly detected variable region to infer the expansion pattern in Korea, indicates that the main highway, running obliquely between the North-West and South-East regions, appears to be responsible for the current population genetic structure of M. pruinosa in Korea, facilitating gene flow through this highway traffic.

Morphological and Genetic Diversity of Two Forms of Muksun Coregonus muksun (Salmonidae) of the Khatanga River Basin as a Key for Understanding the Phylogenetic Relationships between Muksun and Whitefish C. lavaretus

Morphological analysis of muksun Coregonus muksun of the Khatanga River basin revealed that along with its typical high-density-rakered form, individuals of the low-density-rakered form also live in this region. Analysis of the genetic polymorphism of three marker regions of mitochondrial (ND1 and COI fragments) and nuclear (ITS1) DNA indicates the polyphyletic origin of these forms. The revealed low level of genetic differentiation of muksun and whitefish C. lavaretus, along with the available data on the absence of clear diagnostic species characteristics, suggest that they belong to the same biological species C. lavaretus.