COI Dataset Research Articles

Estimating the biodiversity of terrestrial invertebrates on a forested island using DNA barcodes and metabarcoding data.

Invertebrates are a major component of terrestrial ecosystems, however, estimating their biodiversity is challenging. We compiled an inventory of invertebrate biodiversity along an elevation gradient on the temperate forested island of Hauturu, New Zealand, by DNA barcoding of specimens obtained from leaf litter samples and pitfall traps. We compared the barcodes and biodiversity estimates from this data set with those from a parallel DNA metabarcoding analysis of soil from the same locations, and with pre-existing sequences in reference databases, before exploring the use of combined data sets as a basis for estimating total invertebrate biodiversity. We obtained 1,282 28S and 1,610 COI barcodes from a total of 1,947 invertebrate specimens, which were clustered into 247 (28S) and 366 (COI) OTUs, of which≤10% were represented in GenBank. Coleoptera were most abundant (730 sequenced specimens), followed by Hymenoptera, Diptera, Lepidoptera, and Amphipoda. The most abundant OTU from both the 28S (153 sequences) and COI (140 sequences) data sets was an undescribed beetle from the family Salpingidae. Based on the occurrences of COI OTUs along the elevation gradient, we estimated there are ~1,000 arthropod species (excluding mites) on Hauturu, including 770 insects, of which 344 are beetles. A DNA metabarcoding analysis of soil DNA from the same sites resulted in the identification of similar numbers of OTUs in most invertebrate groups compared with the DNA barcoding, but less than 10% of the DNA barcoding COI OTUs were also detected by the metabarcoding analysis of soil DNA. A mark-recapture analysis based on the overlap between these data sets estimated the presence of approximately 6,800 arthropod species (excluding mites) on the island, including ~3,900 insects. Estimates of New Zealand-wide biodiversity for selected arthropod groups based on matching of the COI DNA barcodes with pre-existing reference sequences suggested over 13,200 insect species are present, including 4,000 Coleoptera, 2,200 Diptera, and 2,700 Hymenoptera species, and 1,000 arachnid species (excluding mites). These results confirm that metabarcoding analyses of soil DNA tends to recover different components of terrestrial invertebrate biodiversity compared to traditional invertebrate sampling, but the combined methods provide a novel basis for estimating invertebrate biodiversity.

Coleoptera of Canada.

The beetle fauna of Canada was assessed, including estimates of yet unreported diversity using information from taxonomists and COI sequence clusters in a BOLD (Barcode of Life Datasystems) COI dataset comprising over 77,000 Canadian records. To date, 8302 species of Coleoptera have been recorded in Canada, a 23% increase from the first assessment in 1979. A total of 639 non-native beetle species have become established in Canada, with most species in the Staphylinidae (153 spp.), Curculionidae (107 spp.), Chrysomelidae (56 spp.) and Carabidae (55 spp.). Based on estimates from the taxonomic community and our BOLD dataset, we estimate that slightly more than 1000 beetle species remain to be reported from Canada, either as new records or undescribed species. Renewed enthusiasm toward and financial support for surveys, especially in the central and western provinces of Canada will be critical for detecting, documenting and describing these species. The Barcode of Life database is still far from comprehensive for Canadian Coleoptera but substantial progress has been made and the number of Barcode Index Numbers (BINs) (as candidate species) has reached nearly 70% of the number of species reported from Canada. Comparison of BINs to observed species in a group of Canadian Staphylinidae suggests that BINs may provide a good estimate of species diversity within the beetles. Histeridae is a diverse family in Canada that is notably underrepresented in BOLD. Families such as Mordellidae, Scraptiidae, Latridiidae, Ptiliidae and Scirtidae are poorly known taxonomically in Canada and are represented in our BOLD dataset by many more BINs than recorded species.

Assessing the effectiveness of molecular data for species identification and diversity studies of Nigerian herpetofauna

We evaluated the effectiveness of molecular data (mitochondrial cytochrome c oxidase subunit I – COI and 16S ribosomal RNA – 16S) for species identification and diversity studies of Nigerian herpetofauna. We collected 189 specimens consisting of 91 amphibians and 98 reptiles from the largest and under-studied national park in Nigeria – Gashaka Gumti National Park (GGNP), eastern Nigeria. The resulting COI and 16S data allowed us to compare our sequences with others in global databases for species identification. During query searches, we observed poor representation of COI sequences for African herpetofauna in the GenBank and BOLD databases. Neighbour-joining (NJ) trees constructed based on COI and 16S datasets for all specimens sequenced for this study clustered most individuals of the same species. On the other hand, morphologically unidentified amphibian (Leptopelis sp and Ptychadena sp) and reptile (Hemidactylus sp and Lycophidion sp) species formed strongly supported lineages in the NJ trees, and were clearly separated from their sister species. Further, the NJ tree-based analyses recovered at least two well supported divergent lineages within Agama agama and Trachylepis maculilabris. Our study confirms the efficiency of integrating morphological and DNA barcoding approaches in identification and diversity studies of Nigerian herpetofauna. Finally, we recommend development of comprehensive and reliable DNA barcode reference libraries for global amphibians and reptiles.

Molecular phylogenetic investigations of Triops granarius (Lucas, 1864 (Branchiopoda: Notostraca) from the type locality of the former Apus orientalis Tiwari, 1952 and three other localities in the Western Ghats of India.

We investigated the phylogenetic position of Triops granarius populations from four localities in the Western Ghats using partial sequences of three mitochondrial genes (COI, 12S rRNA and 16S rRNA) publicly available on the GenBank database. One of these localities, Panchgani, is particularly important since it is the type locality of the former Apus orientalis which is currently treated as a junior synonym of T. granarius. Phylogenetic analyses reveal that populations from all the four localities (Kolhapur, Chalkewadi, Panchgani, and Dighi) form a single lineage, which is here named 'Maharashtra lineage'. One of the two previously published samples from India, treated as lineage 'Triops granarius 4' is nested within this clade. The 'Maharashtra lineage' is separated from other lineages by mean maximum likelihood distance ≥ 11.9% in the COI gene. This distance is suggestive of a separation on species level from other lineages of T. granarius. This interpretation is further supported by a conservative genus-wide species delimitation analysis performed in the present study upon application of the Automatic Barcode Gap Discovery method. The 'Maharashtra lineage' branches out in two sub-lineages of Panchgani+Kolhapur and Dighi+L4+Chalkewadi samples, separated by 5.9% mean ML distance (uncorrected p-distance = 5.4%) in COI. The application of a 5% threshold to the COI dataset would thus even suggest a possible differentiation of both sub-lineages on species level. Comparative morphological data is presently not available because most vouchers associated with the sequences were depleted for DNA extraction. Further studies are needed in order to prepare a sound taxonomic revision. Thus, in the current study we refrain from re-instating Apus orientalis to full species status (likewise, for other names of Asian taxa in this morphogroup, including Apus sinensis Uéno, we retain the status as junior synonym of T. granarius). Nonetheless, our study highlights the fact that still there may be undescribed cryptic species associated with the specific name in this part of Western Ghats (Linnean Shortfall) and paves the way for future taxonomic investigations and conservation strategies for the genus Triops in India.

Delimiting species of marine gastropods (Turridae, Conoidea) using RAD sequencing in an integrative taxonomy framework.

Species delimitation in poorly known and diverse taxa is usually performed based on monolocus, DNA-barcoding-like approaches, while multilocus data are often used to test alternative species hypotheses in well-studied groups. We combined both approaches to delimit species in the Xenuroturris/Iotyrris complex, a group of venomous marine gastropods from the Indo-Pacific. First, COI sequences were analysed using three methods of species delimitation to propose primary species hypotheses. Second, RAD sequencing data were also obtained and a maximum-likelihood phylogenetic tree produced. We tested the impact of the level of missing data on the robustness of the phylogenetic tree obtained with the RAD-seq data. Alternative species partitions revealed with the COI data set were also tested using the RAD-seq data and the Bayes factor species delimitation method. The congruence between the species hypotheses proposed with the mitochondrial nuclear data sets, together with the morphological variability of the shell and the radula and the distribution pattern, was used to turn the primary species hypotheses into secondary species hypotheses. Allopatric primary species hypotheses defined with the COI gene were interpreted to correspond to intraspecific structure. Most of the species are found sympatrically in the Philippines, and only one is confidently identified as a new species and described as Iotyrris conotaxis n. sp. The results obtained demonstrate the efficiency of the combined monolocus/multilocus approach to delimit species.

DNA Barcodes for Forensically Important Fly Species in Brazil.

Here, we analyze 248 DNA barcode sequences of 35 fly species of forensic importance in Brazil. DNA barcoding can be effectively used for specimen identification of these species, allowing the unambiguous identification of 31 species, an overall success rate of 88%. Our results show a high rate of success for molecular identification using DNA barcoding sequences and open new perspectives for immature species identification, a subject on which limited forensic investigations exist in Tropical regions. We also address the implications of building a robust forensic DNA barcode database. A geographic bias is recognized for the COI dataset available for forensically important fly species in Brazil, with concentration of sequences from specimens collected mainly in sites located in the Cerrado, Mata Atlântica, and Pampa biomes.

Molecular taxonomy and evolutionary relationships in the Oswaldoi-Konderi complex (Anophelinae: Anopheles: Nyssorhynchus) from the Brazilian Amazon region.

Recent studies have shown that Anopheles oswaldoi sensu lato comprises a cryptic species complex in South America. Anopheles konderi, which was previously raised to synonymy with An. oswaldoi, has also been suggested to form a species complex. An. oswaldoi has been incriminated as a malaria vector in some areas of the Brazilian Amazon, Colombia, Peru and Venezuela, but was not recognized as a vector in the remaining regions in its geographic distribution. The role of An. konderi as a malaria vector is unknown or has been misattributed to An. oswaldoi. The focus of this study was molecular identification to infer the evolutionary relationships and preliminarily delimit the geographic distribution of the members of these complexes in the Brazilian Amazon region. The specimens were sampled from 18 localities belonging to five states in the Brazilian Amazon and sequenced for two molecular markers: the DNA barcode region (COI gene of mitochondrial DNA) and Internal Transcribed Spacer 2 (ITS2 ribosomal DNA). COI (83 sequences) and ITS2 (27 sequences) datasets generated 43 and 10 haplotypes, respectively. Haplotype networks and phylogenetic analyses generated with the barcode region (COI gene) recovered five groups corresponding to An. oswaldoi s.s., An. oswaldoi B, An. oswaldoi A, An. konderi and An. sp. nr. konderi; all pairwise genetic distances were greater than 3%. The group represented by An. oswaldoi A exhibited three strongly supported lineages. The molecular dating indicated that the diversification process in these complexes started approximately 2.8 Mya, in the Pliocene. These findings confirm five very closely related species and present new records for these species in the Brazilian Amazon region. The paraphyly observed for the An. oswaldoi complex suggests that An. oswaldoi and An. konderi complexes may comprise a unique species complex named Oswaldoi-Konderi. Anopheles oswaldoi B may be a potential malaria vector in the extreme north of the Brazilian Amazon, whereas evidence of sympatry for the remaining species in other parts of the Brazilian Amazon (Acre, Amazonas, Pará and Rondônia) precluded identification of probable vectors in those areas.

Molecular Characterization of the Horn Fly Haematobia irritans Infesting Horses in Central Anatolia Region in Turkey

This study reports intense horn fly infestations of horses raised in an important wetland ecosystem, Sultan Marshes in Central Anatolia, Turkey. In total, seven horses raised together were found to be harbored over 500 flies per site of each animal. Totally, 376 fly specimens were collected from the horses by using the nets and were subjected to the laboratory for species identification. All flies were morphologically identified as the adults of Haematobia irritans. Partial fragments of mitochondrial cytochrome oxidase I (mt-COI) gene from totally 50 isolates were amplified for sequence and phylogenetic analyses. The mt-COI sequence analyses revealed no polymorphism among the isolates and explored a unique haplotype for H. irritans. A mean haplotype and nucleotide diversities of 0.8571 and 0.00695 were determined, respectively, within the COI data set of H. irritans, and newly characterized haplotype from Turkey exhibited a mean intraspecific genetic difference of 1.0 to the all published sequences of the isolates from several countries. COI data set also revealed a mean interspecific genetic difference of 1.7 between H. irritans and Haematobia exigua.

DNA barcoding for species identification in deep-sea clams (Mollusca: Bivalvia: Vesicomyidae)

Deep-sea clams (Bivalvia: Vesicomyidae) have been found in reduced environments over the world oceans, but taxonomy of this group remains confusing at species and supraspecific levels due to their high-morphological similarity and plasticity. In the present study, we collected mitochondrial COI sequences to evaluate the utility of DNA barcoding on identifying vesicomyid species. COI dataset identified 56 well-supported putative species/operational taxonomic units (OTUs), approximately covering half of the extant vesicomyid species. One species (OTU2) was first detected, and may represent a new species. Average distances between species ranged from 1.65 to 29.64%, generally higher than average intraspecific distances (0–1.41%) when excluding Pliocardia sp.10 cf. venusta (average intraspecific distance 1.91%). Local barcoding gap existed in 33 of the 35 species when comparing distances of maximum interspecific and minimum interspecific distances with two exceptions (Abyssogena southwardae and Calyptogena rectimargo-starobogatovi). The barcode index number (BIN) system determined 41 of the 56 species/OTUs, each with a unique BIN, indicating their validity. Three species were found to have two BINs, together with their high level of intraspecific variation, implying cryptic diversity within them. Although fewer 16 S sequences were collected, similar results were obtained. Nineteen putative species were determined and no overlap observed between intra- and inter-specific variation. Implications of DNA barcoding for the Vesicomyidae taxonomy were then discussed. Findings of this study will provide important evidence for taxonomic revision in this problematic clam group, and accelerate the discovery of new vesicomyid species in the future.

DNA barcoding, phylogeny and phylogeography of the cyst nematode species of the Avenae group from the genus Heterodera (Tylenchida: Heteroderidae)

Summary Among the recognised species groups of Heterodera, the Avenae group is one of the largest with a total of 12 species. Ten of them, H. arenaria, H. aucklandica, H. australis, H. avenae, H. filipjevi, H. mani, H. pratensis, H. riparia, H. sturhani and H. ustinovi, are morphologically closely related and represent the H. avenae species complex, and the other two, H. hordecalis and H. latipons, are morphologically more distinct from this complex. In this study we provide comprehensive phylogenetic analyses of several hundred COI and ITS rRNA gene sequences from the Avenae group using Bayesian inference, maximum likelihood and statistical parsimony. Some 220 COI and 11 ITS rRNA new gene sequences from 147 nematode populations collected in 26 countries were obtained in this study. Our study showed that the COI gene is a powerful DNA barcoding marker for identification of populations and species from the Avenae group. A putatively new cyst nematode species related to H. latipons was revealed from the analysis of COI and ITS rRNA gene datasets. COI gene sequences allow distinguishing H. arenaria, H. australis and H. sturhani from each other and other species. Problems of species delimiting of these species are discussed. The results of the analysis showed that COI haplotypes corresponded to certain pathotypes of the cereal cyst nematodes. It is recommended that information on COI haplotypes of studied populations be included in research with these nematodes. Based on the results of phylogeographical analysis and age estimation of clades with a molecular clock approach, it was hypothesised that several species of the Avenae group primarily originated and diversified in the Irano-Anatolian hotspot during the Pleistocene and Holocene periods and then dispersed from this region across the world. Different geographic barriers, centres and times of origin might explain current known distribution patterns for species of the Avenae group. Possible pathways, including a long distance trans-Atlantic dispersal, and secondary centres of diversification are proposed and discussed.

A new species of the hysius species-group of Calisto Hübner (Lepidoptera, Nymphalidae, Satyrinae) and insights into the status of different populations currently attributed to C. grannus Bates

A species belonging to the hysius group of Calisto is newly described. Calisto bahoruco new species inhabits the easternmost area of the southern Hispaniola Sierra de Bahoruco. The species is closely related to C. hysius Godart, another endemic from the southern mountains. The two species differ in average forewing length (larger in C. hysius), the relative size of ocelli (larger in C. bahoruco), and in the darker ground color with more contrasted paler edges of lines at underside of wings in C. bahoruco compared to C. hysius. Their male genitalia differ in the shape of the uncus and in the heavier sclerotization in C. bahoruco. COI barcodes bear a minimum of 15 differences between the two species. All species delimitation methods applied to the COI dataset, ABGD, BIN, bPTP, mPTP, and PTP yielded 8 species. The exception was the GMYC that failed to recognize the new taxon and oversplitted several other taxa. COI gene tree obtained by Maximum Likelihood and Bayesian have identical topologies agreeing with a multilocus phylogeny reconstructed in a previous work on Calisto and placing the new taxon as sister of C. hysius. Additionally, we provide new evidence of the conspecific nature of all named populations within Calisto grannus group.

Systematics of the Culex coronator complex (Diptera: Culicidae): morphological and molecular assessment

AbstractThe Culex coronator complex of the mosquito subgenus Culex includes five currently recognized species: Cx. camposi, Cx. coronator, Cx. ousqua, Cx. usquatissimus and Cx. usquatus. Because of the confusing taxonomic history of the complex, we aimed to clarify the specific status of these nominal forms based on an examination of holotypes and lectotypes and molecular data from other specimens. Critical assessment of published descriptions and study of type specimens revealed that the known distributions of the five species overlap considerably and exhibit biotic sympatry in some areas. Sequences from the COI barcode region and complete mitochondrial genomes were used to assess the relationships and degree of genetic divergence of the species and two newly discovered morphological forms, Cx. coronator Forms 1 and 2. Genetic distances in the COI dataset varied from 0.00 to 2.67%, with the largest relative divergence being 4.41 between specimens of Cx. coronator and Cx. coronator Form 1. Bayesian Poisson tree process analysis of the COI barcode region also failed to provide support for the nominal species. Evidence from the morphological and molecular data thus leads us to conclude (at least provisionally) that the Cx. coronator complex is a single polymorphic species. The forms constitute a monophyletic group but there is no support for the specific status of the five nominal forms.

Molecular phylogenetic analysis of genus Osteobrama Heckel, 1843 and discovery of Osteobrama serrata sp. nov. from North East India

Genus Osteobrama Heckel is characterized by strongly compressed, fairly deep body with abdominal edge sharp and trenchant entirely or from the pelvic fin base to vent; anal fin long with 14–36 rays, of which 11–13 are branched rays. The fishes of this genus are distributed in Pakistan, India, Bangladesh, Burma, and Yunnan (China). Present study involve six Osteobrama species viz. O. belangeri, O. cotio, O. cunma, O. feae, O. serrata sp. nov., and O. vigorsii. Out of the 655 positions analyzed in the COI sequence dataset, 171 positions (26.1%) were variable, and 151 positions (23%) were parsimoniously informative. The genetic distance between Osteobrama species ranges from 7.10% (between O. cotio and O. serrata sp. nov.) to 22.86% (between O. belangeri and O. feae) for COI sequences and 3.04% (between O. serrata sp. nov. and O. cunma) to 9.89% (between O. serrata sp. nov. and O. belangeri) for 16S sequences. Osteobrama belangeri show very high interspecies K2P distance with all other Osteobrama species for COI and 16S datasets. Best fit models for COI and 16S rRNA dataset were HKY + G + I and K2 + G, respectively. The maximum-likelihood (ML) phylogenetic trees were constructed using the COI and 16S rRNA sequences.

Population Genetics of Overwintering Monarch Butterflies, Danaus plexippus (Linnaeus), from Central Mexico Inferred from Mitochondrial DNA and Microsatellite Markers.

Population genetic variation and demographic history in Danaus plexippus (L.), from Mexico were assessed based on analyses of mitochondrial cytochrome c oxidase subunit I (COI; 658 bp) and subunit II (COII; 503 bp) gene segments and 7 microsatellite loci. The sample of 133 individuals included both migratory monarchs, mainly from 4 overwintering sites within the Monarch Butterfly Biosphere Reserve (MBBR) in central Mexico (states of Michoacán and México), and a nonmigratory population from Irapuato, Guanajuato. Haplotype (h) and nucleotide (π) diversities were relatively low, averaging 0.466 and 0.00073, respectively, for COI, and 0.629 and 0.00245 for COII. Analysis of molecular variance of the COI data set, which included additional GenBank sequences from a nonmigratory Costa Rican population, showed significant population structure between Mexican migratory monarchs and nonmigratory monarchs from both Mexico and Costa Rica, suggesting limited gene flow between the 2 behaviorally distinct groups. Interestingly, while the COI haplotype frequencies of the nonmigratory populations differed from the migratory, they were similar to each other, despite the great physical distance between them. Microsatellite analyses, however, suggested a lack of structure between the 2 groups, possibly owing to the number of significant deviations from Hardy–Weinberg equilibrium resulting from heterzoygote deficiencies found for most of the loci. Estimates of demographic history of the combined migratory MBBR monarch population, based on the mismatch distribution and Bayesian skyline analyses of the concatenated COI and COII data set (n = 89) suggested a population expansion dating to the late Pleistocene (~35000–40000 years before present) followed by a stable effective female population size (Nef) of about 6 million over the last 10000 years.

DNA Barcoding of Mealybugs (Hemiptera: Coccoidea: Pseudococcidae) From Mainland China

Mealybugs (Hemiptera: Pseudococcidae) are an insect group that feeds on plant sap; many are major pests of ornamental plants and crops worldwide. The difficulty of morphological identification of mealybugs points to a need for a rapid and effective identification method, like DNA barcoding, to assist morphological taxonomy. Here, we employed diverse methods (best close match [BCM], Neighbor-Joining [NJ] tree, Barcoding with LOGic formulas [BLOG], Poisson Tree Process [PTP] Species Delimitation Method) to test the efficiency of two molecular markers (mitochondrial cytochrome c oxidase I [COI] and large ribosomal subunit gene [28S]) that could be used for species identification of 54 mealybug species that commonly occur in China. Two hundred six COI barcodes (47 species) and 242 28S sequences (53 species) were recovered from 246 individuals. In both the COI and 28S data sets, species except for Planococcus citri and P. minor were unambiguously identified by all the methods. The PTP analysis based on COI sequences generated more putative species in Antonina tesquorum , Atrococcus paludinus , and Formicococcus sp. than morphological identification. Among these three cases, the sequences of At. paludinus showed 3.55% variation at the 28S locus, possibly reflecting cryptic diversity in this taxon. Our study corroborates the utility of the COI and 28S genes in the rapid identification of mealybugs, and the barcode library we provide will create an effective identification system for mealybug pest management in China.

First time DNA barcoding of the common shipworm Teredo navalis Linnaeus 1758 (Mollusca: Bivalvia: Teredinidae): Molecular-taxonomic investigation and identification of a widespread wood-borer

The common shipworm Teredo navalis is one of the most widespread marine wood-boring bivalves of the world and probably one of the most wood destructive and cost-incurring marine invertebrates. First reports on T. navalis for Europe date back to 1731 for the North Sea (The Netherlands) and to 1835 for the Baltic Sea (Germany). It is still unclear, however, where this species originates from. Therefore, T. navalis is considered cryptogenic for European waters, including the Baltic Sea.In this study, 181 specimens of Teredinidae from six different sampling areas all over Europe and North America were molecular-taxonomically investigated using several molecular markers, two nuclear (18S/28S) as well as one mitochondrial marker (cytochrome c oxidase subunit I, hereafter COI). For the COI gene amplification, a new specific primer pair (Ter fw II/Ter rev I) for T. navalis was developed, which allowed sequencing of a 675bp COI gene fragment for the first time. For amplifying the COI gene fragment of other examined teredinids than T. navalis, a third primer (Ter fw III) was designed. These three new primers are valuable tools to identify teredinid species with the DNA barcoding approach.Classification of T. navalis into the system of wood-boring bivalves using a combined 18S/28S dataset showed no differentiation between specimens from Europe and the North American East Coast. The results of the COI dataset analyses showed high haplotype diversity in combination with a low nucleotide diversity and a star-shaped network with a predominant haplotype occurring in all investigated regions. Moreover, no indications have been found on a sibling species in the Baltic Sea. The data indicate a recent population expansion for the examined sampling sites whereas the origin of the assumed worldwide distributed species T. navalis remains open.

Scorpion speciation in the Holy Land: Multilocus phylogeography corroborates diagnostic differences in morphology and burrowing behavior among Scorpio subspecies and justifies recognition as phylogenetic, ecological and biological species

Scorpio Linnaeus, 1758 (family Scorpionidae Latreille, 1802) was considered monotypic for over a century, and comprised a single species, Scorpio maurus Linnaeus, 1758, with 19 subspecies, distributed from West Africa, throughout the Maghreb and the Middle East, to Iran. Two parapatric subspecies, Scorpio maurus fuscus (Ehrenberg, 1829) and Scorpio maurus palmatus (Ehrenberg, 1828), have long been recognized in the eastern Mediterranean region. We examined morphological variation, burrow architecture and genetic divergence among 39 populations across the distribution of the two subspecies to assess whether they are conspecific and, if not, how many species might be involved. Cuticle coloration, pedipalp chela digital carina condition, and selected measurements were recorded. Sixty burrows were excavated and examined for burrow structure and depth. A multilocus dataset comprising concatenated fragments of one nuclear (28S rDNA) and three mitochondrial (12S rDNA, 16S rDNA, Cytochrome c Oxidase Subunit I) loci, totaling ca. 2400 base-pairs, was produced for 41 individuals, and a single-locus dataset comprising 658 base-pairs of the COI locus for 156 individuals. Despite overlapping ranges in morphometric characters of pedipalp chela shape, the putative subspecies were easily distinguished by cuticle coloration and condition of the pedipalp chela digital carina, and were also found to differ significantly in burrow architecture and depth. Phylogeographical analyses of the COI and multilocus datasets recovered seven distinct clades. Separate analyses of mitochondrial sequences, and combined analyses of mitochondrial and nuclear sequences support most clades. The two major clades corresponded with the geographical distributions of S. m. fuscus and S. m. palmatus in the region. Specimens from these clades were genetically distinct, and exhibited different burrow structure in geographically-proximate localities, suggesting reproductive isolation. The palmatus clade included two distinct subclades of specimens from localities adjacent to the Dead Sea. Three other clades, comprising specimens from the most northeastern localities, were tentatively assigned to subspecies previously recorded in neighboring Jordan and Syria. The morphological, behavioral and genetic evidence supports previous suggestions that Scorpio maurus is a species complex and justifies the following taxonomic emendations: Scorpio fuscus (Ehrenberg, 1829), stat. nov.; Scorpio kruglovi Birula, 1910, stat. nov.; Scorpio palmatus (Ehrenberg, 1828), stat. nov.; Scorpio propinquus (Simon, 1872), stat. nov.

Two distinct evolutionary lineages of the Astacus leptodactylus species-complex (Decapoda : Astacidae) inferred by phylogenetic analyses

Narrow-clawed crayfish (Astacus leptodactylus Eschscholtz, 1823 species-complex) is one of five European freshwater crayfish species. Even though widely distributed, it hasn’t been frequently studied and its taxonomy and systematics are unresolved. The results of a recent comparative morphometric character study revealed that morphometry of Asian and European populations differ significantly. In this research, for the first time, mitochondrial molecular markers (16S rRNA and COI) were used with the aim of elucidating the phylogenetic relationship between European and Asian populations of the narrow-clawed crayfish. Analyses included crayfish from Croatia, Bulgaria, Armenia, Russia, Poland and Turkey, and three different optimality criteria were applied. Phylogenetic relationships were reconstructed using the COI dataset, as well as the concatenated one (COI + 16S rRNA). For both datasets, congruent topologies were obtained and trees were characterised by the existence of two well supported phylogroups, one that included European populations, and the other Asian. Results indicate the presence of distinct evolutionary lineages within the A. leptodactylus species-complex, and corroborate previous results obtained using morphometric analyses.

Systematics of the oswaldoi complex (Anopheles, Nyssorhynchus) in South America.

BackgroundEffective malaria control relies on accurate identification of those Anopheles mosquitoes responsible for the transmission of Plasmodium parasites. Anopheles oswaldoi s.l. has been incriminated as a malaria vector in Colombia and some localities in Brazil, but not ubiquitously throughout its Neotropical range. This evidence together with variable morphological characters and genetic differences supports that An. oswaldoi s.l. compromises a species complex. The recent fully integrated redescription of An. oswaldoi s.s. provides a solid taxonomic foundation from which to molecularly determine other members of the complex.MethodsDNA sequences of the Second Internal Transcribed Spacer (ITS2 - rDNA) (n = 192) and the barcoding region of the Cytochrome Oxidase I gene (COI - mtDNA) (n = 110) were generated from 255 specimens of An. oswaldoi s.l. from 33 localities: Brazil (8 localities, including the lectotype series of An. oswaldoi), Ecuador (4), Colombia (17), Trinidad and Tobago (1), and Peru (3). COI sequences were analyzed employing the Kimura-two-parameter model (K2P), Bayesian analysis (MrBayes), Mixed Yule-Coalescent model (MYC, for delimitation of clusters) and TCS genealogies.ResultsSeparate and combined analysis of the COI and ITS2 data sets unequivocally supported four separate species: two previously determined (An. oswaldoi s.s. and An. oswaldoi B) and two newly designated species in the Oswaldoi Complex (An. oswaldoi A and An. sp. nr. konderi). The COI intra- and inter-specific genetic distances for the four taxa were non-overlapping, averaging 0.012 (0.007 to 0.020) and 0.052 (0.038 to 0.064), respectively. The concurring four clusters delineated by MrBayes and MYC, and four independent TCS networks, strongly confirmed their separate species status. In addition, An. konderi of Sallum should be regarded as unique with respect to the above. Despite initially being included as an outgroup taxon, this species falls well within the examined taxa, suggesting a combined analysis of these taxa would be most appropriate.ConclusionsThrough novel data and retrospective comparison of available COI and ITS2 DNA sequences, evidence is shown to support the separate species status of An. oswaldoi s.s., An. oswaldoi A and An. oswaldoi B, and at least two species in the closely related An. konderi complex (An. sp. nr. konderi, An. konderi of Sallum). Although An. oswaldoi s.s. has never been implicated in malaria transmission, An. oswaldoi B is a confirmed vector and the new species An. oswaldoi A and An. sp. nr. konderi are circumstantially implicated, most likely acting as secondary vectors.

Phylogeography and conservation genetics of the Amazonian freshwater stingray Paratrygon aiereba Müller & Henle, 1841 (Chondrichthyes: Potamotrygonidae)

The family Potamotrygonidae is monophyletic comprising three genera: Paratrygon Duméril, Potamotrygon Garman and Plesiotrygon Rosa, Castello & Thorson. The distribution of most species in this family is restricted to a single basin or fluvial system. Only Potamotrygon motoro, Potamotrygon orbignyi and Paratrygon aiereba are found in more than one river basin. In this study we investigate genetic structuring of Paratrygon aiereba, from five rivers of the Amazon region: Negro, Solimões-Amazon-Estuary system, Tapajós, Xingu and Araguaia. Sixty-three individuals were sequenced for ATPase 6, and a representative subsample of 27 individuals was sequenced for COI. The COI dataset analysis indicated that Paratrygon is sister to all other potamotrygonid genera and species. Population parameters inferred from the analysis of ATPase 6 sequences revealed that the populations of this species are structured within each river, with no or nearly non-existent gene flow occurring between rivers and a positive correlation between geographic and genetic distances. Paratrygon aiereba is comprised of three geographically restricted clades with K2P interclade distances of at least 2%. Intraspecific divergence within P. aiereba is similar to the interspecific divergence observed in Potamotrygon spp. sampled throughout the same geographic area. Using the premises of COI barcoding and the allopatric distribution of the three P. aiereba clades, the taxon P. aiereba most likely comprises three distinct biological species. Since freshwater stingrays of the family Potamotrygonidae are highly exploited for the aquarium trade, management and conservation strategies need to be implemented at the level of each river basin, rather than at the level of the Amazon basin.