Morphometric and meristic diversity of the species Scolopendra cingulata Latreille, 1829 (Chilopoda: Scolopendridae) in the Mediterranean region

Scolopendra cingulata Latreille, 1829 is the commonest scolopendromorph species in the Mediterranean area. However, its morphological and geographic variation has not been examined so far, and therefore robust hypotheses about the factors that have shaped that variability are lacking. We examined, using multivariate methods for 19 morphometric and meristic characters, the morphological variation of that species using a comprehensive sample of 503 adult specimens from 130 restricted geographic localities. The localities were distributed in three major geographic areas (Balkans, Asia Minor-Middle East, and Italy) in order to discern biological entities and to estimate the morphological relationships between populations and geographic regions. Results showed significant differences between the three geographic groups. Characters such as the distance between the paramedian sulci of the 7th and 8th tergite, the number of antennal segments, the number of spines on the dorsal side of 21st prefemur and number of spines on the 21st prefemoral process significantly discriminated populations of S. cingulata along a west-east geographic gradient. Both eastern (Balkan) and easternmost populations (Asia Minor-Middle East) showed higher mean values of antennal articles and spines of 21st prefemur than the Italian populations. No significant morphological variation was discovered between the sexes of S. cingulata apart from cephalic width and distance between the two paramedian sulci of the 7th and 8th tergite. Based on certain morphometric differences among the three major geographic regions, we suggest that S. cingulata originated in the east and colonized south European area from two different geographic routes. It is suggested that west-east morphological trend of S. cingulata is related either to the prominent palaeogeographic events of the area or to the competition with S. oraniensis Lucas, 1846 in the western Mediterranean.     

The evolutionary history of the Mediterranean centipede Scolopendra cingulata (Latreille, 1829) (Chilopoda: Scolopendridae) across the Aegean archipelago

In this study we investigate the evolutionary relationships of Scolopendra cingulata (Latreille, 1829) within insular Greece. Our main goal is to infer the time frame of the differentiation of the species in the study area. In this regard, sequence data originating from three mitochondrial genes are used to reconstruct the evolutionary history of 47 insular populations of S. cingulata from the Aegean archipelago. Within the phylogenetic framework and by implementing a relaxed molecular clock methodology, we infer the time estimates of separations of the S. cingulata lineages. The results of the phylogenetic analysis support the presence of three distinct S. cingulata groups in the region. The first group accommodates populations from the eastern Aegean islands, and is closely related to the second group that hosts mainly populations of northern and central Cyclades. The third group is composed of insular populations originating from southern Cyclades. Different temporal splitting scenarios have been evaluated. Based on the scenario strongly supported by the data, we propose a biogeographical scenario that could account for the contemporary distribution of the species' lineages. The splitting events of S. cingulata are estimated to have occurred within the late Miocene. The historical events of the last 13.77 Myr have shaped, through a series of mostly vicariant and dispersal incidents, the present‐day biogeographical pattern of the species. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 105 , 507–521.     

First genetic characterization of the brown dog tick <Emphasis Type="Italic">Rhipicephalus sanguineus</Emphasis> sensu lato in Peninsular Malaysia

The brown dog tick Rhipicephalus sanguineus sensu lato (s.l.) is a species complex comprising three main mitochondrial lineages, namely tropical, temperate and southeast European lineages. Despite its medical and veterinary importance, little attention has been paid to the genetic lineage of this species in Southeast Asia. Rhipicephalus sanguineus s.l. from Malaysia was investigated genetically, for the first time, using the mitochondria-encoded cytochrome c oxidase subunit I (COI) and 16S ribosomal RNA (16S) genes. Specifically, a pair of primers was developed to amplify the COI sequences in the present study. Both genes unambiguously assigned Malaysian material into the tropical lineage of R. sanguineus s.l. The 16S sequences were highly conserved; no variation site was observed. The COI sequences revealed slightly higher variation by recovering four haplotypes, one of which is restricted to the northernmost of Peninsular Malaysia. This finding will be a stepping stone in promoting more biological studies of this species complex in this region.     

Mitochondrial and nuclear markers reveal a lack of genetic structure in the entocommensal nemertean Malacobdella arrokeana in the Patagonian gulfs

Malacobdella arrokeana is an entocommensal nemertean exclusively found in the bivalve geoduck Panopea abbreviata, and it is the only representative of the genus in the southern hemisphere. To characterize its genetic diversity, population structure and recent demographic history, we conducted the first genetic survey on this species, using sequence data for the cytochrome oxidase I gene (COI), 16S rRNA (16S) and the internal transcribed spacer (ITS2). Only four different ITS2 genotypes were found in the whole sample, and the two main haplotypes identified in the mitochondrial dataset were present among all localities with a diversity ranging from 0.583 to 0.939. Nucleotide diversity was low (π = 0.001–0.002). No significant genetic structure was detected between populations, and mismatch distribution patterns and neutrality tests results are consistent with a population in expansion or under selection. Analysis of molecular variance (AMOVA) revealed that the largest level of variance observed was due to intrapopulation variation (100, 100 and 94.39 % for 16S, COI and ITS2, respectively). F _st values were also non-significant. The observed lack of population structure is likely due to high levels of genetic connectivity in combination with the lack or permeability of biogeographic barriers and episodes of habitat modification.     

Molecular cloning and characterization of a new cDNA sequence encoding a venom peptide from the centipede <Emphasis Type="Italic">Scolopendra subspinipes</Emphasis> mutilans

Many studies have been performed on venomous peptides derived from animals. However, little of this research has focused on peptides from centipede venoms. Here, a venom gland cDNA library was suc-cessfully constructed for the centipede Scolopendra subspinipes mutilans. A new cDNA encoding the precursor of a venom peptide, named SsmTx, was cloned from the venomous gland cDNA library of the centipede S. subspinipes mutilans. The full-length SsmTx cDNA sequence is 465 nt, including a 249 nt ORF, a 45 nt 5′ UTR and a 171 nt 3′ UTR. There is a signal tail AATAAA 31 nt upstream of the poly (A) tail. The precursor nucleotide sequence of SsmTx encodes a signal peptide of 25 residues and a mature peptide of 57 residues, which is bridged by two pairs of disulfide bonds. SsmTx displays a unique cysteine motif that is completely different from that of other venomous animal toxins. This is the first reported cDNA sequence encoding a venom peptide from the centipede S. subspinipes mutilans.     

Molecular insights into the taxonomy of Hypanis (Bivalvia, Cardiidae, Lymnocardiinae) in the Black Sea lagoons

In this study, we used data from morphology and three DNA markers to assess the taxonomic status of the putative bivalve species Hypanis colorata and Hypanis angusticostata in a Black Sea lagoon, the Razelm Lake in Romania. The morphological data (the shape of shell ribs and the multivariate analysis of morphometric variance of three variables constructed as the ratios between the main dimensions of the shell) confirmed that the two analyzed species are distinct morphological entities. Three molecular markers, one from the nuclear genome (18S rRNA) and two from the mitochondrial genome (16S rRNA and COI), showed extremely reduced sequence divergence (0?C0.1%) between the two putative species. Based on these results, we suggest that H. angusticostata and H. colorata are morphotypes of a single species.     

Integrative revision of the giant pill-millipede genus Sphaeromimus from Madagascar,with the description of seven new species (Diplopoda,Sphaerotheriida, Arthrosphaeridae)

The Malagasy giant pill-millipede genus Sphaeromimus de Saussure & Zehntner, 1902 is revised. Seven new species, S. titanus sp. n., S. vatovavy sp. n., S. lavasoa sp. n., S. andohahela sp. n., S. ivohibe sp. n., S. saintelucei sp. n., and S. andrahomana sp. n. were discovered, in one case with the help of sequence data, in the rainforests of southeastern Madagascar. The species are described using light- and scanning electron microscopy. A key to all 10 species of the genus is presented. All but one (S. andohahela) of the newly discovered species are microendemics each occurring in isolated forest fragments. The mitochondrial COI barcoding gene was amplified and sequenced for 18 Sphaeromimus specimens, and a dataset containing COI sequences of 28 specimens representing all Sphaeromimus species (except S. vatovavy) was analyzed. All species are genetically monophyletic. Interspecific uncorrected genetic distances were moderate (4–10%) to high (18–25%), whereas intraspecific variation is low (0–3.5%). Sequence data allowed the correct identification of three colour morphs of S. musicus, as well as the identity of a cave specimen, which although aberrant in its morphology and colouration, was genetically identical to the holotype of S. andrahoma.     

Towards resolving the identities of the Graphium butterflies (Lepidoptera: Papilionidae) of Peninsular Malaysia

Graphium butterflies are famous in Peninsular Malaysia for their colourful wings, yet their taxonomy remains unresolved. The popular guides to Malaysian butterflies, place the species in one, two or three genera and identification to species using obscure morphological characters can be difficult, especially for the closely related species, G. bathycles bathycloides and G. chironides malayanum. We sequenced the COI mtDNA barcode for Graphium specimens in the Museum of Zoology, University of Malaya to test the utility of DNA barcoding for the identification of Graphium species. Additionally, we sequenced 28S rRNA to examine, in conjunction with COI, the phylogenetic relationships of these species and investigate the validity of Pathysa and Paranticopsis as distinct genera. We found that all species of Graphium possessed a distinctive cluster of DNA barcodes with the exception of specimens originally identified as G. bathycles bathycloides and G. chironides malayanum which shared DNA barcodes. On further examination we found that the morphological determinations were ambiguous as the specimens overlapped for diagnostic characters reported for each species. The COI and 28S rRNA phylogenetic trees showed a similar topology with Paranticopsis species forming a clade nested within a larger clade also comprising Pathysa species. Based on this topology, in order for Pathysa to be a valid genus, at least three other clades within Graphium s.l. would also have to be raised as genera.     

Review of the Parasa undulata (Cai, 1983) species group with the first conifer-feeding larva for Limacodidae and descriptions of two new species from China and Taiwan (Lepidoptera,Limacodidae)

Although the caterpillars are well-known for the stings and magnificent coloration, the systematics of Limacodidae is historically neglected and chaotic due to the difficulty in matching the larval with adult stages as well as the very conservative and convergent adult morphology. One of the biggest taxonomic problems surrounds a collective group from Southeastern Asia, termed the “green limacodid moths”, which harbours at least 90 species placed in the genus Parasa Walker, 1859 and 14 “subunits”. The P. undulata group was previously composed of 3 species from China and Taiwan, and characterized only by wing pattern. This species group is extensively studied herein with two new species described, i.e. P. viridiflamma sp. n. (Taiwan) and P. minwangi sp. n. (S. China), and discovery of female genitalia of three species, presenting new phylogenetic insights in this potentially paraphyletic genus. In addition, one limacodid larva was found to be feeding exclusively on Picea (Pinaceae) in Taiwan. Its identity, Parasa pygmy Solovyev, 2010 in P. undulata group, is confirmed through matching its COI sequence to the adult. This discovery is also biologically significant because the previous known host breadth of Parasa was of polyphagy on various angiosperm plant families. This case, therefore, represents the first record of conifer-feeding behavior in this family as well as the first of specialized herbivory in the genus. Meanwhile, the background match between Picea leaves and larval coloration is shared with other Picea-feeding insects. This phenomenon is worth of further investigation in the aspect of convergent evolution of crypsis associated with a particular plant.     

Invasion genetics of American cherry fruit fly in Europe and signals of hybridization with the European cherry fruit fly

The American cherry fruit fly is an invasive pest species in Europe, of serious concern in tart cherry production as well as for the potential to hybridize with the European cherry fruit fly, Rhagoletis cerasi L. (Diptera: Tephritidae), which might induce new pest dynamics. In the first European reports, the question arose whether only the eastern American cherry fruit fly, Rhagoletis cingulata (Loew) (Diptera: Tephritidae), is present, or also the closely related western American cherry fruit fly, Rhagoletis indifferens Curran. In this study, we investigate the species status of European populations by comparing these with populations of both American species from their native ranges, the invasion dynamics in German (first report in 1993) and Hungarian (first report in 2006) populations, and we test for signals of hybridization with the European cherry fruit fly. Although mtDNA sequence genealogy could not separate the two American species, cross‐species amplification of 14 microsatellite loci separated them with high probabilities (0.99–1.0) and provided evidence for R. cingulata in Europe. German and Hungarian R. cingulata populations differed significantly in microsatellite allele frequencies, mtDNA haplotype and wing pattern distributions, and both were genetically depauperate relative to North American populations. The diversity suggests independent founding events in Germany and Hungary. Within each country, R. cingulata displayed little or no structure in any trait, which agrees with rapid local range expansions. In cross‐species amplifications, signals of hybridization between R. cerasi and R. cingulata were found in 2% of R. cingulata individuals and in 3% of R. cerasi. All putative hybrids had R. cerasi mtDNA indicating that the original between‐species mating involved R. cerasi females and R. cingulata males.     

Antifungal effect and pore-forming action of lactoferricin B like peptide derived from centipede Scolopendra subspinipes mutilans

The centipede Scolopendra subspinipes mutilans has been a medically important arthropod species by using it as a traditional medicine for the treatment of various diseases. In this study, we derived a novel lactoferricin B like peptide (LBLP) from the whole bodies of adult centipedes, S. s. mutilans, and investigated the antifungal effect of LBLP. LBLP exerted an antifungal and fungicidal activity without hemolysis. To investigate the antifungal mechanism of LBLP, a membrane study with propidium iodide was first conducted against Candida albicans. The result showed that LBLP caused fungal membrane permeabilization. The assays of the three dimensional flow cytometric contour plot and membrane potential further showed cell shrinkage and membrane depolarization by the membrane damage. Finally, we confirmed the membrane-active mechanism of LBLP by synthesizing model membranes, calcein and FITC-dextran loaded large unilamellar vesicles. These results showed that the antifungal effect of LBLP on membrane was due to the formation of pores with radii between 0.74 nm and 1.4 nm. In conclusion, this study suggests that LBLP exerts a potent antifungal activity by pore formation in the membrane, eventually leading to fungal cell death.     

Can Environment Predict Cryptic Diversity? The Case of Niphargus Inhabiting Western Carpathian Groundwater

In the last decade, several studies have shown that subterranean aquatic habitats harbor cryptic species with restricted geographic ranges, frequently occurring as isolated populations. Previous studies on aquatic subterranean species have implied that habitat heterogeneity can promote speciation and that speciation events can be predicted from species’ distributions. We tested the prediction that species distributed across different drainage systems and karst sectors comprise sets of distinct species. Amphipods from the genus Niphargus from 11 caves distributed along the Western Carpathians (Romania) were investigated using three independent molecular markers (COI, H3 and 28S). The results showed that: 1) the studied populations belong to eight different species that derive from two phylogenetically unrelated Niphargus clades; 2) narrow endemic species in fact comprise complexes of morphologically similar species that are indistinguishable without using a molecular approach. The concept of monophyly, concordance between mitochondrial and nuclear DNA, and the value of patristic distances were used as species delimitation criteria. The concept of cryptic species is discussed within the framework of the present work and the contribution of these species to regional biodiversity is also addressed.     

Comparative Phylogeography of Direct-Developing Frogs (Anura: Craugastoridae: Pristimantis) in the Southern Andes of Colombia

The Andes of South America hosts perhaps the highest amphibian species diversity in the world, and a sizable component of that diversity is comprised of direct-developing frogs of the genus Pristimantis (Anura: Craugastoridae). In order to better understand the initial stages of species formation in these frogs, this study quantified local-scale spatial genetic structuring in three species of Pristimantis. DNA sequences of two mitochondrial gene fragments (16S and COI) were obtained from P. brevifrons, P. palmeri and P. jubatus at different locations in the Cordillera Occidental. We found high levels of genetic diversity in the three species, with highly structured populations (as measured by F _ST) in P. brevifrons and P. palmeri while P. jubatus showed panmixia. Large effective population sizes, inferred from the high levels of genetic diversity, were found in the three species and two highly divergent lineages were detected within P. jubatus and P. palmeri. Estimated divergence times among populations within P. brevifrons and P. palmeri coincide with the Pleistocene, perhaps due to similar responses to climatic cycling or recent geological history. Such insights have important implications for linking alpha and beta diversity, suggesting regional scale patterns may be associated with local scale processes in promoting differentiation among populations in the Andes.     

Genetic diversity of genus Vespa including an invaded species of V. velutina (Hymenoptera: Vespidae) in Korea inferred from DNA barcoding data

With about 5000 known species, the Vespidae is a large family belongs to order Hymenoptera. The genus Vespa with 22 species is one of the four genera of the subfamily Vespinae. In Korea, 10 species and subspecies are recognized. Because of their social behavior, their treat to human health and their impact in apiculture, the reliable and sometimes automated identification of these insects to species level are important. To test the efficacy of DNA barcoding method for identification of species of the genus Vespa in Korea, 30 samples of eight Korean species of genus Vespa were collected and mitochondrial DNAs of 658 bp fragment cytochrome oxidase subunit 1 (CO1) region were sequenced. A Bayesian Inference based on COI gene of the Korean Vespa species was constructed. The phylogenetic tree shoed that identification of all specimens is possible based on COI gene and we found strong relation between the sequences of the collected species from different localities in South Korea which clustered together with 100% support with sequences of the same species in GenBank. The results demonstrate that DNA barcoding is a useful technique for rapid and accurate species recognition in Korean Vespa species. The DNA barcode part of COI for V. binghami is provided for the first time that can help for identification of this species through DNA barcoding. Also, the genetic diversity among Korean Vespa velutina was zero suggests that the invasion might have occurred in a single event with small number of founders.     

A new species of Newportia Gervais, 1847 from Puerto Rico,with a revised key to the species of the genus (Chilopoda,Scolopendromorpha, Scolopocryptopidae)

A new species of the centipede genus Newportia, Newportia stoevi sp. n., is described from Rio Encantado Cave, Puerto Rico. It differs from all congeners by having sternites distinctly margined laterally and ultimate legs bearing 4 spinous processes on both prefemur and femur, and 2 on tibia. The value of some terms used in the taxonomy of the genus have been analyzed and an amended identification key to the species of Newportia is provided.     

Eretes diving beetles (Coleoptera: Dytiscidae) in Central Europe – witnesses of climate change?

Records of the predaceous diving beetles of the genus Eretes Laporte, 1833 (Coleoptera: Dytiscidae) in Central Europe are summarised. While old records from the beginning of the twentieth century from the Czech Republic, Hungary, and Romania belong to Eretes griseus (Fabricius, 1781), a species which has not been recaptured in Central Europe for nearly the last hundred years, recently collected specimens from Hungary and Slovakia belong to E. sticticus (Linnaeus, 1767) and represent its first records in these countries as well as in Central Europe. The first specimens were collected at light during hot summer nights and may document a recent spreading of the species from the Mediterranean. In addition, the occurrence of E. sticticus is formally confirmed in Bulgaria, Croatia, Greece, Israel, Libya, South European Territory of Russia, and Tunisia.     

Molecular Phylogeny of Weakfish Species of the Stellifer Group (Sciaenidae,Perciformes) of the Western South Atlantic Based on Mitochondrial and Nuclear Data

The phylogenetic relationships within the Stellifer group of weakfishes (Stellifer, Odontoscion, Ophioscion, and Bairdiella) were evaluated using 2723 base pairs comprising sequences of nuclear (rhodopsin, TMO-4C4, RAG-1) and mitochondrial (16S rRNA and COI) markers obtained from specimens of nine species. Our results indicate a close relationship between Bairdiella and Odontoscion, and also that the genus Stellifer is not monophyletic, but rather that it consists of two distinct lineages, one clade containing S. microps/S. naso/S. brasiliensis and the other, S. rastrifer/S. stellifer/Stellifer sp. B, which is closer to Ophioscion than the former clade. The O. punctatissimus populations from the northern and southern Brazilian coast were also highly divergent in both nuclear (0.8% for rhodopsin and 0.9% for RAG-1) and mitochondrial sequences (2.2% for 16S rRNA and 7.3% for COI), which we conclude is consistent with the presence of two distinct species. The morphological similarities of the members of the Stellifer group is reinforced by the molecular data from both the present study and previous analyses, which have questioned the taxonomic status of the Stellifer group. If, on the one hand, the group is in fact composed of four genera (Stellifer, Ophioscion, Odontoscion, and Bairdiella), one of the two Stellifer clades should be reclassified as a new genus. However, if the close relationship and the reduced genetic divergence found within the group is confirmed in a more extensive study, including representatives of additional taxa, this, together with the morphological evidence, would support downgrading the whole group to a single genus. Obviously, these contradictory findings reinforce the need for a more systematic taxonomic revision of the Stellifer group as a whole.