The evolution of female-biased genital diversity in bedbugs (Cimicidae).

Rapid genitalia evolution is believed to be mainly driven by sexual selection. Recently, noncopulatory genital functions have been suggested to exert stronger selection pressure on female genitalia than copulatory functions. In bedbugs (Cimicidae), the impact of the copulatory function can be isolated from the noncopulatory impact. Unlike in other taxa, female copulatory organs have no function in egg-laying or waste-product expulsion. Males perform traumatic mating by piercing the female integument, thereby imposing antagonistic selection on females and suspending selection to morphologically match female genitalia. We found the location of the copulatory organ evolved rapidly, changing twice between dorsal and ventral sides, and several times along the anteroposterior and the left-right axes. Male genital length and shape varied much less, did not appear to follow the positional changes seen in females, and showed no evidence for coevolution. Female genitalia position evolved 1.5 times faster than male genital length and shape and showed little neutral or geographic signals. Instead, we propose that nonmorphological male traits, such as mating behavior, may drive female genitalia morphology in this taxon. Models of genitalia evolution may benefit from considering morphological genital responses to nonmorphological stimuli, such as male mating behavior or copulatory position.

Benthic invertebrates in Svalbard fjords-when metabarcoding does not outperform traditional biodiversity assessment.

To protect and restore ecosystems and biodiversity is one of the 10 challenges identified by the United Nations's Decade of the Ocean Science. In this study we used eDNA from sediments collected in two fjords of the Svalbard archipelago and compared the taxonomic composition with traditional methods through metabarcoding, targeting mitochondrial CO1, to survey benthos. Clustering of 21.6 mill sequence reads with a d value of 13 in swarm, returned about 25 K OTU reads. An identification search with the BOLD database returned 12,000 taxonomy annotated sequences spanning a similarity range of 50% to 100%. Using an acceptance filter of minimum 90% similarity to the CO1 reference sequence, we found that 74% of the ca 100 taxon identified sequence reads were Polychaeta and 22% Nematoda. Relatively few other benthic invertebrate species were detected. Many of the identified sequence reads were extra-organismal DNA from terrestrial, planktonic, and photic zone sources. For the species rich Polychaeta, we found that, on average, only 20.6% of the species identified from morphology were also detected with DNA. This discrepancy was not due to missing reference sequences in the search database, because 90-100% (mean 96.7%) of the visually identified species at each station were represented with barcodes in Boldsystems. The volume of DNA samples is small compared with the volume searched in visual sorting, and the replicate DNA-samples in sum covered only about 2% of the surface area of a grab. This may considerably reduce the detection rate of species that are not uniformly distributed in the sediments. Along with PCR amplification bias and primer mismatch, this may be an important reason for the limited congruence of species identified with the two approaches. However, metabarcoding also identified 69 additional species that are usually overlooked in visual sample sorting, demonstrating how metabarcoding can complement traditional methodology by detecting additional, less conspicuous groups of organisms.

Phylogeny and Cryptic Diversity of Diopatra (Onuphidae, Annelida) in the East Atlantic.

Diopatra Audouin & Milne-Edwards, 1833 is a species rich genus that is common in tropical and subtropical regions. The genus is readily identified by its striking, spiral branchiae, but species identification has historically been challenging due to a high variation in diagnostic characters used. This study aims to reconstruct the phylogeny of Diopatra with molecular markers and assess the species diversity of West African Diopatra with the species delimitation programs bPTP and BPP. Specimens were collected from Morocco to Angola, and the markers COI, 16S and 28S were sequenced from 76 specimens. The constructed phylogeny retrieved Diopatra as monophyletic, as well as five well supported clades within the genus. All clades were defined by morphological characters, some of which have previously not been considered to have high phylogenetic or taxonomical value. Species delimitation analyses recovered 17 new species, several of which were not readily identified morphologically. One species complex comprising between one and 12 species was left unresolved due to incongruence between the species delimitation methods and challenging morphology. Our results indicate that the diversity of Diopatra is significantly underestimated, where this regional study near to doubled the number ofknown species from the East Atlantic.

Scanning Electron Microscopy Observations of Loa loa (Nematoda).

We present a case report of periocular Loa loa. The key feature of L. loa distinguishing it from other human filarial parasites are cuticular bosses, which are presented in images from a light microscope and a scanning electron microscope. The cuticular bosses could be divided into three subtypes not previously described.

Bedbugs Evolved before Their Bat Hosts and Did Not Co-speciate with Ancient Humans.

All 100+ bedbug species (Cimicidae) are obligate blood-sucking parasites [1, 2]. In general, blood sucking (hematophagy) is thought to have evolved in generalist feeders adventitiously taking blood meals [3, 4], but those cimicid taxa currently considered ancestral are putative host specialists [1, 5]. Bats are believed to be the ancestral hosts of cimicids [1], but a cimicid fossil [6] predates the oldest known bat fossil [7] by >30 million years (Ma). The bedbugs that parasitize humans [1, 8] are host generalists, so their evolution from specialist ancestors is incompatible with the "resource efficiency" hypothesis and only partially consistent with the "oscillation" hypothesis [9-16]. Because quantifying host shift frequencies of hematophagous specialists and generalists may help to predict host associations when vertebrate ranges expand by climate change [17], livestock, and pet trade in general and because of the previously proposed role of human pre-history in parasite speciation [18-20], we constructed a fossil-dated, molecular phylogeny of the Cimicidae. This phylogeny places ancestral Cimicidae to 115 mya as hematophagous specialists with lineages that later frequently populated bat and bird lineages. We also found that the clades, including the two major current urban pests, Cimex lectularius and C. hemipterus, separated 47 mya, rejecting the notion that the evolutionary trajectories of Homo caused their divergence [18-21]. VIDEO ABSTRACT.

DNA barcode reference libraries for the monitoring of aquatic biota in Europe: Gap-analysis and recommendations for future work.

Effective identification of species using short DNA fragments (DNA barcoding and DNA metabarcoding) requires reliable sequence reference libraries of known taxa. Both taxonomically comprehensive coverage and content quality are important for sufficient accuracy. For aquatic ecosystems in Europe, reliable barcode reference libraries are particularly important if molecular identification tools are to be implemented in biomonitoring and reports in the context of the EU Water Framework Directive (WFD) and the Marine Strategy Framework Directive (MSFD). We analysed gaps in the two most important reference databases, Barcode of Life Data Systems (BOLD) and NCBI GenBank, with a focus on the taxa most frequently used in WFD and MSFD. Our analyses show that coverage varies strongly among taxonomic groups, and among geographic regions. In general, groups that were actively targeted in barcode projects (e.g. fish, true bugs, caddisflies and vascular plants) are well represented in the barcode libraries, while others have fewer records (e.g. marine molluscs, ascidians, and freshwater diatoms). We also found that species monitored in several countries often are represented by barcodes in reference libraries, while species monitored in a single country frequently lack sequence records. A large proportion of species (up to 50%) in several taxonomic groups are only represented by private data in BOLD. Our results have implications for the future strategy to fill existing gaps in barcode libraries, especially if DNA metabarcoding is to be used in the monitoring of European aquatic biota under the WFD and MSFD. For example, missing species relevant to monitoring in multiple countries should be prioritized for future collaborative programs. We also discuss why a strategy for quality control and quality assurance of barcode reference libraries is needed and recommend future steps to ensure full utilisation of metabarcoding in aquatic biomonitoring.

Molecular phylogeny of Caudofoveata (Mollusca) challenges traditional views.

The shell-less, worm-shaped Caudofoveata (=Chaetodermomorpha) is one of the least known groups of molluscs. The taxon consists of 141 recognized species found from intertidal environments to the deep-sea where they live burrowing in sediment. Evolutionary relationships of the group have been debated, but few studies based on morphological or molecular data have investigated the phylogeny of the group. Here we use molecular phylogenetics to resolve relationships among and within families of Caudofoveata. Phylogenetic analyses were performed using selected mitochondrial and nuclear genes from species from all recognized families of Caudofoveata. In resulting trees and contrary to traditional views, Prochaetodermatidae forms the sister clade to a clade containing the other two currently recognized families, Chaetodermatidae and Limifossoridae. The monophyly of Prochaetodermatidae is highly supported, but Limifossoridae and Chaetodermatidae are not recovered as monophyletic. Most of the caudofoveate genera are also not recovered as monophyletic in our analyses. Thus results from our molecular data suggest that the current classification of Caudofoveata is in need of revision, and indicate evolutionary scenarios that differ from previously proposed hypotheses based on morphology.

Rhachotropis (Eusiroidea, Amphipoda) from the North East Atlantic.

The genus Rhachotropis has the widest geographic and bathymetric distribution of all amphipod genera worldwide. Molecular and morphological investigations of specimens sampled around Iceland and off the Norwegian coast allow the first insights into the relationships of North East Atlantic Rhachotropis. The 31 cytochrome oxidase subunit I (COI) sequences generated for this study were assigned 13 Barcode Index Numbers (BINs) in the Barcode of Life database (BOLD), of which 12 are new to the database. Molecular analyses of COI and 16S sequences could not confirm a theory that depth has a greater influence on the phylogeny of Rhachotropis than geographic distance. Although the North East Atlantic is a well-studied area, our molecular investigations revealed the genus Rhachotropis may contain cryptic species, which indicates a higher biodiversity than currently known. For example, the specimens which key to Rhachotropis helleri is a complex of three COI clades, two of which cannot be identified with morphological traits. One specimen of each of the clades in the cladogram was documented by high definition photographs. A special focus was on the visual morphology of the eyes, as this character shows interspecific differences within the genus Rhachotropis in response to fixation in ethanol. Detailed morphological investigation showed that some clades thought to be indistinguishable can be separated by minute but consistent morphological characters. Datamining Genbank to examine all registered COI-sequences of R. aculeata, the only previously known Rhachotropis BIN in the North Atlantic and sub-Arctic, showed R. aculeata to be subdivided by an Arctic and a North Atlantic population.

Phylogenetic evidence that both ancient vicariance and dispersal have contributed to the biogeographic patterns of anchialine cave shrimps.

Cave shrimps from the genera Typhlatya, Stygiocaris and Typhlopatsa (Atyidae) are restricted to specialised coastal subterranean habitats or nearby freshwaters and have a highly disconnected distribution (Eastern Pacific, Caribbean, Atlantic, Mediterranean, Madagascar, Australia). The combination of a wide distribution and a limited dispersal potential suggests a large-scale process has generated this geographic pattern. Tectonic plates that fragment ancestral ranges (vicariance) has often been assumed to cause this process, with the biota as passive passengers on continental blocks. The ancestors of these cave shrimps are believed to have inhabited the ancient Tethys Sea, with three particular geological events hypothesised to have led to their isolation and divergence; (1) the opening of the Atlantic Ocean, (2) the breakup of Gondwana, and (3) the closure of the Tethys Seaway. We test the relative contribution of vicariance and dispersal in the evolutionary history of this group using mitochondrial genomes to reconstruct phylogenetic and biogeographic scenarios with fossil-based calibrations. Given that the Australia/Madagascar shrimp divergence postdates the Gondwanan breakup, our results suggest both vicariance (the Atlantic opening) and dispersal. The Tethys closure appears not to have been influential, however we hypothesise that changing marine currents had an important early influence on their biogeography.

When molecules support morphology: Phylogenetic reconstruction of the family Onuphidae (Eunicida, Annelida) based on 16S rDNA and 18S rDNA.

Onuphid polychaetes are tubicolous marine worms commonly reported worldwide from intertidal areas to hadal depths. They often dominate in benthic communities and have economic importance in aquaculture and recreational fishing. Here we report the phylogeny of the family Onuphidae based on the combined analyses of nuclear (18S rDNA) and mitochondrial (16S rDNA) genes. Results of Bayesian and Maximum Likelihood analyses supported the monophyly of Onuphidae and its traditional subdivision into two monophyletic subfamilies: Onuphinae and Hyalinoeciinae. Ten of 22 recognized genera were monophyletic with strong node support; four more genera included in this study were either monotypic or represented by a single species. None of the genera appeared para- or polyphyletic and this indicates a strong congruence between the traditional morphology-based systematics of the family and the newly obtained molecular-based phylogenetic reconstructions. Intergeneric relationships within Hyalinoeciinae were not resolved. Two strongly supported monophyletic groups of genera were recovered within Onuphinae: ((Onuphis, Aponuphis), Diopatra, Paradiopatra) and (Hirsutonuphis, (Paxtonia, (Kinbergonuphis, Mooreonuphis))). A previously accepted hypothesis on the subdivision of Onuphinae into the Onuphis group of genera and the Diopatra group of genera was largely rejected.

Mitogenomic sequences and evidence from unique gene rearrangements corroborate evolutionary relationships of myctophiformes (Neoteleostei).

BACKGROUND: A skewed assemblage of two epi-, meso- and bathypelagic fish families makes up the order Myctophiformes - the blackchins Neoscopelidae and the lanternfishes Myctophidae. The six rare neoscopelids show few morphological specializations whereas the divergent myctophids have evolved into about 250 species, of which many show massive abundances and wide distributions. In fact, Myctophidae is by far the most abundant fish family in the world, with plausible estimates of more than half of the oceans combined fish biomass. Myctophids possess a unique communication system of species-specific photophore patterns and traditional intrafamilial classification has been established to reflect arrangements of photophores. Myctophids present the most diverse array of larval body forms found in fishes although this attribute has both corroborated and confounded phylogenetic hypotheses based on adult morphology. No molecular phylogeny is available for Myctophiformes, despite their importance within all ocean trophic cycles, open-ocean speciation and as an important part of neoteleost divergence. This study attempts to resolve major myctophiform phylogenies from both mitogenomic sequences and corroborating evidence in the form of unique mitochondrial gene order rearrangements. RESULTS: Mitogenomic evidence from DNA sequences and unique gene orders are highly congruent concerning phylogenetic resolution on several myctophiform classification levels, corroborating evidence from osteology, larval ontogeny and photophore patterns, although the lack of larval morphological characters within the subfamily Lampanyctinae stands out. Neoscopelidae is resolved as the sister family to myctophids with Solivomer arenidens positioned as a sister taxon to the remaining neoscopelids. The enigmatic Notolychnus valdiviae is placed as a sister taxon to all other myctophids and exhibits an unusual second copy of the tRNA-Met gene - a gene order rearrangement reminiscent of that found in the tribe Diaphini although our analyses show it to be independently derived. Most tribes are resolved in accordance with adult morphology although Gonichthyini is found within a subclade of the tribe Myctophini consisting of ctenoid scaled species. Mitogenomic sequence data from this study recognize 10 reciprocally monophyletic lineages within Myctophidae, with five of these clades delimited from additional rearranged gene orders or intergenic non-coding sequences. CONCLUSIONS: Mitogenomic results from DNA sequences and unique gene orders corroborate morphology in phylogeny reconstruction and provide a likely scenario for the phylogenetic history of Myctophiformes. The extent of gene order rearrangements found within the mitochondrial genomes of myctophids is unique for phylogenetic purposes.

Ecology of twelve species of Thyasiridae (Mollusca: Bivalvia).

Benthic samples from coastal locations off Southwestern Norway were examined and the specimens of Thyasiridae were identified to species. A multivariate analysis based on 13 parameters was carried out and the environmental preferences of all thyasirid species present were determined. The potential of the Thyasiridae as indicators of organic enrichment was investigated by using direct canonical correspondence analyses to identify correlations between selected environmental parameters and the collected biological data. The presence of Thyasira sarsi together with a low biodiversity is a good indicator of organic enrichment. High thyasirid species diversity seems to indicate good environmental conditions, and single thyasirid species that lack symbiotic bacteria might also be useful as indicators of good environmental conditions.

Phylogenetic utility of five genes for dipteran phylogeny: a test case in the Chironomidae leads to generic synonymies.

In this study we examine the utility of three mitochondrial (COI, COII, 16S) and two nuclear (CAD and EF-1a) markers for estimating lower-level phylogenetic relationships within the dipteran family Chironomidae. As a test case we use species of the genus Micropsectra and the putatively closely related genera Krenopsectra, Parapsectra and Paratanytarsus. We also examine the phylogenetic evidence for the currently accepted species groups within the genus Micropsectra. In our results, highly variable EF-1a sequences within some species indicate the first find of paralogous gene copies in nematocerous Diptera. Among the other genes, COI is found to have the weakest while CAD contains the strongest phylogenetic signal. The resulting phylogeny displays a well-supported, but paraphyletic Micropsectra with regard to Krenopsectra acuta and five Parapsectra species, indicating taxonomic synonymy of these genera with 100% posterior probability. The genus Parapsectra is polyphyletic within Micropsectra while Paratanytarsus remains monophyletic although with low posterior probability. Micropsectra acuta, M. bumasta, M. fallax, M. nohedensis, M. mendli, M. uliginosa, M. chionophila, M. nana, M. styriaca and M. wagneri will all be new combinations as a consequence of the synonymy.

Vibrio tapetis-like strain isolated from introduced Manila clams Ruditapes philippinarum showing symptoms of brown ring disease in Norway.

The Manila clam Ruditapes philippinarum was introduced to Norway in 1987 and was produced in 2 hatcheries until 1991. Clam seed was planted at 6 sites. Two sites were on the Island of Tysnes, south of Bergen. Surviving adult Manila clams were recovered in 1995 and 1996. In the present study, Manila clams from the original seeding that displayed morphological signs of brown ring disease (BRD) were recovered in June 2003 (n=7) and in June 2004 (n=17). Samples from extrapallial fluid, tissues and haemolymph were inoculated on marine agar. Replicate subcultures on selective media were used to select potential Vibrio tapetis strains, and in total, 190 bacterial strains were isolated. One of these strains clustered within the V tapetis clade and was named NRP 45. DNA:DNA hybridisation with the type strain CECT4600 showed 52.7 and 57.3% DNA:DNA similarity. Hybridisation of NRP 45 and the V tapetis LP2 strain, isolated from corkwing wrasse Symphodus melops, produced 46.6 and 44.4% re-association. Partial gene segments encoding 16S rRNA, gyrase B protein (GyrB) and chaperonin 60 protein (Cpn60) were characterised and compared to CECT 4600. NRP 45 showed 5 differences in the 1416 nucleotides (nt) of the 16S rRNA encoding gene (99.6% similarity), while the GyrB encoding gene had 62 substitutions of 1181 nt compared (94.8% similarity) and the Cpn60 encoding gene had 22 substitutions out of 548 nt compared (96% similarity). This is the first finding of BRD and the first isolation of a V. tapetis-like bacterial strain from a bivalve in Norway.

The disunity of "Mysidacea" (Crustacea).

New studies on malacostracan relationships have drawn attention to issues concerning monophyly of the order Mysidacea, manifested in recent crustacean classifications that treat the taxon as two separate orders, Lophogastrida and Mysida. We present molecular phylogenies of these orders based on complete sequences of nuclear small-subunit ribosomal DNA (18S rRNA), and morphological evidence is used to revise the classification of the order Mysida to better reflect evolutionary history. A secondary structure model for 18S rRNA was constructed and used to assign putative stem and loop regions to two groups of partitions for phylogenetic analyses. Phylogenies were estimated by maximum-likelihood, Bayesian inference, and maximum-parsimony. The analyses gave strong support for three independently derived lineages, represented by three monophyletic groups, Lophogastrida, Stygiomysida, and Mysida. The family Petalophthalmidae is considered as sister group to the family Mysidae, and Boreomysinae and Rhopalophthalminae are the most early derived of the Mysidae. The tribes contained in the current classification of the subfamily Mysinae are not well-supported by either molecular data or morphology.

A comprehensive DNA sequence library is essential for identification with DNA barcodes.

In this study we examine the possibility of utilising partial cox1 gene sequences as barcodes to identify non-biting midges (Diptera: Chironomidae). We analysed DNA from 97 specimens of 47 species in the genera Cladotanytarsus, Micropsectra, Parapsectra, Paratanytarsus, Rheotanytarsus, Tanytarsus and Virgatanytarsus with a main focus on Micropsectra, Parapsectra and Paratanytarsus. Our findings show that (1) cox1 is easily amplified from extracts from different life stages with the standard barcoding primers. (2) Although K2P-distances between con-specific sequences varied up to 4.9%, con-specifics clustered together with 91-100% bootstrap support in maximum parsimony analysis. This indicates that barcodes may be excellent tools to identify species that are already in a cox1 library. (3) Both neighbour joining and maximum parsimony failed to reconstruct monophyletic genera. Thus, if a well-matching cox1 sequence is not already available in the library, the prospects of approximately identifying an unknown taxon, even to the correct genus of subtribe Tanytarsina, are not good.