Mitochondrial genomes are readily sequenced with recent technology and thus evolutionary lineages can be densely sampled. This permits better phylogenetic estimates and assessment of potential biases resulting from heterogeneity in nucleotide composition and rate of change. We gathered 245 mitochondrial sequences for the Coleoptera representing all 4 suborders, 15 superfamilies of Polyphaga, and altogether 97 families, including 159 newly sequenced full or partial mitogenomes. Compositional heterogeneity greatly affected 3rd codon positions, and to a lesser extent the 1st and 2nd positions, even after RY coding. Heterogeneity also affected the encoded protein sequence, in particular in the nad2, nad4, nad5, and nad6 genes. Credible tree topologies were obtained with the nhPhyML (“nonhomogeneous”) algorithm implementing a model for branch-specific equilibrium frequencies. Likelihood searches using RAxML were improved by data partitioning by gene and codon position. Finally, the PhyloBayes software, which allows different substitution processes for amino acid replacement at various sites, produced a tree that best matched known higher level taxa and defined basal relationships in Coleoptera. After rooting with Neuropterida outgroups, suborder relationships were resolved as (Polyphaga (Myxophaga (Archostemata + Adephaga))). The infraorder relationships in Polyphaga were (Scirtiformia (Elateriformia ((Staphyliniformia + Scarabaeiformia) (Bostrichiformia (Cucujiformia))))). Polyphagan superfamilies were recovered as monophyla except Staphylinoidea (paraphyletic for Scarabaeiformia) and Cucujoidea, which can no longer be considered a valid taxon. The study shows that, although compositional heterogeneity is not universal, it cannot be eliminated for some mitochondrial genes, but dense taxon sampling and the use of appropriate Bayesian analyses can still produce robust phylogenetic trees.

We investigated whether small RNA (sRNA) sequenced from field-collected mosquitoes and chironomids (Diptera) can be used as a proxy signature of viral prevalence within a range of species and viral groups, using sRNAs sequenced from wild-caught specimens, to inform total RNA deep sequencing of samples of particular interest. Using this strategy, we sequenced from adult Anopheles maculipennis s.l. mosquitoes the apparently nearly complete genome of one previously undescribed virus related to chronic bee paralysis virus, and, from a pool of Ochlerotatus caspius and Oc. detritus mosquitoes, a nearly complete entomobirnavirus genome. We also reconstructed long sequences (1503-6557 nt) related to at least nine other viruses. Crucially, several of the sequences detected were reconstructed from host organisms highly divergent from those in which related viruses have been previously isolated or discovered. It is clear that viral transmission and maintenance cycles in nature are likely to be significantly more complex and taxonomically diverse than previously expected.

Mitochondrial genome sequences are important markers for phylogenetics but taxon sampling remains sporadic because of the great effort and cost required to acquire full-length sequences. Here, we demonstrate a simple, cost-effective way to sequence the full complement of protein coding mitochondrial genes from pooled samples using the 454/Roche platform. Multiplexing was achieved without the need for expensive indexing tags (‘barcodes’). The method was trialled with a set of long-range polymerase chain reaction (PCR) fragments from 30 species of Coleoptera (beetles) sequenced in a 1/16th sector of a sequencing plate. Long contigs were produced from the pooled sequences with sequencing depths ranging from ∼10 to 100× per contig. Species identity of individual contigs was established via three ‘bait’ sequences matching disparate parts of the mitochondrial genome obtained by conventional PCR and Sanger sequencing. This proved that assembly of contigs from the sequencing pool was correct. Our study produced sequences for 21 nearly complete and seven partial sets of protein coding mitochondrial genes. Combined with existing sequences for 25 taxa, an improved estimate of basal relationships in Coleoptera was obtained. The procedure could be employed routinely for mitochondrial genome sequencing at the species level, to provide improved species ‘barcodes’ that currently use the cox1 gene only.

Phylogeny of mosquitoes of tribe Culicini (Diptera: Culicidae) based on morphological diversity. -Zoologica Scripta, 41, 499-514. Relationships among taxa of the mosquito tribe Culicini are explored using 169 morphological characters from 86 exemplar species representing the four genera and 26 subgenera of Culicini, most species groups and subgroups of subgenus Culex and an outgroup of five species from five other tribes. We analysed the data set, with multistate characters treated as unordered, under implied weights with values of K ranging from 1 to 20, implemented by TNT. Each analysis, except K = 4, produced a single most parsimonious (fittest) cladogram (MPC). The topology of the ingroup was identical for K = 6-11, whereas the MPCs for K = 14-20 differed only in the position of a single species, which occupied the same position in the K = 16 and K = 6-11 topologies. The K = 9 and K = 16 trees were given further consideration. In both these cladograms, Lutzia is sister to a clade comprising genera Culex, Deinocerites and Galindomyia. The two topologies have 13 clades in common, but their arrangements differ primarily because of Culex (Culex) duttoni acting as a 'rogue' taxon. We evaluated the effect of removing this species from the K = 9 and K = 16 analyses and chose the refined K = 9 topology as the best hypothesis of relationships within Culicini. Genus Culex is not monophyletic because it includes Deinocerites and Galindomyia as derived members of the New World subgenera. With the exception of subgenera Culex, Eumelanomyia and Neoculex, there is strong support for the monophyly of all genera and subgenera. Subgenus Culex would be monophyletic were four other subgenera included and three other taxa (the Afrotropical Cx. duttoni, Neotropical Cx. apicinus and the Australasian Atriceps Group) excluded.

No abstract

To successfully implement surveillance or control strategies for mosquitoes, up-to-date knowledge of regional species composition is vital. The last report regarding mosquitoes (Diptera: Culicidae) in the Åland archipelago, southwestern Finland listed 19 species (Utrio, 1979). To determine the current species diversity, one collection trip was made to mainland Åland in 2015 and three in 2016. Mosquitoes (n = 3286) were collected as both adult and immature life stages from 88 collections within 29 1-km areas. Fifteen of the 19 previously reported species were obtained, leaving the current status of four species uncertain. At least 11 species previously not reported from Åland, but confirmed on the Finnish mainland, were collected. Aedes geminus Peus was identified based on examination of the gonostylus, and represents a new species distribution for Finland. Anopheles maculipennis s.s. Meigen was confirmed from cytochrome c oxidase subunit I (COI) and internal transcribed spacer 2 (ITS2) sequence data and is reinstated on the list of Finnish species, along with Ochlerotatus sticticus (Meigen). Dahliana geniculata (Olivier) was found in two locations, in 2 months, indicating that there is an established population in Åland. The present data confirm that at least 27 species inhabit mainland Åland, rising to 31 when historical data are included. The Finnish mosquito fauna is increased from 38 to 41 species.

Background Culicoides obsoletus is an abundant and widely distributed Holarctic biting midge species, involved in the transmission of bluetongue virus (BTV) and Schmallenberg virus (SBV) to wild and domestic ruminants. Females of this vector species are often reported jointly with two morphologically very close species, C. scoticus and C. montanus, forming the Obsoletus/Scoticus Complex. Recently, cryptic diversity within C. obsoletus was reported in geographically distant sites. Clear delineation of species and characterization of genetic variability is mandatory to revise their taxonomic status and assess the vector role of each taxonomic entity. Our objectives were to characterize and map the cryptic diversity within the Obsoletus/Scoticus Complex. Methods Portion of the cox1 mitochondrial gene of 3763 individuals belonging to the Obsoletus/Scoticus Complex was sequenced. Populations from 20 countries along a Palaearctic Mediterranean transect covering Scandinavia to Canary islands (North to South) and Canary islands to Turkey (West to East) were included. Genetic diversity based on cox1 barcoding was supported by 16S rDNA mitochondrial gene sequences and a gene coding for ribosomal 28S rDNA. Species delimitation using a multi-marker methodology was used to revise the current taxonomic scheme of the Obsoletus/Scoticus Complex. Results Our analysis showed the existence of three phylogenetic clades (C. obsoletus clade O2, C. obsoletus clade dark and one not yet named and identified) within C. obsoletus. These analyses also revealed two intra-specific clades within C. scoticus and raised questions about the taxonomic status of C. montanus. Conclusions To our knowledge, our study provides the first genetic characterization of the Obsoletus/Scoticus Complex on a large geographical scale and allows a revision of the current taxonomic classification for an important group of vector species of livestock viruses in the Palaearctic region.

Mosquitoes (Diptera: Culicidae) were collected in Finland between 2012 and 2018 to determine the species present and their distributions. In total, 52 466 specimens from 1031 collections formed the basis for the preparation of distribution maps for each of the 40 species that were collected. Anopheles maculipennis s.s., An. claviger, Aedes geminus and Ochlerotatus sticticus are confirmed on mainland Finland after previous records were uncertain or absent. Coquillettidia richiardii, Culiseta morsitans, Cs. ochroptera, Culex territans, Cx. torrentium, Oc. leucomelas, Oc. nigrinus, Oc. pullatus and Oc. punctodes occur more widely than previously reported. Three species, Ae. rossicus, Cs. subochrea and Oc. cyprius were not collected, although Ae. rossicus was subsequently found in Lapland by another researcher. No invasive species were collected. Ochlerotatus communis, an aggressive biter, was the most commonly encountered species. Larval collection data suggest that several species may have up to three generations per year in Finland, with Cx. torrentium and Cx. pipiens having at least two, and Oc. communis and Oc. punctor regularly found as larvae across the summer. These data, especially when coupled with historical records, are vital for monitoring species which have significant vector potential, particularly when faced with a warming climate.