Phylogeny of the genus Chironomus (Diptera) inferred from DNA sequences of mitochondrial cytochrome b and cytochrome oxidase I

Two mitochondrial genes, Cytochrome b (Cytb) and Cytochrome c oxidase subunit I (COI), have been used as phylogenetic markers in Chironomids. The nucleotide sequences of 685 bp from Cytb and 596 bp from COI have been determined for 36 Chironomus species from the Palearctic, or Holarctic, and Australasia. The concatenated sequence of 1281 bp from both genes was used to investigate the phylogenetic relationships among these species. The nucleotide sequence alignments were used for construction of phylogenetic trees based on maximum-parsimony and neighbor-joining methods. Both techniques produced similar phylogenies. Monophyly of the genus Chironomus is supported by a bootstrap value of 100% at the basal branch. Six clusters of species have been revealed with high bootstrap values supporting both monophyly of each cluster and the validity of the branching order within each cluster. Four species, C. circumdatus, C. nepeanensis, C. dorsalis, and C. crassiforceps, cannot be placed into any cluster. Cytological phylogenies were constructed using the same set of species, except for C. biwaprimus. These trees showed many similarities to that obtained from the mitochondrial (mt) sequence analysis, but also a number of significant differences. When compared with the tree constructed from the sequence of 23 species available for one of the globin genes, globin 2b (gb2b), there was better support for the mt tree than for the cytological trees. An intron, which varies in its occurrence and position in gb2b, was also investigated and the distribution of the introns supports the phylogenetic history of the genus Chironomus obtained with mt data. The differences observed in the cytological trees seem to be attributable more to the retention of the same chromosome banding sequence across several species, rather than convergent evolutionary events. An important question is the determination of the position of the subgenus Camptochironomus in relation to the representatives of the nominal subgenus Chironomus, since it has been suggested that this is a separate genus. The Camptochironomus species are internal to the trees and have arisen more recently than some of the species of the subgenus Chironomus, indicating that they are not sufficiently differentiated to be considered more than a subgenus.     

Phylogeny and life history evolution of the genus Chrysoritis within the Aphnaeini (Lepidoptera: Lycaenidae), inferred from mitochondrial cytochrome oxidase I sequences

Phylogenetic relationships among 26 South African species in the tribe Aphnaeini (Lepidoptera: Lycaenidae) were inferred from DNA characters of the mitochondrial gene cytochrome oxidase I (COI), using maximum-parsimony methods. The resulting phylogenetic estimate supports the systematic hypothesis made by Heath (1997, Metamorphosis, supplement 2), based on morphological characters, that at least three preexisting genera (Chrysoritis, Poecilmitis, and Oxychaeta) should be collapsed into the single monophyletic genus Chrysoritis. Two of the species groups described by Heath within Chrysoritis are also monophyletic, while one is paraphyletic and thus unsupported by the molecular data. Strong node support and skewed transition/transversion ratios suggest that two Chrysoritis clades contain synonymous species. Aphytophagy appears as a derived feeding strategy. Evolutionary patterns of ant association indicate lability at the level of ant genus, while association with different ant subfamilies may have played an ancestral and chemically mediated role in the diversification of South African aphnaeines.     

Phylogeny and historical biogeography of Agabinae diving beetles (Coleoptera) inferred from mitochondrial DNA sequences

The Agabinae, with more than 350 species, is one of the most diverse lineages of diving beetles (Dytiscidae). Using the mitochondrial genes 16S rRNA and cytochrome oxidase I we present a phylogenetic analysis based on 107 species drawn mostly from the four main Holarctic genera. Two of these genera (Ilybius and Ilybiosoma) are consistently recovered as monophyletic with strong support, Platambus is never recovered as monophyletic, and Agabus is found paraphyletic with respect to several of the species groups of Platambus. Basal relationships among the main lineages are poorly defined, although within each of them relationships are in general robust and very consistent across the parameter space, and in agreement with previous morphological analyses. In each of the two most diverse lineages (Ilybius and Agabus including part of Platambus) there is a basal split between Palearctic and Nearctic clades, estimated to have occurred in the late Eocene. The Palearctic clade in turn splits into a Western Palearctic clade and a clade containing mostly Eastern Palearctic species, and assumed to be ancestrally Eastern Palearctic but with numerous transitions to a Holarctic or Nearctic distribution. These results suggest an asymmetry in the colonization routes, as there are very few cases of transcontinental range expansions originating from the Nearctic or the Western Palearctic. According to standard clock estimates, we do not find any transcontinental shift during the Pliocene, but numerous speciation events within each of the continental or subcontinental regions.     

Phylogeny of the genus Aphis Linnaeus, 1758 (Homoptera: Aphididae) inferred from mitochondrial DNA sequences

Aphis is the largest aphid genus in the world and contains several of the most injurious aphid pests. It is also the most reluctant aphid genus to any comprehensive taxonomic treatment: while most species are easily classified into "species groups" that form well defined entities, numerous species within these groups are difficult to tell apart morphologically and identification keys remain ambiguous and mostly rely on host plant affiliation. In this paper, we used partial sequences of COI/COII and CytB genes to reconstruct the first phylogeny of Aphis and discuss the present systematics. The monophyly of the subgenus Bursaphis and of the tree major species groups, Black aphid, Black backed aphid and frangulae-like species was recovered by all phylogenetic analyses. However our data suggested that the nominal subgenus was not monophyletic. Relationships between major species groups were often ambiguous but "Black" and "Black backed" species groups appeared as sister clades. The most striking result of this study was that our molecular data met the same limits as the morphological characters used in classifications: mitochondrial DNA did not allow the differentiation of species that are difficult to identify. Further, interspecies relationships within groups of species for which taxonomic treatment is difficult stayed unresolved. This suggests that species delineation in the genus Aphis is often ambiguous and that diversification might have been a rapid process.     

Phylogeny of the mud worm genus Marenzelleria (Polychaeta, Spionidae) inferred from mitochondrial DNA sequences

The taxonomy of the cryptic morphospecies of the mud worm genus Marenzelleria is particularly difficult and the phylogenetic relationship within the genus is unknown. Herein we reconstructed the phylogeny of all five species of this genus using sequence data of three mitochondrial genes (16SrDNA, cytochrome b, cytochrome oxidase subunit I) from 104 specimens out of 26 populations. For the three invasive species of the genus, Marenzelleria neglecta, M. viridis and M. arctia , individuals from native populations as well as from recently invaded populations were included. Nuclear 18S rDNA sequences were used to evaluate the appropriate outgroup taxon among several spionid polychaete species. The results supported the monophyly of Marenzelleria , and Malacoceros fuliginosus was found to be a suitable outgroup for the analysis of the mitochondrial gene segments. All phylogenetic reconstructions revealed a basal position of M. arctia and M. wireni , which have primarily Arctic distribution, with M. arctia obtaining the most basal position. Together with the present-day distribution of the species, this indicates an origin of the genus in the Arctic region. The relationship of the species M. neglecta , M. viridis and M. bastropi could not be resolved sufficiently due to genealogical discordance that might reflect relatively young cladogenetic events.     

Phylogeny and evolution of Digitulati ground beetles (Coleoptera, Carabidae) inferred from mitochondrial ND5 gene sequences

Genealogical trees have been constructed using mitochondrial ND5 gene sequences of 87 specimens consisting of 32 species which have been believed to belong to the division Digitulati (one of the lineages of the subtribe Carabina) of the world. There have been recognized six lineages, which are well separated from each other. Each lineage contains the following genus: (1) the lineage A: Ohomopterus from Japan; (2) the lineage B: Isiocarabus from eastern Eurasian Continent; (3) the lineage C: Carabus from China which are further subdivided into three sublineages; (4) the lineage D: Carabus from USA; (5) the lineage E: Carabus from the Eurasian Continent, Japan and North America; and (6) the lineage F: Eucarabus from the Eurasian Continent. Additionally, the genus Acrocarabus which had been treated as a constituent of the division Archicarabomorphi has been recognized to be the 7th lineage of the division Digitulati from the ND5 genealogical analysis as well as morphology. These lineages are assumed to have radiated within a short period and are largely linked to their geographic distribution.     

Phylogeny of Australian Coptotermes (Isoptera: Rhinotermitidae) species inferred from mitochondrial COII sequences

Six Australian species of Coptotermes are traditionally recognized, but recent cuticular hydrocarbon studies suggest that some of these may represent more than one species. An understanding of the phylogenetic diversity of Australian Coptotermes, particularly the pest species, is likely to be important for the improvement of termite management strategies. A study of phylogenetic relationships among species of this genus was performed, based on the mitochondrial cytochrome oxidase (COII) gene, comparing the data with recent data from Asian species. Representatives of the species C. lacteus (Froggatt), C. frenchi Hill and C. michaelseni Silvestri were each found to form closely related monophyletic groups, however representatives of C. acinaciformis (Froggatt) were not. For C. acinaciformis, representatives from northern mound-building populations were found to form a distinct group to southern, tree-nesting forms. Among southern C. acinaciformis, two Western Australian representatives were found to be divergent from other populations. The results suggest that C. acinaciformis probably represents a complex of species rather than one, as has been suggested previously. One unidentified Coptotermes sp. taxon from Melbourne was found to be divergent from other taxa. Notably, some Australian species were more closely related to Asian species than other Australian species.     

Phylogeny of the eelpout genus Lycodes (Pisces,Zoarcidae) as inferred from mitochondrial cytochrome b and 12S rDNA

The bottom-dwelling and species-rich eelpout genus Lycodes Reinhardt has a great potential for the study of Arctic marine speciation. Subdivision of the genus has been based on single or few morphological characters (e.g., lateral line configuration) with contradicting results and phylogenetic approaches have not been attended. Here we present the first phylogenetic analysis of the genus employing DNA sequences of the mitochondrial genes cytochrome b and 12S rDNA (714 bp). The analysis with the two genes combined resulted in two equally parsimonious trees. In both cladograms most of the previously suggested subgroups are para- or polyphyletic, except for the so-called short-tailed Lycodes spp., with a short tail, a single mediolateral lateral line configuration and a shallow or filled otolith sulcus. The group of long-tailed Lycodes spp., with ventral or ventro-medio-lateral types of lateral line configuration and a deep otolith sulcus, appears to be paraphyletic, since Pacific and Atlantic species in this group are not each other's closest relatives. Thus, the short-tailed species are placed in a derived clade, indicating a secondary shortening of the tail, and a "slope to shore" type of evolution. This is not in accordance with earlier assumptions of the more elongate, deeper living species being the more derived. The basal position of long-tailed Pacific species supports earlier theories of Pacific origin of the genus/family. Small genetic differences between Arctic/Atlantic species indicate a rather recent radiation in these areas after the opening of the Bering Strait 3.0-3.5 million years ago.     

Phylogeny of the intertidal aleocharine tribe Liparocephalini (Coleoptera: Staphylinidae)

The tribe Liparocephalini (genera Liparocephalus Mäklin, 1853;Diaulota Casey, 1893 [ = Genoplectes Sawada, 1955];Paramblopusa Ahn & Ashe, Amblopusa Casey, 1893;Salinamexus Moore & Legner, 1977 [ = Biophytosus Moore & Legner, 1977]), all of which are exclusively restricted to the Pacific coasts of the Holarctic Region, is hypothesized to be a monophyletic group based on the following synapomorphies: seta v absent (inferred to be lost) from mentum (reversed one time in the Liparocephalus lineage), setae distributed only on mesal surface of galea and apex with setae, one medial seta present on prementum, and contiguous mesocoxal cavities. Natural history and history of the classification of the Liparocephalini are discussed. Phylogenetic analyses of the species of the Liparocephalini are presented based on larval (21 characters, 57 states), adult (49 characters, 115 states), and a combination of larval and adult characters (70 characters, 172 states). Analysis of the combined larval and adult data sets with successive approximation resulted in a single most parsimonious tree (length = 937, CI = 0.885, RI = 0.930) with the following patterns of generic relationships (outgroup (Salinamexus + Biophytosus (Amblopusa (Paramblopusa (Diaulota + Genoplectes, Liparocephalus))))).     

Molecular systematics of pikas (genus Ochotona) inferred from mitochondrial DNA sequences

The phylogenetic relationships among worldwide species of genus Ochotona were investigated by sequencing mitochondrial cytochrome b and ND4 genes. Parsimony and neighbor-joining analyses of the sequence data yielded congruent results that strongly indicated three major clusters: the shrub-steppe group, the northern group, and the mountain group. The subgeneric classification of Ochotona species needs to be revised because each of the two subgenera in the present classification contains species from the mountain group. To solve this taxonomic problem so that each taxon is monophyletic, i.e. , represents a natural clade, Ochotona could be divided into three subgenera, one for the shrub-steppe species, a second for the northern species, and a third for the mountain species. The inferred tree suggests that the differentiation of this genus in the Palearctic Region was closely related to the gradual uplifting of the Tibet (Qinghai-Xizang) Plateau, as hypothesized previously, and that vicariance might have played a major role in the differentiation of this genus on the Plateau. On the other hand, the North American species, O. princeps, is most likely a dispersal event, which might have happened during the Pliocene through the opening of the Bering Strait. The phylogenetic relationships within the shrub-steppe group are worth noting in that instead of a monophyletic shrub-dwelling group, shrub dwellers and steppe dwellers are intermingled with each other. Moreover, the sequence divergence within the sister taxa of one steppe dweller and one shrub dweller is very low. These findings support the hypothesis that pikas have entered the steppe environment several times and that morphological similarities within steppe dwellers were due to convergent evolution.     

Phylogeny of finescale shiners of the genus Lythrurus (Cypriniformes: Cyprinidae) inferred from four mitochondrial genes

We infer the phylogenetic relationships of finescale shiners of the genus Lythrurus, a group of 11 species of freshwater minnows widely distributed in eastern North America, using DNA sequences from the ND2 (1047 bp), ATPase8 and 6 (823 bp), and ND3 (421 bp) mitochondrial protein-coding genes. The topologies resulting from maximum parsimony, Bayesian, and maximum likelihood tree building methods are broadly congruent, with two distinct clades within the genus: the L. umbratilis clade (L. umbratilis + L. lirus + (L. fasciolaris + (L. ardens, L. matutinus))) and the L. bellus clade (L. fumeus + L. snelsoni + (L. roseipinnis + (L. atrapiculus + (L. bellus, L. algenotus)))). Support is weak at the base of several clades, but strongly supported nodes differ significantly from prior investigations. In particular, our results confirm and extend earlier studies recovering two clades within Lythrurus corresponding to groups with largely "northern" and "southern" geographic distributions. Several species in this genus are listed in the United States as threatened or of special concern due to habitat degradation or limited geographic ranges. In this study, populations assigned to L. roseipinnis show significant genetic divergence suggesting that there is greater genetic diversity within this species than its current taxonomy reflects. A full accounting of the biodiversity of the genus awaits further study.     

Ultrastructure and motility pattern of the spermatozoa of Aleochara curtula (Coleoptera, Staphylinidae)

Ultrastructure and motility pattern of spermatozoa of the rove beetle Aleochara curtula were examined using electron and light microscopic methods. The spermatozoon is about 100 microm long and filiform. The head piece comprises a 5 microm long triple layered acrosome and 10 microm long nucleus. The flagellum consists of a 9+9+2 axoneme, two accessory bodies and two mitochondrial derivatives about equal in size but of different shape in their cross sections. In both derivatives there are paracrystalline inclusions. The flagellum is attached to the head by a 2 microm long centriole adjunct which is characterized by its electron dense material that forms a three layered folded lamellar structure. When liberated in buffer solution the sperm flagella assume a coiled hook-like form with the excentric stiff head protruding in front. The spermatozoa are driven through the medium by a small helicoidal wave of high frequency superimposed to the bent flagella. The maximum speed measured was 15.2 microm/s. The sperm architecture of A. curtula is similar to that of other Aleochara species but differs in total length and dimensions of the mitochondrial derivatives. For that reason Aleochara sperm can certainly prove useful to study the effect of the mitochondrial derivatives on sperm motility.     

Numerous transposed sequences of mitochondrial cytochrome oxidase I-II in aphids of the genus Sitobion (Hemiptera: Aphididae)

Polymerase chain reaction (PCR) products corresponding to 803 bp of thecytochrome oxidase subunits I and II region of mitochondrial DNA (mtDNACOI-II) were deduced to consist of multiple haplotypes in three Sitobionspecies. We investigated the molecular basis of these observations. PCRproducts were cloned, and six clones from one individual per species weresequenced. In each individual, one sequence was found commonly, but alsotwo or three divergent sequences were seen. The divergent sequences wereshown to be nonmitochondrial by sequencing from purified mtDNA and Southernblotting experiments. All seven nonmitochondrial clones sequenced tocompletion were unique. Nonmitochondrial sequences have a high proportionof unique sites, and very few characters are shared betweennonmitochondrial clones to the exclusion of mtDNA. From these data, weinfer that fragments of mtDNA have been transposed separately (probablyinto aphid chromosomes), at a frequency only known to be equalled inhumans. The transposition phenomenon appears to occur infrequently or notat all in closely related genera and other aphids investigated. Patterns ofnucleotide substitution in mtDNA inferred over a parsimony tree are verydifferent from those in transposed sequences. Compared with mtDNA,nonmitochondrial sequences have less codon position bias, more evenexchanges between A, G, C and T, and a higher proportion of nonsynonymousreplacements. Although these data are consistent with the transposedsequences being under less constraint than mtDNA, changes in thenonmitochondrial sequences are not random: there remains significantposition bias, and probable excesses of synonymous replacements and ofconservative inferred amino acid replacements. We conclude that aproportion of the inferred change in the nonmitochondrial sequencesoccurred before transposition. We believe that Sitobion aphids (and otherspecies exhibiting mtDNA transposition) may be important for studying themolecular evolution of mtDNA and pseudogenes. However, our data highlightthe need to establish the true evolutionary relationships between sequencesin comparative investigations.     

Korean species of the genus Bisnius Stephens (Coleoptera: Staphylinidae)

Two Korean species of the genus Bisnius Stephens are studied. Bisnius macies (Sharp), found along the seashore of some islands, is introduced for the first time in Korea.     

Hydropy and ultrastructure of egg envelopes in Aleochara bilineata (Coleoptera, Staphylinidae)

Aleochara bilineata oviposits in soil microhabitats likely to contain the dipteran pupae that are hosts of its ectoparasitoid first instar larvae. The eggs of A. bilineata have a rigid chorion but they are nonetheless hydropic and, after 30 h of development, start to increase in volume and do so until 50 h. This increase in volume is due to absorption of water. The eggs increase their initial volume by a factor of 1.68 that corresponds to an increase of 44.44% of initial weight. To explain this augmentation in volume, we describe the modifications occurring in the egg chorion during hydropy. The increase in volume in such a rigid egg is made possible through the fragmentation of the chorion which, initially dense and regular, becomes fragmented. Such adaptation enables female A. bilineata to oviposit hydropic eggs in habitats where mechanical resistance is needed. Accepted: 29 October 2000     

Phylogeny of east Asian mitochondrial DNA lineages inferred from complete sequences

The now-emerging mitochondrial DNA (mtDNA) population genomics provides information for reconstructing a well-resolved mtDNA phylogeny and for discerning the phylogenetic status of the subcontinentally specific haplogroups. Although several major East Asian mtDNA haplogroups have been identified in studies elsewhere, some of the most basal haplogroups, as well as numerous minor subhaplogroups, were not yet determined or fully characterized. To fill the lacunae, we selected 48 mtDNAs from >2,000 samples across China for complete sequencing that cover virtually all (sub)haplogroups discernible to date in East Asia. This East Asian mtDNA phylogeny can henceforth serve as a solid basis for phylogeographic analyses of mtDNAs, as well as for studies of mitochondrial diseases in East and Southeast Asia.     

Phylogeny of magpies (genus Pica) inferred from mtDNA data

We investigated the phylogenetic relationships of species and subspecies of the cosmopolitan genus Pica using 813 bp of the mitochondrial genome (including portions of 16s rDNA, tRNA-Leu, and ND1). The phylogenetic relationships within the genus Pica revealed in our molecular analyses can be summarized as follows: (1). the Korean magpie (Pica pica sericea) appears basal within the genus Pica; (2). the European magpie (Pica pica pica) shows a close relationship to the Kamchatkan magpie (Pica pica camtschatica); (3). two North American species (Pica hudsonia and Pica nuttalli) shows a sister-group relationship; (4). most importantly, the European+Kamchatkan clade appears more closely related to the North American clade than to Korean magpies. Based on these results and genetic distance data, it is possible that members of an ancestral magpie lineage in East Asia initially moved north to form Kamchatkan magpies and then crossed the Bering land bridge to found North American taxa. At a later date, a group might have split off from Kamchatkan magpies and migrated west to form the Eurasian subspecies. The divergence between the two North American taxa appears to have happened no later than the divergence of Eurasian subspecies and both processes appear to have been relatively rapid. Rather than the formation of P. hudsonia by re-colonization from an Asian magpie ancestor, as suggested by, our data suggest a shared ancestry between P. hudsonia and P. nuttalli. Based on the above findings, including phylogenetic placement of P. hudsonia and P. nuttalli as nested within the larger Pica pica clade, and the lack of evidence suggesting reproductive isolation within the genus Pica, we believe that the current classification may be inaccurate. A more conservative classification would recognize one monophyletic species (i.e., P. pica) and treat P. nuttalli and P. hudsonia as subspecies (i.e., P. p. nuttalli and P. p. hudsonia). More extensive studies on the population genetics and biogeography of magpies should be conducted to better inform any taxonomic decisions.     

Phylogeny and molecular evolution of the tribe Harpalini (Coleoptera, Carabidae) inferred from mitochondrial cytochrome-oxidase I

The tribe Harpalini is a group of ground beetles with a world-wide distribution that comprises approximately 2000 species and about 238 genera and subgenera. Hypotheses about the phylogenetic relationships of the subtribes of Harpalini are implicit within the systematic criteria put forward by different authors. A 759 bp fragment of the mitochondrial COI was sequenced in 119 specimens (107 species) of 52 genera and subgenera that represent the main lineages of Harpalines, and 3 species of other tribes used as outgroups. A hierarchical study of sequence divergence (under uncorrected and corrected models) and ts:tv ratio pattern analyses were carried out at different taxonomic levels. A low saturation rate was detected at first and second codon positions, whereas A+T richness causes a low transitions:transversions ratio, which suggests--a priori--a high rate of saturation at the third codon position. A progressive accumulation of sequence divergence and a decreasing ts:tv ratio were found from lower to higher taxonomic levels. MP strict consensus, ML, and minimum evolution distance (under ts+tv and tv only schemes) trees showed similar major clades within the tribe. The subtribe Ditomina is a monophyletic lineage with close affinities to the subtribe Harpalina. Harpalina is a polyphyletic lineage as the genus Daptus is always related to members of the subtribe Stenolophina, and the Selenophorines resulted a polyphyletic group related to the subtribe Anisodactylina. Main lineages proposed by Noonan [Quaest. Entomol. 9 (1973) 266] within the subtribe Anisodactylina have been corroborated in this study. The Australian genus Phorticosomus is not related to Ditomina but to the Australian Notiobioids lineage. Most taxa of the subtribe Stenolophina are always included in the same clade, together with taxa of the subtribe Pelmatellina, which might be considered as a lineage of Stenolophina related to Bradycellus and Dicheirotrichus. The subtribe Amblystomina lacks a well-supported relationship to the other subtribes of Harpalini and could not be consistently related to any of them.