The Anopheles albimanus white gene: molecular characterization of the gene and a spontaneous white gene mutation

We have cloned and characterized the white gene of Anopheles albimanus. Comparison of the deduced amino acid sequence of this white gene with its homologs from six species of Diptera show that the An. albimanus gene is most similar to the white gene of An. gambiae (92% identity). A spontaneous white-eyed mutant An. albimanus was caused by an approximately 10 kb insertion into a CT dinucleotide repeat region of intron 2 of the white locus. The flanks of this insertion are long (at least 400 bp), nearly perfect inverted terminal repeat sequences. This cloned white gene should be useful as a marker for germ line transformation of An. albimanus. This revised version was published online in July 2006 with corrections to the Cover Date.     

Two Corbicula (Corbiculidae: Bivalvia) mitochondrial lineages are widely distributed in Asian freshwater environment

We investigated the biogeography of Asian Corbicula using mitochondrial gene sequence variation for Corbicula members sampled from 24 localities of eight Asian regions. A total of 210 individuals were genetically characterized by examining sequence variations of a 614 bp fragment of the mitochondrial cytochrome oxidase I (COI) gene. Phylogenetic analyses of the COI dataset revealed that Corbicula members are subdivided into two well-supported clades: estuarine and freshwater. A robust dichotomy between the Japanese/Korean (Corbicula japonica) and Chinese (Corbicula fluminalis) estuarine forms was evident, suggesting that these two regional populations represent a deep phylogeographic split. Our mitochondrial gene tree showed that among the freshwater members, two Corbicula mitochondrial lineages are the most common, having attained extensive geographic distribution in the Asian freshwater environment. While the present study provides significant biogeographic information on Asian Corbicula, a comprehensive phylogenetic study by cross-referencing the mitochondrial-based Corbicula phylogeny with nuclear gene data is required to fully understand the evolutionary origin(s) of triploidy/clonality in this genus.     

High mitochondrial diversity in geographically widespread butterflies of Madagascar: A test of the DNA barcoding approach

The standardized use of mitochondrial cytochrome c oxidase subunit I (COI) gene sequences as DNA barcodes has been widely promoted as a high-throughput method for species identification and discovery. Species delimitation has been based on the following criteria: (1) monophyletic association and less frequently (2) a minimum 10× greater divergence between than within species. Divergence estimates, however, can be inflated if sister species pairs are not included and the geographic extent of variation within any given taxon is not sampled comprehensively. This paper addresses both potential biases in DNA divergence estimation by sampling range-wide variation in several morphologically distinct, endemic butterfly species in the genus Heteropsis, some of which are sister taxa. We also explored the extent to which mitochondrial DNA from the barcode region can be used to assess the effects of historical rainforest fragmentation by comparing genetic variation across Heteropsis populations with an unrelated forest-associated taxon Saribia tepahi. Unexpectedly, generalized primers led to the inadvertent amplification of the endosymbiont Wolbachia, undermining the use of universal primers and necessitating the design of genus-specific COI primers alongside a Wolbachia-specific PCR assay. Regardless of the high intra-specific genetic variation observed, most species satisfy DNA barcoding criteria and can be differentiated in the nuclear phylogeny. Nevertheless, two morphologically distinguishable candidate species fail to satisfy the barcoding 10× genetic distance criterion, underlining the difficulties of applying a standard distance threshold to species delimitation. Phylogeographic analysis of COI data suggests that forest fragmentation may have played an important role in the recent evolutionary diversification of these butterflies. Further work on other Malagasy taxa using both mitochondrial and nuclear data will provide better insight into the role of historical habitat fragmentation in species diversification and may potentially contribute to the identification of priority areas for conservation.     

DNA barcoding of endoparasitoid wasps in the genus Anicetus reveals high levels of host specificity (Hymenoptera: Encyrtidae)

The genus Anicetus includes economically important biocontrol agents that are introduced for control of soft and wax scale insect agricultural pests (Ceroplastes spp.). Understanding of host–parasitoid associations is critical to the successful outcome of their utilization in biological control projects. However, identification of these parasitoids is often difficult because of their small size and generally similar morphological features, and hence, studies on the host–parasitoid associations. Here, nucleotide sequence data were generated from the mitochondrial COI gene and the D2 region of 28S rRNA to assess genetic variation within and between species of Anicetus occurring in China. The results of this study support the use of the COI and the D2 region of 28S rRNA gene as useful markers in separating species of Anicetus, even in cases where morphological differences are subtle. On the other hand, the COI gene is also useful in recognizing species with much variation in morphology. DNA barcoding reveals high levels of host specificity of endoparasitoids wasps in the genus Anicetus. Our results indicate that each Anicetus species is adapted to a limited set of host species, or even are monospecific in their host choice.     

Celebrity with a neglected taxonomy: molecular systematics of the medicinal leech (genus Hirudo)

The medicinal leech is the most famous representative of the Hirudinea. It is one of few invertebrates widely used in medicine and as a scientific model object. It has recently been given considerable conservation effort. Despite all attention there is confusion regarding the taxonomic status of different morphological forms, with many different species described in the past, but only two generally accepted at present. The results of the phylogenetic analysis of a nuclear (ITS2+5.8S rRNA) and two mitochondrial gene sequences (12S rRNA, COI) suggest that the genus Hirudo is monophyletic. It consists, apart form the type Hirudo medicinalis and the East Asian Hirudo nipponia, of three other, neglected species. All of them have already been described either as species or morphological variety, and can readily be identified by their coloration pattern. The type species is in weakly supported sister relation with Hirudo sp. n. (described as variety orientalis) from Transcaucasia and Iran. Sister to them stands Hirudo verbana from southeastern Europe and Turkey, which is nowadays predominantly bred in leech farms and used as 'medicinal leech.' The North African Hirudo troctina is the sister taxon to this group of Western Eurasian species, whereas the basal split is between H. nipponia and the Western Palaearctic clade.     

Cophylogeny and disparate rates of evolution in sympatric lineages of chewing lice on pocket gophers

Although molecular-based phylogenetic studies of hosts and parasites are increasingly common in the literature, no study to date has examined two congeneric lineages of parasites that live in sympatry on the same lineage of hosts. This study examines phylogenetic relationships among chewing lice (Phthiraptera: Trichodectidae) of the Geomydoecus coronadoi and Geomydoecus mexicanus species complexes and compares these to phylogenetic patterns in their hosts (pocket gophers of the rodent family Geomyidae). Sympatry of congeneric lice provides a natural experiment to test the hypothesis that closely related lineages of parasites will respond similarly to the same host. Sequence data from the mitochondrial COI and the nuclear EF-1alpha genes confirm that the two louse complexes are reciprocally monophyletic and that individual clades within each species complex parasitize a different species of pocket gopher. Phylogenetic comparisons reveal that both louse complexes show a significant pattern of cophylogeny with their hosts. Comparisons of rates of nucleotide substitution at 4-fold degenerate sites in the COI gene indicate that both groups of lice have significantly higher basal mutation rates than their hosts. The two groups of lice have similar basal rates of mutation, but lice of the G. coronadoi complex show significantly elevated rates of nucleotide substitution at all sites. These rate differences are hypothesized to result from population-level phenomena, such as effective population size, founder effects, and drift, that influence rates of nucleotide substitution.     

In Memoriam: Professor Walter James Harman, PhD (1928–2002)

The Mediterranean spiny lobsters, the common spiny lobster Palinurus elephas (Fabricius, 1758) and the pink spiny lobster P. mauritanicus Gruvel, 1911, are important target species for commercial fisheries. In this study, we focus our attention on the DNA sequence variation of the mitochondrial cytochrome oxydase I gene (COI) in the two species of Palinurus. Spiny lobster DNA samples from four Mediterranean localities were analysed to examine the genetic variability at both the intra- and interspecific level. Furthermore, the phylogenetic relationships within the family Palinuridae (among the two species of Palinurus, most of the species of Panulirus and all the species of Jasus) are examined.     

Mitochondrial cytochrome C oxidase subunit I of Manduca sexta and a comparison with other invertebrate genes

A cDNA encoding mitochondrial cytochrome c oxidase subunit I (mt COI) from Manduca sexta (Lepidoptera: Sphingidae) was cloned and sequenced. AT (adenine-thymine) content is high and codon usage is biased and likely reflects the role of mt COI in electron transport. The encoded protein is 514 amino acids long, contains seven invariant His residues observed in COIs in all organisms and would be predicted to be composed of 12 transmembrane regions.     

A PCR-based diagnostic tool for distinguishing grape skin color mutants

Berry skin color mutants are phenotypically different from their original cultivars, but they show identical molecular profile if analysed by using microsatellite markers. This work gives an easy, inexpensive and quick diagnostic tool to discriminate these somatic variants. We distinguished some grape (Vitis vinifera L.) skin color mutants from white to red or pink and from black to grey, pink or white and we investigated their molecular bases by single-strand conformational polymorphism (SSCP), single base primer extension and coding sequence analysis of anthocyanin biosynthetic enzyme genes and by polymerase chain reaction (PCR) analysis of VvmybA1 regulatory gene. Analyses of structural genes did not reveal polymorphisms between wild type and mutant cultivars but only among different varieties, whereas the study of VvmybA1 regulatory gene has given important outcomes for color mutants characterisation. The discrimination between white wild type and its derived colored mutant and between black wild type and white mutant has been obtained through a simple test of amplification for presence/absence. The discrimination between black wild type and less colored mutant has occurred through a quantitative result on agarose gel confirmed by real-time PCR analysis: the amount of functional allele in less colored somatic variants genome was about one-fourth of the correspondent quantity in original black cultivars genome.     

Taxonomic re-evaluation of the Taiwanese montane earthworm Amynthas wulinensis Tsai,Shen & Tsai, 2001 (Oligochaeta: Megascolecidae): Polytypic species or species complex?

Body size and colouration are two characters commonly used in the taxonomy of many animal taxa. However, they are seldom used by earthworm taxonomists because they are subject to environmental influences and tend to vary intraspecifically. In the present study, DNA sequences of the mitochondrial COI gene are used to evaluate whether specimens of the megascolecid earthworm Amynthas wulinensis Tsai, Shen & Tsai, 2001 that differ in body size and/or colouration belong to different genetic lineages. Phylogenetic analyses and morphological comparisons indicate that A. wulinensis in the previous broad sense is a species complex composed of three species differing in body size, colouration, and genital markings. Consequently, two new species, Amynthas lini and Amynthas meishanensis, are described. Taxonomic affinities of the A. wulinensis species complex are discussed, as is the feasibility of using body size and colouration in earthworm taxonomy.     

Phylogeography of Asian weaver ants, Oecophylla smaragdina

The phylogeography of Oecophylla smaragdina was studied using the mitochondrial cytochrome b gene (Cytb), cytochrome oxidase subunit I (COI), and nuclear long-wavelength opsin gene (LW Rh). Weaver ants were collected from 35 localities and from one to nine colonies per locality. Neighbor-joining trees inferred from 647 bp of Cytb and 1,026 bp of COI using Oecophylla longinoda as an outgroup indicated that the haplotypes of O. smaragdina were clearly separated into seven groups: group 1 of India excluding West Bengal; group 2 of Bengal, Indochinese Peninsula, Malay Peninsula and Greater Sunda Islands, including Lombok and Sumbawa; group 3 of the Philippines; group 4 of Flores; group 5 of Sulawesi; group 6 of Halmahera; and group 7 of New Guinea and Australia. This grouping was also supported by a strict consensus tree derived from maximum parsimony and maximum likelihood trees. In addition, two haplotypes of LW Rh were found in O. smaragdina: one in group 2 and another in all the other groups. Comparison to haplotypes in other hymenopteran species suggests that group 2 is younger than other groups of O. smaragdina. The clustering of the seven groups was coincident with geological evidence of the distribution of continents, islands, and seas during glacial periods.     

Evolution, biogeography, and the utility of mitochondrial 16s and COI genes in phylogenetic analysis of the crab genus Austinixa (Decapoda: Pinnotheridae)

This study used molecular data (mitochondrial 16s and COI) for the first time to explore evolutionary relationships among species of the pinnotherid crab genus Austinixa. Low levels of phylogenetic signal were detected for COI. High levels of phylogenetic signal were detected for 16s, indicating it is a more useful marker for inferring species level phylogenies in Austinixa. Phylogeographic patterns among species of Austinixa are consistent with allopatric speciation due to numerous climatic and oceanographic fluctuations during the last 5-6 my. In addition, all but two species have been derived since the closure of the Isthmus of Panama, a pattern consistent with hypotheses that the marine biota of the Caribbean and southeastern North America underwent a pulse of biotic turnover within the last 2-3 my. Austinixa aidae and Austinixa hardyi had identical 16s sequences, and differed by only 2 bp in COI, raising questions about the validity of A. hardyi as a distinct species.     

Molecular phylogenetic investigations of the Viviparidae (Gastropoda: Caenogastropoda) in the lakes of the Rift Valley area of Africa

The freshwater gastropod family Viviparidae is nearly cosmopolitan, but absent from South America. On the African continent, two genera are recognized; the widespread Bellamya and the monotypic Neothauma, which is confined to Lake Tanganyika. Most of the African Bellamya species are confined to the major lakes of the Rift Valley area in Africa, i.e. Lake Albert, Lake Malawi, Lake Mweru, and Lake Victoria. The phylogenetic analyses of mitochondrial (COI and 16S) and nuclear (H3, 18S and 28S) DNA inferred three major lake-clades; i.e. Lake Victoria/Kyoga/Albert, Lake Malawi and Lake Mweru/Bangweulu. The endemic B. rubicunda from Lake Albert and B. unicolor from Lake Kyoga were inferred to be part of the Lake Victoria clade. Bellamya capillata as identified by shell characters was polyphyletic in gene trees. The monophyletic Bellamya species radiation in Lake Malawi was most nearly related to the Lake Victoria/Kyoga/Albert-clade. Taxa from the Zambian lakes, Mweru and Bangweulu, were inferred together and placed ancestral to the other lakes. Neothauma tanganyicense was inferred as the sister-group to the Zambian Bellamya. Within the lake-clades the endemic radiations show very low genetic diversities (0–4.1% in COI), suggesting much faster morphological divergence than molecular divergence. Alternatively, Bellamya in Africa constitutes only a few species with several sub-species or eco-phenotypic morphs. The African viviparids were inferred to be the sister-group to a clade comprising Asian species, and the relatively low genetic diversity between the clades (12.6–15.5% in COI) makes a recent Miocene dispersal event from Asia to Africa much more likely than an ancient Gondwana vicarience distribution.     

The white-phase-specific gene<Emphasis Type="Italic"> WH11</Emphasis> is not required for white-opaque switching in<Emphasis Type="Italic"> Candida albicans</Emphasis>

Phenotypic switching between white and opaque cells is important for adaptation to different host environments and for mating in the opportunistic fungal pathogen Candida albicans. Genes that are specifically activated in one of the two cell types are likely to be important for their phenotypic characteristics. The WH11 gene is a white-phase-specific gene that has been suggested to be involved in the maintenance of the white-phase phenotype. To elucidate the role of WH11 in white-opaque switching, we constructed mutants of the C. albicans strain WO-1 in which the WH11 gene was deleted. The wh11 mutants were still able to form both white and opaque cells whose cellular and colony phenotypes were indistinguishable from those of the wild type. Deletion of WH11 also did not affect the activation and deactivation of the white-phase-specific WH11 promoter and the opaque-phase-specific OP4 and SAP1 promoters in the appropriate cell type. Finally, switching from the white to the opaque phase and vice versa occurred with the same frequency in wild-type and wh11 mutants. Therefore, the WH11 gene is not required for phenotypic switching, and its protein product seems to have other roles in white cells, which are dispensable after the switch to the opaque phase.Communicated by E. Cerdá-Olmedo     

The white-line-in-agar test is not specific for the two cultivated mushroom associated pseudomonads, Pseudomonas tolaasii and Pseudomonas "reactans"

A sharply defined white line in vitro forms between the pathogenic form of Pseudomonas tolaasii and another Pseudomonas bacterium, referred to as "reactans". This interaction has been considered as highly specific. However, results presented in this study rise doubt about the strict specificity of this interaction, as some other pseudomonads, associated with the cultivated mushroom Agaricus bisporus, also yielded a white line precipitate when they were streaked towards Pseudomonas tolaasii LMG 2342T. Moreover, some Finnish isolates inducing brown blotch symptoms on mushrooms like P. tolaasii(T), produced a typical white precipitate when streaked towards P. "reactans" LMG5329, even though phenotypical and genotypical features exclude these isolates from the species P. tolaasii. We propose that the white-line-in-agar (WLA) test should no longer be considered as an unequivocal diagnostic trait of P. tolaasii.     

Molecular phylogeny of Allograpta (Diptera, Syrphidae) reveals diversity of lineages and non-monophyly of phytophagous taxa

Phylogenetic relationships of genera Allograpta, Sphaerophoria and Exallandra (Diptera, Syrphidae) were analyzed based on sequence data from the mitochondrial protein-coding gene cytochrome c oxidase subunit I (COI) and the nuclear 28S and 18S ribosomal RNA genes. The three genera are members of the subfamily Syrphinae, where nearly all members feed as larvae on soft-bodied Hemiptera and other arthropods. Phytophagous species have recently been discovered in two subgenera of Allograpta, sg Fazia and a new subgenus from Costa Rica. Phylogenetic analyses of the combined datasets were performed using parsimony, under static alignment and direct optimization, maximum likelihood and Bayesian inference. Congruent topologies obtained from all the analyses indicate paraphyly of the genus Allograpta with respect to Sphaerophoria and Exallandra. Exallandra appears embedded in the genus Sphaerophoria, and both genera are placed within Allograpta. The distribution of phytophagous taxa in Allograpta indicates that plant feeding evolved at least twice in this group.     

Intraspecific diversity of the Cassava Green Mite Mononychellus progresivus (Acari: Tetranychidae) using comparisons of mitochondrial and nuclear ribosomal DNA sequences and cross-breeding

Intraspecific diversity in Cassava Green Mite Mononychellus progresivus Doreste was examined using individuals collected in Benin and the Congo and in Columbia and Brazil. Comparisons were based on mitochondrial and ribosomal DNA sequences and the results of several cross-breeding experiments. Sequence variation was examined in a total of 1139 base pairs (bp) constituting the ITS2 ribosomal DNA (805 bp) and a fragment of the Cytochrome Oxidase I (COI) gene (334 bp). Sequence divergence is low, ranging from 0% to 2.1% for COI and from 0% to 0.4% for ITS2. Inter-strain comparisons have shown that the two African populations appear to be identical. They were similar to the Colombian population while the Brazilian population was clearly different. The data support the hypothesis of a single introduction of the species in the two African populations. Crossing experiments have shown partial hybrid sterility, suggesting a genetic incompatibility consistent with differences detected by sequence data. The results show the usefulness of molecular markers as a tool for determining taxonomic status and dispersion paths in spider mites.     

Hyperparasitism by Mesochorus spp. (Hymenoptera: Ichneumonidae) in Peristenus sp. (Hymenoptera: Braconidae) and development of PCR primers for hyperparasitoid detection

Peristenus sp. pupae collected from Lygus spp. nymphs in 2001 and 2002 were over-wintered in the laboratory. In both years, more than 30% of adults emerging from over-wintering pupae were identified as ichneumonid hyperparasitoids, Mesochorus curvulus Thomson and Meschorus sp. (Hymenoptera: Ichneumonidae). At the end of the over-wintering period, Peristenus sp. males emerged first followed by Peristenus sp. females and finally Mesochorus spp. The male:female ratio in emerging Peristenus sp. adults was skewed towards males. The Internal Transcribed Spacer (ITS) region and the cytochrome oxidase I (COI) gene from Mesochorus spp. were sequenced. ITS sequences were used to develop PCR primers to detect Mesochorus spp. hyperparasitism in the primary host, Lygus spp. PCR analysis of field-collected Lygus spp. nymphs gave similar estimates of Mesochorus spp. hyperparasitism to the rearing protocols (25–28%). Sequence analysis of COI and ITS regions and subsequent restriction endonuclease analysis of ITS PCR products from Mesochorus spp. indicate the presence of two genotypes in the population. The possibility that these two genotypes represent separate or cyrptic species is discussed.     

Planarian mitochondria I. Heterogeneity of cytochrome c oxidase subunit I gene sequences in the freshwater planarian,Dugesia japonica

Summary We have detected sequence heterogeneity in the cytochrome c oxidase subunit I (COI) gene of a freshwater planarian, Dugesia japonica, collected in one locality. A part of the COI gene was amplified via the polymerase chain reaction (PCR) using template DNA prepared from a mixture of 500 individuals or from each of 18 individuals. Analyses of DNA sequences by standard strategies for cloning and sequencing or by direct sequencing clearly show that (1) considerable sequence heterogeneity exists in DNA prepared from the mixed individuals, (2) 11 individuals have almost identical sequences (type A), and (3) 7 individuals have sequences different from one another (Seq-D 1 to SeqD7; collectively called type D). Each of the Seq-D1-D7 sequences except for Seq-D5 shows some heterogeneity even in a single individual (heteroplasmy). A possible cause of the sequence heterogeneities is discussed.Offprint requests to: Y. Bessho