Existence of two strains of <Emphasis Type="Italic">Habrobracon hebetor</Emphasis> (Hymenoptera: Braconidae): a complex in Thailand and Japan

Habrobracon hebetor Say (Hymenoptera: Braconidae) is a cosmopolitan gregarious ectoparasitoid that attacks larvae of several species of Lepidoptera. Although there are two genetically different strains within H. hebetor, distribution of the strains has been poorly understood. In 2010, in Thailand, where H. hebetor has been known as a parasitoid of stored grain pests, it was found that H. hebetor attacked Opisina arenosella Walker (Lepidoptera: Oecophoridae), which is an invasive pest of coconut palm. For correct identification of this H. hebetor, we conducted DNA analysis and cross tests using populations collected from O. arenosella and stored grain pests in Thailand and populations in Japan known as H. hebetor. We obtained 413 bp of mitochondrial cytochrome oxidase I (COI) sequences and 414 bp of 16S rRNA gene sequences, and both indicated that there are two distinct clades within H. hebetor: one contains insects from Thailand, Spain, India, and Barbados; the other contains insects from Japan and the USA. There were no genetic differences or sexual isolation between Thai populations from different hosts. Our results also showed that populations in Thailand were sexually isolated from a H. hebetor population in Japan.     

A new species of the genus <Emphasis Type="Italic">Morellia</Emphasis> Robineau-Desvoidy (Diptera: Muscidae) from Yunnan,China, with analysis of available DNA barcoding sequences

A new species of the genus Morellia Robineau-Desvoidy, 1830, Morellia (Morellia) trifurcata sp. n., collected from Yunnan, China is described. Four DNA sequences of the partial mitochondrial cytochrome c oxidase subunit I (mtCOI) gene of this new species are provided. In order to evaluate the availability of DNA barcoding for identifying Morellia species, 38 currently available, non-identical COI sequences of 16 Morellia species are involved in a molecular analysis using the neighbor-joining (NJ) method. The intra- and interspecific p-distances are summarized.     

Pelagic larval dispersal habits influence the population genetic structure of clam <Emphasis Type="Italic">Gomphina aequilatera</Emphasis> in China

Pelagic larval dispersal habits influence the population genetic structure of marine mollusk organisms via gene flow. The genetic information of the clam Gomphina aequilatera (short larval stage, 10 days) which is ecologically and economically important in the China coast is unknown. To determine the influence of planktonic larval duration on the genetic structure of G. aequilatera. Mitochondrial markers, cytochrome oxidase subunit i (COI) and 12S ribosomal RNA (12S rRNA), were used to investigate the population structure of wild G. aequilatera specimens from four China Sea coastal locations (Zhoushan, Nanji Island, Zhangpu and Beihai). Partial COI (685 bp) and 12S rRNA (350 bp) sequences were determined. High level and significant F_ST values were obtained among the different localities, based on either COI (F_ST?=?0.100–0.444, P?<?0.05) or 12S rRNA (F_ST?=?0.193–0.742, P?<?0.05), indicating a high degree of genetic differentiation among the populations. The pairwise N_m between Beihai and Zhoushan for COI was 0.626 and the other four pairwise N_m values were >?1, indicating extensive gene flow among them. The 12S rRNA showed the same pattern. AMOVA test results for COI and 12S rRNA indicated major genetic variation within the populations: 77.96% within and 22.04% among the populations for COI, 55.73% within and 44.27% among the populations for 12S rRNA. A median-joining network suggested obvious genetic differentiation between the Zhoushan and Beihai populations. This study revealed the extant population genetic structure of G. aequilatera and showed a strong population structure in a species with a short planktonic larval stage.     

Complete mitochondrial genome of <Emphasis Type="Italic">Ampittia dioscorides</Emphasis> (Lepidoptera: Hesperiidae) and its phylogenetic analysis

The complete mitochondrial genome of Ampittia dioscorides (Lepidoptera: Hesperiidae) was determined. The sequenced genome is a circular molecule of 15313 bp, containing 13 protein-coding genes (PCGs), 22 tRNA genes, two rRNA genes, and an A + T-rich region. The gene arrangements and transcribing directions are identical to those in most of the reported lepidopteran mitogenomes. The base composition of the whole genome and genes or regions are also similar to those in other lepidopteran species. All the PCGs are initiated by typical ATN codons; the exception being COI, which begins with a CGA codon. Eight genes (ND2, ATPase8, ATPase6, COIII, ND5, ND4L, ND6, and Cytb) end with a TAA stop codon, and two genes (ND1 and ND3) end with TAG. The remaining three genes (COI and COII, which end with TA-, and ND4, which ends with T-) have incomplete stop codons. All tRNAs have the typical clover-leaf structure of mitochondrial tRNAs, with the exception of tRNA^Ser(AGY). On the basis of the concatenated nucleotide and amino acid sequences of the 13 PCGs and wingless gene of 22 butterfly species, maximum parsimony (MP) and Bayesian inference (BI) trees were constructed, respectively. Both MP and BI trees had the same topological structure: ((((Nymphalidae + Danaidae) + Lycaenidae) + Pieridae) + Papilionidae) + Hesperiidae). The results provide support for Hesperiidae as a superfamily-level taxon.     

Taxonomic position of the family Porcellanasteridae within the class Asteroidea

The phylogenetic relationships and taxonomic position of sea stars of the family Porcellanasteridae have been a subject of debate for over a hundred years. In this paper, the methods of molecular phylogenetic analysis were used to solve these issues. The nucleotide sequences of fragments of the mitochondrial gene of subunit 1 of cytochrome oxidase (COI) and those of the 16S rRNA gene were obtained for 9 and 6 species of porcellanasterids for the first time, respectively. The phylogenetic trees including these sequences indicate the nearest relationships of the family Porcellanasteridae with the families Ctenodiscidae and Goniopectinidae, thereby supporting the validity of the allocation of these three families in the suborder Cribellina.     

Molecular genetic analysis of the suprageneric system of casebearer moths,with a description of a new genus from the tribe Carpochenini Cǎpuşe, 1973 (Lepidoptera,Coleophoridae)

Based on a complex approach including morphological and molecular genetic methods, the taxonomic position of two casebearer moth species Coleophora tsherkesi Falkovitsh, 1970 and C. isabellina Falkovitsh, 1970 (Lepidoptera, Coleophoridae) is discussed. On the phylogenetic tree based on the cytochrome oxidase subunit I gene (COI), two species form a common cluster with species of the genera Ionescumia, Carpochena, and Goniodoma. Data on the molecular phylogeny and morphology justify the placement of Coleophora tsherkesi and C. isabellina in a separate genus Falkmisa Anikin et Dyomin, gen. n. Thus, the tribe Carpochenini includes three genera: Ionescumia, Carpochena, and Falkmisa.     

Molecular genetic markers of intra- and interspecific divergence within starfish and sea urchins (Echinodermata)

A fragment of the mitochondrial COI gene from isolates of several echinoderm species was sequenced. The isolates were from three species of starfish from the Asteriidae family (Asterias amurensis and Aphelasterias japonica collected in the Sea of Japan and Asterias rubens collected in the White Sea) and from the sea urchin Echinocardium cordatum (family Loveniidae) collected in the Sea of Japan. Additionally, regions including internal transcribed spacers and 5.8S rRNA (ITS1–5.8S rDNA–ITS2) were sequenced for the three studied starfish species. Phylogenetic analysis of the obtained COI sequences together with earlier determined homologous COI sequences from Ast. forbesii, Ast. rubens, and Echinocardium laevigaster from the North Atlantic and E. cordatum from the Yellow and North Seas (GenBank) placed them into strictly conspecific clusters with high bootstrap support (99% in all cases). Only two exceptions–Ast. rubens DQ077915 sequence placed with the Ast. forbesii cluster and Aph. japonica DQ992560 sequence placed with the Ast. amurensis cluster–were likely results of species misidentification. The intraspecific polymorphism for the COI gene within the Asteriidae family varied within a range of 0.2-0.9% as estimated from the genetic distances. The corresponding intrageneric and intergeneric values were 10.4-12.1 and 21.8-29.8%, respectively. The interspecific divergence for the COI gene in the sea urchin of Echinocardium genus (family Loveniidae) was significantly higher (17.1-17.7%) than in the starfish, while intergeneric divergence (14.6-25.7%) was similar to that in asteroids. The interspecific genetic distances for the nuclear transcribed sequences (ITS1–5.8S rDNA–ITS2) within the Asteriidae family were lower (3.1-4.5%), and the intergeneric distances were significantly higher (32.8-35.0%), compared to the corresponding distances for the COI gene. These results suggest that the investigated molecular-genetic markers could be used for segregation and identification of echinoderm species.     

Genetic variability of wildlife-derived <Emphasis Type="Italic">Sarcoptes scabiei</Emphasis> determined by the ribosomal ITS-2 and mitochondrial 16S genes

Infestation by the ectoparasitic mite Sarcoptes scabiei (Acari: Sarcoptidae) has important implications for global wildlife conservation and both animal and human health. Ribosomal and mitochondrial DNA sequences of parasites are useful to determine genetic diversity and to describe their likely dynamic evolution. In this study, we described the genetic diversity of S. scabiei individuals collected from wild animals in China by sequencing the ribosomal ITS-2 and mitochondrial 16S rRNA genes. A total of 13 Sarcoptes isolates of wildlife, coupled with one of rabbit origin, were subjected to genetic characteristics. After cloning and sequencing, 14 ITS-2 sequences and 12 16S rRNA sequences were obtained and analyzed. Further analysis of haplotype network and population genetic structure revealed that there were 79 haplotypes in ITS-2 (main haplotype H2) and 31 haplotypes in 16S rRNA (main haplotype C10). The phylogenetic trees showed some partial clustering by location and host, and the analysis of gene polymorphism may prompt that all isolates of S. scabiei have a similar origin. We speculate that the genetic evolution of S. scabiei may be related with that of the hosts, but more research is necessary to better understand the host-parasite co-evolutionary relationship in S. scabiei. These results provide new insights into understanding the population genetics and evolutionary biology of S. scabiei and therefore a better understanding of controlling its infestation pathways worldwide.     

Genotypic characterization of Lactic acid bacteria in gut microbiome of freshwater fish

The present study focused on identification and genotypic characterization of Lactic acid bacteria (LAB) in the intestine of freshwater fish. 76 strains of LAB were isolated and identified by 16S rRNA gene sequences and hsp60 gene sequences as different strains of Lactobacillus plantarum, Lactobacillus pentosus, Lactobacillus fermentum, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus brevis, Lactobacillus reuteri, Lactobacillus salivarius, Pediococcus pentosaceus, Pediococcus acidilactici, Weissella paramesenteroides, Weissella cibaria, Enterococcus faecium, and Enterococcus durans. The hsp60 gene showed a higher level of sequence variation among the isolates examined, with lower interspecies sequence similarity providing more resolutions at the species level than the 16S rRNA gene. Phylogenetic tree derived from hsp60 gene sequences with higher bootstrap values at the nodal branches was more consistent as compared to phylogenetic tree constructed from 16S rRNA gene sequences. Closely related species L. plantarum and L. pentosus as well as species L. delbrueckii subsp. bulgaricus and L. fermentum were segregated in different cluster in hsp60 phylogenetic tree whereas such a distribution was not apparent in 16S rRNA phylogenetic tree. In silico restriction analysis revealed a high level of polymorphism within hsp60 gene sequences. Restriction pattern with enzymes AgsI and MseI in hsp60 gene sequences allowed differentiation of all the species including closely related species L. plantarum and L. pentosus, E. faecium and E. durans. In general, hsp60 gene with higher evolutionary divergence proved to be a better phylogenetic marker for the group LAB.     

Molecular characterization and phylogenetic comparisons of three <Emphasis Type="Italic">Mayetiola</Emphasis> species (Diptera: Cecidomyiidae) infesting cereals in Tunisia

Species from the genus Mayetiola are observed in the main cereal cultures of Tunisia. Some researchers have studied M. destructor that attacks wheat and M. hordei that attacks barley. However, a third important species observed in oat, M. avenae, has not been studied and is not well documented in Tunisia. A method to easily separate the species is needed to clarify the occurrences of these gall midge species. This study aimed to first distinguish between the three species of gall midges by molecular characterization and second to reveal the phylogenetic relationships within and between the three species of Mayetiola collected from 5 different regions of northern Tunisia. To achieve these purposes, two regions of the mitochondrial DNA, cytochrome oxidase subunit I gene, and the 16S rRNA gene were amplified by polymerase chain reaction and sequenced. For each marker, a set of 75 individuals were used for DNA analysis. Phylogenetic trees were created using the DNA sequences of all samples from the 3 species. Results showed significant separation of the three different species into dissimilar clades. Each clade contained only specimens from the same species. Differences were observed between DNA sequences of the same species. The differences within the same species were not representative of geographical variations but coexisted within a population Therefore, using the COI and 16S rRNA genes as markers can clearly separate M. avenae, M. destructor and M. hordei.     

Molecular genetic and karyological analysis of antlered sculpins of <Emphasis Type="Italic">Enophrys diceraus</Emphasis> group (Cottidae)

Molecular genetic and karyological analyses of antlered sculpin, Enophrys diceraus, from the Sea of Japan and the Sea of Okhotsk were carried out. The karyotype of this species was studied for the first time. On the basis of karyological analysis, it was established that E. diceraus from the Sea of Japan and the Sea of Okhotsk was polymorphic in terms of the number of chromosomes and their morphology (2n = 36, 35, and 37, NF = 40). It was suggested that the karyotype with 35 chromosomes could have been produced as a result of Robertsonian translocation; the karyotype with 37 chromosomes could have been produced by crossing of individuals with different number of chromosomes. On the basis of the molecular genetic analysis of the mitochondrial COI and 16S rRNA genes, considerable differences between E. diceraus from the Sea of Japan and the Sea of Okhotsk corresponding to the level of interspecies genetic variability were established. It is concluded that E. diceraus from the Sea of Japan belongs to another species, most likely, E. namiyei.     

The Calibrated Phylogeny of the <Emphasis Type="Italic">Drosophila fasciola</Emphasis> Subgroup (<Emphasis Type="Italic">D. repleta</Emphasis> Group Wasserman) Indicates Neogene Diversification of Its Internal Branches

The species of the Drosophila fasciola subgroup Wasserman represent the dominant section of the Drosophila repleta group Wasserman in the American rainforests and have a broad geographical distribution in the New World. However, despite of its wide range, the D. fasciola subgroup is one of the most overlooked D. repleta subgroups. Here, we report a molecular phylogenetic analysis focused on the D. fasciola subgroup using two mitochondrial [cytochrome oxidase subunit I (COI), cytochrome oxidase subunit II (COII)] and two nuclear [elongation factor-1alpha F1 (EF-alphaF1) and transformer (tra)] genes. Overall, we found that this subgroup is a monophyletic taxon, subdivided into two main internal branches: named Fas1 and Fas2 clades. The diversification of these clades is estimated to have begun in the middle Miocene, around 12 Ma [95% high posterior density (HPD) 9.0–15 Ma], and might be associated with the colonization of South America by Central America populations after the closure of Isthmus of Panama due to the temporal congruence between these events. The terminal branches had their origins estimated to be in the Pliocene or the Plio-Pleistocene transition. For the later estimates, both the geomorphological influences and the climatic oscillations of the Pleistocene may have played a role in shaping the diversification of the D. fasciola group.     

Mitochondrial DNA divergence and phylogenetic relationships in mullets (Pisces: Mugilidae) of the Sea of Japan and the Sea of Azov revealed by PCR-RFLP-analysis

Three Mugilid species: Mugil cephalus (Linnaeus, 1758) and Liza haematocheila (Temminck et Schlegel, 1845; syn. Mugil soiuy, M. haematocheilus, L. soiuy, Chelon haematocheilus) from the Sea of Japan, as well as M. cephalus and Liza aurata (Risso, 1810) from the Sea of Azov were investigated on the basis of PCR-RFLP analysis of mitochondrial DNA (mtDNA) fragments, which included 12S/16S rRNA, and ND3/ND4L/ND4 genes. Among 61 individuals of three Mugilid species thirteen different haplotypes were detected. Eight and thirteen restriction endonucleases were found to be species-specific in 12S/16SrRNA and ND3/ND4L/ND4 respectively. This method may be useful for species identification. M. cephalus showed the largest genetic divergence while L. haematocheila and L. aurata were closely related and clustered together. The level of mtDNA differentiation between the two M. cephalus samples from the Sea of Japan and the Sea of Azov, i.e., nucleotide substitutions of approximately 3%, appeared to be relatively high.     

Genetic relationships of Chukchi charr <Emphasis Type="Italic">Salvelinus andriashevi</Emphasis> and Taranetz charr <Emphasis Type="Italic">Salvelinus taranetzi</Emphasis>

On the basis of sequence analysis of the mitochondrial DNA (mtDNA) control region (CR), cytochrome b (Cytb), and cytochrome oxidase-1 (CoI) genes, the relationships of endemic species Salvelinus andriashevi Berg, 1948, represented by the only population from Lake Estikhed (Chukotka), were estimated. The data on the genealogical analysis of mtDNA haplotypes supported phylogenetic closeness of S. andriashevi and S. taranetzi. It was also demonstrated that the specimens of Chukchi charr, along with Salvelinus sp. 4 (Lake Nachikinskoe), S. krogiusae (Lake Dal’nee), S. boganidae and S. elgyticus (Lake El’gygytgyn), and S. a. erythrinus from Canada’s Northwest Territories (NWT) belonged to the Arctic group of Taranetz charr. The problem of coincidence of taxonomic differentiation of charrs of the genus Salvelinus based on morphological and genetic analyses is discussed.     

Variation in the <Emphasis Type="Italic">COI</Emphasis> gene of the freshwater pearl mussel <Emphasis Type="Italic">Margaritifera margaritifera</Emphasis> from River Vuokkijoki

The freshwater pearl mussel Margaritifera margaritifera L. is one of the most endangered freshwater mussels in the world. Effective conservation of threatened species requires not only ecological, but also genetic information from the target species and populations. Since low genetic diversity can reduce the ability of a species to adapt to environmental changes, maintaining genetic diversity has been identified as one of the key elements in successful conservation programs. We examined genetic variation of the freshwater pearl mussel from the River Vuokkijoki, Karelia, Russia. We sequenced a fragment of the cytochrome c oxidase subunit I gene (COI) from 22 individuals and compared the data to 32 previously published COI sequences available in GenBank. We identified 10 different COI haplotypes in the sequenced samples, three of which had not been previously reported. Our results show that the River Vuokkijoki has high genetic diversity and suggest that the colonization of this northern freshwater pearl mussel population might have occurred from multiple and even distant refugia. Therefore, the freshwater pearl mussel population of the River Vuokkijoki is valuable for the conservation of the whole species.     

New Data on MtDNA Variability of Pond Smelt <Emphasis Type="Italic">Hypomesus olidus</Emphasis> (Osmeridae) from the Commander Islands in Comparison with Other Populations of the Species

Sequencing of two regions of mitochondrial DNA, CytB (1124 bp) and COI (629 bp), is conducted for the first time for pond smelt Hypomesus olidus (Osmeridae) from the Commander Islands. The haplotypes identified, H1 and H2, differ by one mutation in the region of the COI gene. Haplotype H1 that belongs to the pond smelt from the Commander Islands was previously massively found in pond smelt of Azabache Lake. Haplotype H2 was not found previously and is probably unique to pond smelt from the Commander Islands.     

Genetic and phenotypic characterizations of <Emphasis Type="Italic">Xenorhabdus</Emphasis> species (Enterobacteriales: Enterobacteriaceae) isolated from steinernematid nematodes (Rhabditida: Steinernematidae) in Japan

The bacterial species of the genus Xenorhabdus in the family Enterobacteriaceae have a mutualistic association with steinernematid entomopathogenic nematodes (EPNs), which have been used as biological control agents against soil insect pests. In this study we present the genetic and phenotypic characterizations of the Xenorhabdus species isolated from steinernematid nematodes in Japan. The 18 Japanese Xenorhabdus isolates were classified into five bacterial species based on 16S ribosomal RNA (16S rRNA) gene sequences: Xenorhabdus bovienii, Xenorhabdus hominickii, Xenorhabdus indica, Xenorhabdus ishibashii, and Xenorhabdus japonica. There was no genetic variation between the 16S RNA sequences among the three X. ishibashii isolates, 0–0.1% variation among the five X. hominickii isolates, and 0–0.5% among the eight X. bovienii isolates. Phenotypic characterization demonstrated that representative isolates of the five bacterial species shared common characteristics of the genus Xenorhabdus, and only X. hominickii isolates produced indole. Symbiotic association and co-speciation of Xenorhabdus bacteria with Steinernema nematodes from Japan are discussed.     

Identification of <Emphasis Type="Italic">Bacillus</Emphasis> Probiotics Isolated from Soil Rhizosphere Using 16S rRNA, <Emphasis Type="Italic">recA</Emphasis>, <Emphasis Type="Italic">rpoB</Emphasis> Gene Sequencing and RAPD-PCR

Some Bacillus species, especially Bacillus subtilis and Bacillus pumilus groups, have highly similar 16S rRNA gene sequences, which are hard to identify based on 16S rDNA sequence analysis. To conquer this drawback, rpoB, recA sequence analysis along with randomly amplified polymorphic (RAPD) fingerprinting was examined as an alternative method for differentiating Bacillus species. The 16S rRNA, rpoB and recA genes were amplified via a polymerase chain reaction using their specific primers. The resulted PCR amplicons were sequenced, and phylogenetic analysis was employed by MEGA 6 software. Identification based on 16S rRNA gene sequencing was underpinned by rpoB and recA gene sequencing as well as RAPD-PCR technique. Subsequently, concatenation and phylogenetic analysis showed that extent of diversity and similarity were better obtained by rpoB and recA primers, which are also reinforced by RAPD-PCR methods. However, in one case, these approaches failed to identify one isolate, which in combination with the phenotypical method offsets this issue. Overall, RAPD fingerprinting, rpoB and recA along with concatenated genes sequence analysis discriminated closely related Bacillus species, which highlights the significance of the multigenic method in more precisely distinguishing Bacillus strains. This research emphasizes the benefit of RAPD fingerprinting, rpoB and recA sequence analysis superior to 16S rRNA gene sequence analysis for suitable and effective identification of Bacillus species as recommended for probiotic products.     

Special traits of the genetic structure and origin of the population of vendace <Emphasis Type="Italic">Coregonus albula</Emphasis> of Pleshcheyevo Lake

Nucleotide sequences of two regions of mitochondrial DNA are analyzed, that of the ND1-fragment and that of the fragment comprising a part of the cytochrome c oxidase gene. In the population of the vendace Coregonus albula from Pleshcheyevo Lake, there are specimens possessing haplotypes considerably differing from common variants of sequences represented both in the water body under consideration and in other water bodies of the European part of Russia. The level of intrapopulation polymorphism exceeds the level between species.