5S rDNA variation and its phylogenetic inference in the genus <Emphasis Type="Italic">Leporinus</Emphasis> (Characiformes: Anostomidae)

5S rDNA sequences have proven to be valuable as genetic markers to distinguish closely related species and also in the understanding of the dynamic of repetitive sequences in the genomes. In the aim to contribute to the knowledge of the evolutionary history of Leporinus (Anostomidae) and also to contribute to the understanding of the 5S rDNA sequences organization in the fish genome, analyses of 5S rDNA sequences were conducted in seven species of this genus. The 5S rRNA gene sequence was highly conserved among Leporinus species, whereas NTS exhibit high levels of variations related to insertions, deletions, microrepeats, and base substitutions. The phylogenetic analysis of the 5S rDNA sequences clustered the species into two clades that are in agreement with cytogenetic and morphological data.     

Ultrastructural characterization and comparative phylogenetic analysis of new microsporidia from Siberian mosquitoes: evidence for coevolution and host switching

A survey of mosquito larvae infected with microsporidia was conducted from 2005 to 2008 in the Tomsk, Kemerovo and Novosibirsk regions of western Siberia, Russia. Twenty-one morphologically and genetically unique species of microsporidia were isolated from nine species of Anopheles, Aedes, Culex and Ochlerotatus mosquitoes including: (1) 14 proposed new species of Amblyospora (A. bakcharia, A. baritia, A. bogashovia, A. chulymia, A. hristinia, A. jurginia, A. kazankia, A. mavlukevia, A. mocrushinia, A. modestium, A. salairia, A. severinia, A. shegaria, and A. timirasia); (2) a newly proposed genus and species, Novothelohaniaovalae and; (3) six species of Amblyospora (A. flavescens, A. kolarovi, A. rugosa), Parathelohania (P. divulgata and P. tomski) and Trichoctosporea (T. pygopellita) from which gene sequences had not been previously obtained. Detailed ultrastructure of meiospores revealed unique cytological features associated with the length, arrangement and ratio of broad to narrow coils of the polar filament, comparative thickness of the exospore and endospore, and overall size of each species reaffirming their value in distinguishing taxonomic relationships. SSU rDNA sequences obtained from each species of microsporidia were unique when compared with GenBank entries. Phylogenetic trees constructed by Maximum Parsimony, Maximum Likelihood and Neighbor Joining analyses yielded similar topologies with a high degree of congruence between parasite and host at the generic level. Species that parasitize Aedes/Ochlerotatus and Culex mosquitoes segregate into distinct monophyletic groupings mirroring their host phylogeny, while species from Anopheles mosquitoes group as a sister clade basal to the entire group of mosquito-parasitic microsporidia as their Anopheles hosts cluster as a sister clade to the entire group of culicine mosquitoes. This provides strong evidence for host-parasite coevolution by descent at the generic level and limited host lineage switching between unrelated taxa. Among parasites of Aedes/Ochlerotatus and Anopheles mosquitoes, we found several instances where a single mosquito species serves as a host for two or more related species of microsporidia, an observation consistent with host switching and independent parasite speciation. Among the microsporidian parasites of Culex mosquitoes, we found only one parasite per host indicating a higher degree of host specificity and less host switching among parasites of this genus. Findings suggest a degree of host-parasite co-speciation with host switching occurring occasionally when the "normal" host is unavailable in the aquatic ecosystem. Frequency of host switching seems to be occurring in proportion to host relatedness and does not cross generic boundaries in this system.     

Phylogenetic reconstructions in the genus Capra (Bovidae, Artiodactyla) based on the mitochondrial DNA analysis

Mitochondrial genome fragments were examined in all species of the genus Capra (Bovidae, Artiodactyla). Phylogenetic analysis was carried out using 59 cytochrome b gene sequences (392 bp), and 22 sequences of the mtDNA variable fragment (402 bp). In the control region, two unique deletions were revealed. One of the deletions was found only in Capra cilindricornis (17 bp), while another one grouped C. caucasica with C. aegagrus (1 bp). The group of Caucasian wild goats splits into two clades, and furthermore, the sequences of C. caucasica demonstrate remarkable similarity to the sequences of C. aegagrus, while C. cylindricus seems to have evolved independently for a long period of time. It was demonstrated that C. pyrenaica and C. ibex were extremely close to one another. Capra sibirica formed an outer group relative to the other species, and according to our data, was the most ancient species of the genus. On the contrary, genetic distance separating C. falconeri (the most independent species of the genus related to its morphology) from the other species is small.     

Sequence heterogeneity in the internal transcribed spacers of two rat ribosomal DNA clones

The sequence of one cloned rat rDNA segment is determined, encompassing the 3'-terminal part of 18 S rDNA (231 bp), ITS 1 (1072 bp), 5.8 S rDNA (156 bp), ITS 2 (771 bp) and the 5'-terminal part of 28 S rDNA (210 bp). Comparison with a distinct clone of the same rDNA segment reveals the identity of the rRNA gene sequences and considerable heterogeneity in both ITS 1 and ITS 2. The heterogeneities include mainly single base-pair changes (23 deletions or insertions and 14 substitutions) distributed all along the ITS sequences.     

Identification of mosquito-parasitic microsporidia, Amblyospora rugosa and Trichoctosporea pygopellita (Microsporidia: Amblysporidae), from Acanthocyclops venustus and Acanthocyclops reductus (Copepoda: Cyclopidae), based on small subunit rDNA analysis

Identical small subunit rDNA sequences were obtained for microsporidia Amblyospora rugosa from blood-sucking mosquitoes larvae Ochlerotatus cantans, O. cataphylla and copepods Acanthocyclops venustus, as well as for Trichoctosporea pygopellita from mosquitoes larvae Ochlerotatus cyprius, O. excrucians and copepods Acanthocyclops reductus. The data on molecular phylogeny and ecological researches show that in Siberia mosquito-parasitic microsporidia of the genera Amblyospora and Trichoctosporea have complex life cycle involving likely intermediate hosts, Acanthocyclops copepods. Life cycle of parasites is synchronized with phenology of their hosts. The phylogenetic analyses shows, that genus Trichoctosporea should be transferred from the family Thelohaniidae to the family Amblyosporidae.     

Use of Microsatellite Locus Flanking Regions for Phylogenetic Analysis? A Preliminary Study of Sebastes subgenera

The systematics of the species-rich genus of Sebastes rockfish has not been resolved, and is unlikely to be resolved using morphological criteria. Using an alternative approach based on DNA sequence variation, we sampled the genome for random, presumably neutral, nuclear DNA sequences, by sequencing microsatellite flanking regions of 15 Sebastes species (representing 15 of 22 extant subgenera) and Hozukius emblemarius. We aligned sequences of flanking regions of eight of the 13 loci that amplified, which presumably are homologous. In aggregate, the aligned sequences included 848 bp, 53 bp of which were polymorphic. The base changes among species included 27 transitions, 20 transversions, three multiple substitutions, and three deletions or insertions. The flanking regions at different loci had different base substitution rates. Nucleotide divergence among Sebastes species ranged from 0.0012 to 0.0216, whereas nucleotide divergence between Sebastes and Hozukius species ranged from 0.0095 to 0.0240. We used maximum parsimony, neighbor-joining, and maximum likelihood methods to examine relationships among the species. H. emblemariusformed a separate group, and five of the western Pacific Ocean species of rockfish clustered together, suggesting that they may have shared an evolutionary history distinct from many North American species. The flanking regions of some microsatellite loci contain DNA sequence variation that distinguishes rockfish species and may prove useful for clarifying relationships among rockfish, or other closely related species.     

Phylogeny of the New World medicinal leech family Macrobdellidae (Oligochaeta: Hirudinida: Arhynchobdellida)

Phylogenetic analyses of the leech family Macrobdellidae were accomplished with all nominal species in the family save one. A total of 17 specimens in nine ingroup species were analysed, along with four outgroup taxa. Twenty-two morphological characters based on jaw dentition, sexual anatomy, and external morphology failed to provide a resolution for many of the relationships in the family. DNA sequence data from nuclear 18S rDNA, nuclear 28S rDNA, mitochondrial 12S rDNA, and mitochondrial cytochrome c oxidase subunit I were examined separately and in combination with morphological characters. The resulting combined analysis strongly corroborated the placement of the genus Philobdella within the family Macrobdellidae and as sister to a monophyletic genus Macrobdella , the typical North American medicinal leeches. Furthermore, sequence divergences among these taxa confirmed the existence of two species, Philobdella gracilis and P. floridana , readily distinguishable on the basis of jaw dentition .     

Molecular phylogeny of the ranid frogs from Southwest India based on the mitochondrial ribosomal RNA gene sequences

The Western Ghats of Southwestern India are known as one of the world's "hotspots" of biodiversity. We collected frog specimens from the family Ranidae and investigated the phylogenetic relationships among ranid species, particularly among the Fejervarya, a genus whose morphological diagnostic characteristics and phylogenetic features remain little known. We analyzed partial sequences of the mitochondrial 12S (428 bp) and 16S rDNAs (549 bp). Results showed that the members of Fejervarya form a monophyletic group with the genera Hoplobatrachus and Euphlyctis among ranid genera. This confirms the recent allocation of Fejervarya, including it within the subfamily Dicroglossinae. The mitochondrial rDNA data in our study also appeared to be useful as a marker to distinguish Fejervarya species without morphological differences. The phylogenetic tree based on the 16S rDNA sequence showed a correlation between Fejervarya phylogenies and their geographic distributions. Lastly, our results suggested the recent occurrence of a radiation event of Fejervarya species in the Indian-Sri Lankan region.     

A new molecular phylogenetic hypothesis for the evolution of freshwater eels

Phylogenetic analysis of a segment of the mitochondrial 16S rDNA of eight Anguilla species from the Indo-Pacific region and from the North Atlantic revealed that the genus Anguilla appears to be surprisingly young, based upon the small observed maximum genetic distance of 4.8% and the high degree of morphological similarity among the species. The placement of A. marmorata as the most ancestral lineage suggests that the genus is likely to have originated in the Indo-Malayian region, from which it quickly spread. Two Pacific species, A. obscura and A. japonica, branched next. A. japonica was placed as sister group to all remaining species, which formed three clades: the first comprising A. australis, the second A. reinhardti and A. mossambica, and the third A. anguilla and A. rostrata. All analyzed specimens of A. rostrata originating from southern New Jersey to Nova Scotia had identical mitotypes, while five mitochondrial genotypes were found in Europe differing by zero to two substitutions. The two Atlantic eel species are very closely related; all surveyed specimens of A. anguilla differ by three to five substitutions from their American allies, corroborating the existence of two distinct biological species. This was also confirmed by restriction analysis of a 350-bp segment of the cytochrome b, in which American specimens were distinct in sharing a single diagnostic restriction site of HinfI. Our results suggest little to no gene flow between the two nominal Atlantic eel species.     

West Nile virus studies in the Sanliurfa Province of Turkey

We attempted to isolate West Nile virus from mosquitoes collected in the field for the first time in Turkey. A total of 6,457 mosquito specimens from Culex pipiens Linnaeus, Ochlerotatus caspius (Pallas) and Aedes spp. species were included in this study. Culex pipiens samples made up 56% of the total species, O. caspius 24% and Aedes spp 20%. There were no positive results after studying mosquito samples using Real-time PCR, VecTest, and Vero cell culture. In serological tests of 181 human serum samples, 29 (16%) were found to be West Nile positive. On the basis of these results, we intend to collect more mosquito samples especially from those areas from which positive serum samples were obtained.     

Segmented Gene Conversion as a Mechanism of Correction of 18S rRNA Pseudogene Located Outside of rDNA Cluster in D. melanogaster

The peculiarities of the sequences of 18S rDNA included in a 90-kb DNA segment cloned in YAC vector are described. This heterochromatic segment is situated on the X chromosome distal to the main rDNA cluster. The pseudo 18S rDNA sequence comprised undamaged stretches of rDNA interspersed with segments characterized by high density of nucleotide substitutions and insertions/deletions. The observed patchwork arrangement of unaltered rDNA sequences was considered as evidence of segmented gene conversion events between the normal and damaged genes which are thought to constitute one of the mechanisms of rDNA array homogenization. The 18S rDNA fragment (510 bp) located nearby, homologous to the internal, undamaged part of pseudo 18S rDNA, carries comparable density of randomly distributed nucleotide substitutions with no evidence of correction. Received: 8 August 1996 / Accepted: 7 December 1996     

Species composition and ecological peculiarities of the blood-sucking mosquito genus Ochlerotatus (cantans and dorsalis species groups) (Diptera, Culicidae) in the Novgorod Region

Seven mosquito species of the genus Ochlerotatus ("cantans" and "dorsalis" species groups, family Culicidae) were found in the Novgorod Region as a result of our field investigations, which have been carried out in 1996-2004. One species, Ochlerotatus annulipes, was for the first time recorded in the Novgorod Region. Distribution, biotopic and seasonal preferences of all discovered species are considered.     

Intraspecific variation in nemerteans (Nemertea): synonymization of genera Paroerstedia and Oerstediella with Oerstedia

Intraspecific variation in internal morphological charactcrs is substantial in many nemertean species, mainly due to the tendency for specimens to contract during fixation. It is shown that variation within the type species for the genus Oerstedia, O. dorsalis (Abildgaard, 1806), encompasses all the character states that have been used to erect the two related genera Oerstediella and Paroerstedia. These genera are synonymized with Oerstedia , and the validity of species previously assigned to these genera is discussed.     

Comparison of the morphology of oocysts and the phylogenetic analysis of four Ascogregarina species (Eugregarinidae: Lecudinidae) as inferred from small subunit ribosomal DNA sequences

This study on the ultrastructure of the oocysts of four isolated species of Ascogregarina (A. taiwanensis (Lien and Levine) (Eugregarinidae: Lecudinidae) from Aedes albopictus (Skuse), A. culicis (Ross) (Eugregarinidae: Lecudinidae) from Aedes aegypti (L.), A. armigerei (Eugregarinidae: Lecudinidae) from Armigeres subalbatus (Coquillet), and Ascogregarina sp. (Eugregarinidae: Lecudinidae) from Ochlerotatus japonicus japonicus (Theobald)) using a scanning electron microscope revealed significant differences in size and in surface structure. The average length of the oocyst was greatest in A. armigerei (13.2+/-0.2 microm) (mean+/-SD) and least in A. culicis (8.8+/-0.4 microm). Oocysts were of moderate length in A. taiwanensis (9.9+/-0.6 microm) and in Ascogregarina sp. (10.7+/-1.1 microm) isolated from O. j. japonicus. The ultrastructure of the surface of the A. culicis oocyst was rough in texture with numerous dense spots and was easily distinguishable from the oocysts of the other three Ascogregarina spp. The maximum likelihood tree inferred from small subunit ribosomal DNA (SSU rDNA) sequences indicated that the four Ascogregarina spp. form a monophyletic cluster among other gregarine parasites. Within the Ascogregarina clade, A. culicis, A. taiwanensis, and Ascogregarina sp. from O. j. japonicus showed a close relationship, whereas A. armigerei was a distantly related species.     

Identification and typing of miso and soy sauce fermentation yeasts, Candida etchellsii and C. versatilis, based on sequence analyses of the D1D2 domain of the 26S ribosomal RNA gene, and the region of internal transcribed spacer 1, 5.8S ribosomal RNA gene and internal transcribed spacer 2

We analyzed sequences of the D1D2 domain of the 26S ribosomal RNA gene (26S rDNA sequence), and the region of internal transcribed spacer 1, 5.8S ribosomal RNA gene and internal transcribed spacer 2 (ITS sequence) of the miso and soy sauce fermentation yeasts, Candida etchellsii and Candida versatilis, in order to evaluate the usefulness of this sequence analysis for identification and typing of these two species. In the 26S rDNA sequence method, the numbers of base substitutions among C. etchellsii strains were up to 2 in 482 bp (99.6% similarity), and they were divided into three types (types A, B, and C). Those of C. versatilis strains were also up to 2 in 521 bp (99.6% similarity) and they were divided into three types (types 1, 2, and 3). In the ITS sequence method, those of C. etchellsii strains were zero in 433 bp (type a, 100% similarity). Those of C. versatilis were 5 in 409 bp (98.8% similarity), divided into 4 types (types I, II, III and IV). It was found that molecular methods based on the sequences of the 26S rDNA D1D2 domain and the ITS region were rapid and precise compared with the physiological method for the identification and typing of these two species.     

Molecular phylogeny and evolution of mosquito parasitic Microsporidia (Microsporidia: Amblyosporidae)

Amblyospora species and other aquatic Microsporidia were isolated from mosquitoes, black flies, and copepods and the small subunit ribosomal RNA gene was sequenced. Comparative phylogenetic analysis showed a correspondence between the mosquito host genera and their Amblyspora parasite species. There is a clade of Amblyospora species that infect the Culex host group and a clade of Amblyospora that infect the Aedes/Ochlerotatus group of mosquitoes. Parathelohania species, which infect Anopheles mosquitoes, may be the sister group to the Amblyospora in the same way that the Anopheles mosquitoes are thought to be the sister group to the Culex and Aedes mosquitoes. In addition, by sequence analysis of small subunit rDNA from spores, we identified the alternate copepod host for four species of Amblyospora. Amblyospora species are specific for their primary (mosquito) host and each of these mosquito species serves as host for only one Amblyospora species. On the other hand, a single species of copepod can serve as an intermediate host to several Amblyospora species and some Amblyospora species may be found in more than one copepod host. Intrapredatorus barri, a species within a monotypic genus with Amblyospora-like characteristics, falls well within the Amblyospora clade. The genera Edhazardia and Culicospora, which do not have functional meiospores and do not require an intermediate host, but which do have a lanceolate spore type which is ultrastructurally very similar to the Amblyospora spore type found in the copepod, cluster among the Amblyospora species. In the future, the genus Amblyospora may be redefined to include species without obligate intermediate hosts. Hazardia, Berwaldia, Larssonia, Trichotuzetia, and Gurleya are members of a sister group to the Amblyospora clades infecting mosquitoes, and may be representatives of a large group of aquatic parasites.     

Phylogenetic reconstructions in the genus <Emphasis Type="Italic">Capra</Emphasis> (Bovidae,Artiodactyla) based on the mitochondrial DNA analysis

Mitochondrial genome fragments were examined in all species of the genus Capra (Bovidae, Artiodactyla). Phylogenetic analysis was carried out using 59 cytochrome b gene sequences (392 bp), and 22 sequences of the mtDNA variable fragment (402 bp). In the control region, two unique deletions were revealed. One of the deletions was found only in Capra cylindricornis (17 bp), while another one grouped C. caucasica with C. aegagrus (1 bp). The group of Caucasian wild goats splits into two clades, and furthermore, the sequences of C. caucasica demonstrate remarkable similarity to the sequences of C. aegagrus, while C. cylindricornis seems to have evolved independently for a long period of time. It was demonstrated that C. pyrenaica and C. ibex were extremely close to one another. Capra sibirica formed an outer group relative to the other species, and according to our data, was the most ancient species of the genus. On the contrary, genetic distance separating C. falconeri (the most independent species of the genus related to its morphology) from the other species is small.     

Identification of Acetobacter strains isolated from Indonesian sources,and proposals of Acetobacter syzygii sp. nov., Acetobacter cibinongensis sp. nov., and Acetobacter orientalis sp. nov

Forty-six strains of acetic acid bacteria newly isolated from flowers, fruits, and fermented foods collected in Indonesia were taxonomically studied. They were Gram-negative rods, produced acetic acid from ethanol, oxidized acetate and lactate to CO(2) and H(2)O, and had Q-9 as the major ubiquinone system. On the basis of DNA-DNA similarity, all strains studied, including type strains and reference strains of the genus Acetobacter, were separated into eleven groups (Groups I to XI). Of the 46 isolates, two isolates were included in Group II and identified as Acetobacter pasteurianus, five in Group IV as A. orleanensis, 16 in Group V as A. lovaniensis, five in Group VII as A. indonesiensis, and three in Group VIII as A. tropicalis. The remaining 15 isolates constituted three new groups based on DNA-DNA similarity; four isolates were included in Group IX, two in Group X, and nine in Group XI. No isolates were identified as A. aceti (Group I), A. peroxydans (Group III), and A. estunensis (Group VI). Phylogenetic analysis based on 16S rDNA sequences of representative strains of the Groups indicated belonging to the strains of the genus Acetobacter. On the basis of DNA base composition, DNA-DNA similarity, and 16S rDNA sequences, three new species of the genus Acetobacter are proposed: Acetobacter syzygii sp. nov. for Group IX, Acetobacter cibinongensis sp. nov. for Group X, and Acetobacter orientalis sp. nov. for Group XI. The distribution of Acetobacter strains in Indonesia is discussed in light of isolation sources.     

Checklist of Iranian mosquitoes (Diptera: Culicidae).

The mosquito fauna of Iran includes seven genera, 64 species, and three subspecies. The records of 12 other species should be verified. There are 24 species in the most recent checklist of Iranian Anopheles. Two species, An. peditaeniatus and An. fluviatilis species V, have been reported since. An. atroparvus, An. labranchiae, and An. martinius of the Maculipennis Group, and An. cinereus, An. nigerrimus, and An. rhodesiensis rupicola were recorded previously but are not included in the checklist. The checklist of Iranian Culicinae includes ten species of the tribe Aedini, but there are some records of four other species: Aedes aegypti, Ochlerotatus berlandi, Oc. chelli, and Oc. dorsalis. The genus Culex includes 19 species, excluding Cx. impudicus, which has not been collected recently, and some doubtful records of Cx. univittatus, Cx. vishnui, and Cx. vagans. The genus Culiseta includes five species and the genera Coquillettidia and Uranotaenia each include one species in Iran. No information is available for the An. subpictus, Oc. caspius, Oc. detritus, and Oc. pulcritaris species complexes in Iran. The An. claviger and Cx. pipiens complexes and the An. hyrcanus group require review.     

Phylogenetic relationships and biodiversity in Hylids (Anura: Hylidae) from French Guiana

We evaluated two biodiversity criteria, higher taxonomic diversity and phylogenetic diversity in French Guiana. For this, we used a recent assessment of the knowledge accumulated since 30 years of study on the amphibian species currently known in French Guiana. We focused on two well-represented genera, Hyla and Scinax, belonging to the subfamily Hylinae. We used partial sequences of two mitochondrial genes (16S rDNA and 12S rDNA, 813 bp) and two nuclear genes (tyrosinase and 18S rRNA, 1590 bp) covering a total of 2403 bp. According to the high bootstrap support in phylogenetic analysis of the complete dataset, the genus Scinax is a homophyletic clade formed by two species groups (rubra and rostrata) in French Guiana. The genus Hyla was confirmed to be a paraphyletic group formed by two species groups as well (30 chromosomes and the 'gladiator frogs'). We confirmed that these genera should be taxonomically reconsidered. Moreover, at the genus, subfamily and family levels, the use of only morphological characters or only molecular DNA markers would hamper estimations of biodiversity. Thus, we strongly advise the combined use of both morphology and molecular data (nuclear and mitochondrial markers).