Molecular phylogenetic study of several eelpout fishes (Perciformes,Zoarcoidei) from Far Eastern seas on the basis of the nucleotide sequences of the mitochondrial cytochrome oxidase 1 gene (<Emphasis Type="Italic">Co-1</Emphasis>)

A total of 95 nucleotide sequences of a Co-1 gene fragment of approximately 650 bp were analyzed for fishes of the orders Perciformes and Scorpaeniformes (outgroup). Gene trees based on four algorithms (BA, NJ, MP, and ML) were similar in topology of solved branches. An emphasis was placed on the species and generic levels, but a significant phylogenetic signal was obtained for higher taxonomic ranks as well. For instance, a monophyletic origin was confirmed for the family Zoarcidae and the subfamily Opisthocentrinae (Stichaeidae). The proportion of different nucleotides in the sequences compared (p-distances) significantly increased with increasing taxonomic rank. The p-distances were estimated for four hierarchic levels and were (1) 0.15 ± 0.06% for the within-species hierarchic level, (2) 6.33 ± 0.37% for the within-genus level, (3) 11.83 ± 0.06% for the within-family level, and (4) 15.22 ± 0.05% for the within-order level. The difference in the Co-1 gene fragments between levels (1) and (2) allows almost errorless species identification on the basis of this kind of a molecular bar code.     

Phylogenetic analysis of Altai osmans of the genus <Emphasis Type="Italic">Oreoleuciscus</Emphasis> (Pisces,Cyprinidae, Leuciscinae), based on the analysis of the cytochrome oxidase 1 gene (<Emphasis Type="Italic">Co-1</Emphasis>) sequence

Molecular genetic analysis of Altai osmans of the genus Oreoleuciscus from two different parts of the range was carried out. In this study, based on the mitochondrial Co-1 gene sequence, a total of 25 fish specimens belonging to four genera were examined: (1) O. humilis, 2 specimens; O. potanini, 13 specimens; (2) Pseudaspius leptocephalus, 1 specimen; (3) Tribolodon brandtii, T. hakonensis, and T. sachalinensis from the GanBank database, 9 speciens; and (4) Leuciscus waleckii, 1 specimen (used as an outgroup). The p-distances were very low both within and between the species: (1) 0.20 ± 0.03%; (2) 0.40 ± 0.12%; and (1–2) 0.80 ± 0.04%. To visualize the relationships among all of the species examined, the neighbor joining (NJ), maximum parsimony (MP), Bayesian (BA), and maximum likelihood (ML) trees were constructed. The results obtained using these methods were very similar. It was demonstrated that species assignment of the individuals (barcoding) with the help Co-1 gene was effective, despite of very low divergence of the two osman taxa, which was comparable with typical intraspecific values in other animal groups. Taxonomic status of O. potanini and O. humilis requires further investigation with paying attention to low genetic distances between these species along with the lack of material from sympatric parts of the ranges.     

Sequence diversity at cytochrome oxidase 1 (Co-1) gene among sculpins (Scorpaeniformes, Cottidae) and some other scorpionfish of Russia Far East with phylogenetic and taxonomic insights

Mitochondrial DNA (mtDNA) atCo-1 gene region was sequenced for 7 scorpion fish species (in total, 16 sequences of at least 552 bp) from the Far East of Russia and compared with 15 other sequences of Scorpaeniformes comprising altogether 29 scorpion-like fish sequences and two outgroup sequences (Cypriniformes). The analysis of the protein-codingCo-1 gene revealed statistically substantiated bias in the (T+C) ∶ (A+G) content, proving basic findings. The average scores ofp-distances for different scales of the evolutionary history atCo-1 gene revealed a clear pattern of increased nucleotide diversity at four different levels: (1) intraspecies, (2) intragenus, (3) intrafamily, and (4) intraorder. The scores of averagep-distances for the compared fish groups were: (1) 1.00±0.20%, (2) 3.80±0.20%, (3) 12.40±1.20%, and (4) 18.00±0.38%, respectively (mean±SE). These data support the concept that speciation in the order Scorpaeniformes, in most cases, follows a geographic mode through accumulation of numerous small genetic changes over a long period of time. However, intraspecies diversity was surprisingly high among scorpionfish. Phylogenetic trees for 29 sequences of scorpionfish and 2 other fishes belonging to ray-finned fishes (Actinopterigii) were developed usingCo-1 gene and four different analytical approaches: Bayesian (BA), maximum likelihood (ML), neighbour-joining (NJ), and maximum parsimony (MP). The analysis revealed a monophyletic origin for the representatives of Cottidae, which is the principal scorpionfish family investigated (100, 96, 98% support level in our BA, MP, and NJ analyses). Similarly, the monophyletic origin of up to the three compared scorpion-like fish genera was supported by molecular phylogenetic data. Species identification on individual basis (barcoding tagging) was high. A few taxonomic complications arose during the analysis and they are discussed here in.     

Cytochrome b gene sequence divergence of seven sisorid species of catfish genus <Emphasis Type="Italic">Glyptothorax</Emphasis> (siluriformes,sisoridae) from India

Samples of seven Sisorid catfish species of the genus Glyptothorax (G. garhwali, G. dakpathari, G. brevipinnis, G. ngapang, G. granulus, G. ventrolineatus, and G. davissinghi) were collected from the Himalayan region and the Western Ghats of India. They were analyzed for the mitochondrial cytochrome b gene (Cyt b). Out of 1152 nucleotide positions analyzed, 269 (23.3%) were found to be variable and 235 (20.3%) were parsimoniously informative. The sequences showed 111 (9.6%) fourfold degenerate sites. The overall transition/transversion bias was R = 3.457. The average proportion of base substitutions measured as P-distance for all sequences of seven Glyptothorax species and other five species comprising four comparison groups was intraspecies, P = 0.17 ± 0.05%, intragenus, P = 10.75 ± 0.48%, intrafamily, P = 20.07 ± 1.43%, and intraorder, P = 21.10 ± 0.45%. Within the Glyptothorax genus maximum divergence was obtained among G. brevipinnis sequences, whereas the least divergence was obtained within G. davissinghi. The phylogenetic trees for 193 and 47 sequences of Sisorid catfishes together were developed using the Cyt b gene and four different analytical approaches: Bayesian (BA), neighbour-joining (NJ), maximum parsimony (MP), and maximum likelihood (ML). The analysis revealed a monophyletic origin for the all investigated representatives of the genus Glyptothorax (99, 100, 99, and 73% support level in our BA, NJ, MP, and ML analyses respectively) and with some reservations for Sisoridae, which is the principal family investigated. The monophyletic origin of the two subfamilies of Sisorid catfish defined in the literature was partly also supported by molecular phylogenetic data.     

Molecular phylogeny of the <Emphasis Type="Italic">Sinocyclocheilus</Emphasis> (Cypriniformes: Cyprinidae) fishes in northwest part of Guangxi,China

The northwestern part of Guangxi is a typical karst landform. There are about ten species of the genus Sinocyclocheilus distributed in the region. To investigate the phylogenetic relationships and resolve the taxonomic status of these species, we determined nucleotide sequences of complete mitochondrial cytochrome b gene of 11 ingroup species and one outgroup species Barbodes laticeps. Phylogenetic trees were reconstructed using maximum parsimony and maximum likelihood analyses. Phylogenetic results showed that these species cluster as one clade with strong support. The 95% credibility interval (CI) of the divergence time between the species in the northwest part of Guangxi and other species in the genus Sinocyclocheilus is 8.28–12.40Mya. For the karst environment, these species show marked, recent genetic differentiation. From the overall consideration of morphology and molecular data Sinocyclocheilus tianeensis should be a valid species.     

Polymorphism of the <Emphasis Type="Italic">cox1</Emphasis> gene in cercariae isolates of bird schistosomes (Trematoda:Schistosomatidae) from ponds of Moscow and Moscow region

Polymorphism of a 810-bp fragment of mitochondrial cox1 gene was studied in 15 cercariae isolates of bird schistosomes (family Schistosomatidae), which were collected in water bodies of Moscow and Moscow oblast and represented three species: Trichobilharzia szidati, T. franki, and T. regenti. A substantial predominance of AT (65.4%) was characteristic of the cox1 sequences in all three species. Rare single nucleotide substitutions determined low (0.2–0.9%) intraspecific nucleotide and amino acid sequence diversity. Haplotype diversity h was high (80–100%) in all three species, suggesting a unique character for almost all cox1 sequences in the sample. Phylogenetic trees based on the nucleotide and amino acid sequence variations were constructed to study the relationships of the three schistosome species. A high support was observed for the main branching node that reflects differentiation of the monophyletic group Trichobilharzia and species of the genera Bilharziella (B. polonica), Dendritobilharzia (D. pulverulenta), and Gigantobilharzia (G. huronensis). Based on the nucleotide substitutions and amino acid polymorphisms, two groups of isolates, which parasitize Lymnaea stagnalis (T. szidati) and snails of the group Radix (T. franki and T. regenti) respectively, were isolated in the genus Trichobilharzia. The time of divergence between the two schistosome groups infecting snails of the genera Radix and Lymnaea was calculated from the cox1 nucleotide substitution rate, which is known for Asian and Indian blood flukes from the genus Schistosoma and is 2–3% per million years on average. Divergence of the three bird schistosome species under study and divergence of the Asian species of mammalian schistosomes were almost concurrent, dating back to 2.5–3.8 Myr ago. Factors responsible for the lack of intraspecific subdivision with respect to the cox1 in bird schistosomes and the lack of separation between two species (T. franki and T. regenti) are discussed.     

Molecular phylogenetic study of several eelpout fishes (Perciformes, Zoarcoidei) from far eastern seas on the basis of the nucleotide sequence of the mitochondrial cytochrome oxidase 1 gene (Co-1)

A total of 95 nucleotide sequences of a Co-1 gene fragment of approximately 650 bp were analyzed for fishes of the orders Perciformes and Scorpaeniformes (outgroup). Gene trees based on four algorithms (BA, NJ, MP, and ML) were similar in topology of solved branches. An emphasis was placed on the species and generic levels, but a significant phylogenetic signal was obtained for higher taxonomic ranks as well. For instance, a monophyletic origin was confirmed for the family Zoarcidae and the subfamily Opisthocentrinae (Stichaeidae). The proportion of different nucleotides in the sequences compared (p-distances) significantly increased with increasing taxonomic rank. The p-distances were estimated for four hierarchic levels and were (1) 0.15 0.06% for the within-species hierarchic level, (2) 6.33 0.37% for the within-genus level, (3) 11.83 0.06% for the within-family level, and (4) 15.22 0.05% for the within-order level. The difference in the Co-1 gene fragments between levels (1) and (2) allows almost errorless species identification on the basis of this kind of a molecular bar code.     

Molecular phylogeny of the New World monkeys (Platyrrhini,primates) based on two unlinked nuclear genes: IRBP intron 1 and ϵ-globin sequences

Nuclear sequences of the 1.8 kilobase (kb) long intron 1 of the interstitial retinol-binding protein gene (IRBP), previously determined for 11 of the 16 extant genera of New World monkeys (superfamily Ceboidea, infraorder Platyrrhini), have now been determined for the remaining 5 genera. The maximum parsimony trees found, first with IRBP sequences alone and then with tandemly combined IRBP and ϵ-globin gene sequences from the same species, supported a provisional cladistic classification with the following clusters. Subtribes Callitrichina (Callithrix, Cebuella), Callimiconina (Callimico), Leontopithecina (Leontopithecus) and Saguina (Saguinus) constitute subfamily Callitrichinae, and subfamilies Callitrichinae, Aotinae (Aotus), and Cebinae (Cebus, Saimiri) constitute family Cebidae. Subtribes Chiropotina (Chiropotes, Cacajao) and Pitheciina (Pithecia) constitute tribe Pitheciini; and tribes Pitheciini and Callicebini (Callicebus) constitute subfamily Pitheciinae. Subtribes Brachytelina (Brachyteles, Lagothrix) and Atelina (Ateles) constitute tribe Atelini, and tribes Atelini and Alouattini (Alouatta) constitute subfamily Atelinae. The parsimony results were equivocal as to whether Pitheciinae should be grouped with Atelinae in family Atelidae or have its own family Pitheciidae. The cladistic groupings of extant ceboids were also examined by different stochastic evolutionary models that employed the same stochastic process of nucleotide substitutions but alternative putative phylogenetic trees on which the nucleotide substitutions occurred. Each model, i.e., each different tree, predicted a different multinomial distribution of nucleotide character patterns for the contemporary sequences. The predicted distributions that were closest to the actual observed distributions identified the best fitting trees. The cladistic relationships depicted in these best fitting trees agreed in almost all cases with those depicted in the maximum parsimony trees. © 1996 Wiley-Liss, Inc.     

Phylogenetic analysis of Altai osmans of the genus Oreoleuciscus (Pisces, Cyprinidae, Leuciscinae), based on the analysis of the cytochrome oxidase 1 gene (Co-1) sequence

Molecular genetic analysis of Altai osmans of the genus Oreoleuciscus from two different parts of the range was carried out. In this study, based on the mitochondrial Co-1 gene sequence, a total of 25 fish specimens belonging to four genera were examined: (1) O. humilis, 2 specimens; O. potanini, 13 specimens; (2) Pseudaspius leptocephalus, 1 specimen; (3) Tribolodon brandtii, T. hakonensis, and T. sachalinensis from the GenBank database, 8 speciens; and (4) Leuciscus waleckii, 1 specimen (used as an outgroup). The p-distances were very low both within and between the species: (1) 0.20 +/- 0.03%; (2) 0.40 +/- 0.12%; and (1-2) 0.80 +/- 0.04%. To visualize the relationships among all of the species examined, the neighbor joining (NJ), maximum parsimony (MP), Bayesian (BA), and maximum likelihood (ML) trees were constructed. The results obtained using these methods were very similar. It was demonstrated that species assignment of the individuals (barcoding) with the help Co-1 gene was effective, despite of very low divergence of the two osman taxa, which was comparable with typical intraspecific values in other animal groups. Taxonomic status of O. potanini and O. humilis requires further investigation with paying attention to low genetic distances between these species along with the lack of material from sympatric parts of the ranges.     

The phylogeny of <Emphasis Type="Italic">Schistidium</Emphasis> (Bryophyta,Grimmiaceae) based on the primary and secondary structure of nuclear rDNA internal transcribed spacers

Phylogeny of Schistidium (Bryophyta, Grimmiaceae) was studied by comparing the nucleotide sequences of internal transcribed spacers ITS1-2 of nuclear rDNA and the trnT-trnD region of chloroplast DNA. Phylogenetic trees constructed based on nuclear and chloroplast sequences were consistent, comprising a basal grade and two large clades. Morphological characteristics specific for these clades were described. Secondary structures of ITS1 and ITS2 Schistidium species were modeled using thermodynamic criteria. Four different structures of the longest ITS1 hairpin were identified. These results were used to analyze possible paths of Schistidium evolution. Characteristics of the ITS2 secondary structure support the two major clades recognized in the phylogenetic trees.     

Population genetic structure of northern Dolly Varden char <Emphasis Type="Italic">Salvelinus malma malma</Emphasis> in Asia and North America

The level of genetic differentiation of northern Dolly Varden char Salvelinus malma malma from Asia and North America was evaluated using the data on mtDNA variation (regions ND1/ND2, ND5/ND6, and Cytb/D-loop) obtained by means of PCR-RFLP analysis. For S. m. malma, the mean values of haplo-type and nucleotide diversity were 0.5261 ± 0.00388 and 0.001558, respectively. The mean estimate of the population nucleotide divergence constituted 0.055%. It was demonstrated that S. m. malma on the most part of the species range examined (drainages of the Beaufort Sea, Chukotka Sea, Bering Sea, and the Sea of Okhotsk) was characterized by the population genetic structure with the low level of genetic differentiation and divergence. At the same time, populations from the Pacific Ocean Gulf of Alaska demonstrated marked genetic differentiation, supported by the high pairwise G4ST values (from 0.4198 to 0.5211) and nucleotide divergence estimates (mean divergence, 0.129%), from Asian and North American populations. Analysis of molecular variance (AMOVA) showed that most of the mtDNA variation in S. m. malma fell in the intrapopulation component (72.5%). At the same time, the differences between the populations (21.1%) and between the regions (6.4%) made lower contribution to the total variation.     

Sequence diversity of a domesticated transposase gene, <Emphasis Type="Italic">MUG1</Emphasis>, in <Emphasis Type="Italic">Oryza</Emphasis> species

MUG1 is a MULE transposon-related domesticated gene in plants. We assessed the sequence diversity, neutrality, expression, and phylogenetics of the MUG1 gene among Oryza ssp. We found MUG1 expression in all tissues analyzed, with different levels in O. sativa. There were 408 variation sites in the 3886 bp of MUG1 locus. The nucleotide diversity of the MUG1 was higher than functionally known genes in rice. The nucleotide diversity (π) in the domains was lower than the average nucleotide diversity in whole coding region. The π values in nonsynonymous sites were lower than those of synonymous sites. Tajima D and Fu and Li D* values were mostly negative values, suggesting purifying selection in MUG1 sequences of Oryza ssp. Genome-specific variation and phylogenetic analyses show a general grouping of MUG1 sequences congruent with Oryza ssp. biogeography; however, our MUG1 phylogenetic results, in combination with separate B and D genome studies, might suggest an early divergence of the Oryza ssp. by continental drift of Gondwanaland. O. longistaminata MUG1 divergence from other AA diploids suggests that it might not be a direct ancestor of the African rice species. These authors contributed equally to this work.     

Radiation of the endemic genus Dendroseris (Asteraceae) on the Juan Fernandez Islands: evidence from sequences of the its regions of nuclear ribosomal DNA

Phylogenetic relationships among nine of the 11 species of the endemic genus Dendroseris on the Juan Fernandez Islands were inferred from nucleotide sequences of the internal transcribed spacer regions (ITS) of the 18-26S nuclear ribosomal DNA. Sequences were determined for 15 populations of Dendroseris and one population for each of two outgroups from the genera Sonchus and Sventenia. Little length variation was detected in the ITS regions of Dendroseris, with ITS 1 253 or 254 bp long and ITS 2 224 or 225 bp. The sequence data provide strong support for the holophyly of Dendroseris despite the distinct morphological differences among the three subgenera. The molecular data also indicate that subg. Dendroseris and Phoenicoseris are holophyletic, but do not support holophyly of subg. Rea. The ITS sequences did not resolve relationships among subgenera, supporting the hypothesis of rapid adaptive radiation of Dendroseris on the islands. Relative rate tests indicate that rates of nucleotide substitutions in the ITS regions are not significantly different among the different lineages of Dendroseris following adaptive radiation. Comparisons of average pairwise sequence divergence of Dendroseris species in the ITS regions and chloroplast genome indicated that ITS sequences have evolved about 38 times faster than cpDNA in the genus. Rates of ITS sequence divergence of Dendroseris were estimated to be faster than (3.94 ± 0.10) × 10^-9 per site per year, and likely (6.06 ±0.15) × 10^-9 per site per year.     

Adaptive intraspecific divergence: An example using the animal cytochrome <Emphasis Type="Italic">b</Emphasis> gene

The topologies of phylogenetic trees characterized by a high level of intraspecific divergence between the phylogenetic DNA groups (clades) are often explained in terms of the theory of Pleistocene refugia. To elucidate the issue of the adaptive role of intraspecific divergence, the changes in the physicochemical properties of amino acids in the course of cladogenesis (MM01 model of the TreeSAAP 3.2 package) were analyzed in this work using as an example the nucleotide sequences of the cytochrome b gene in some species of northern animals (lemmings, redbacked voles, chipmunks, flying squirrels, ermines). It was shown that the process of intraspecific divergence was rarely accompanied by radical amino acid substitutions in cytochrome b caused by adaptation (directional selection). In connection with this, the hypothesis is discussed according to which the adaptive variants formed in the species at the peak of cold were lost with climatic warming due to the drift or selection against individuals adapted to cold.     

Phylogenetic Analysis of Mos1-Like Transposable Elements in the Drosophilidae

We have performed a phylogenetic analysis of 59 mariner elements in 14 Drosophilidae species that are related to the active Drosophila mauritiana Mos1 element. This includes 38 previously described sequences and 21 new sequences amplified by PCR from 10 species. Most of the elements detected are nonfunctional due to several frameshifts and deletions. They have been subdivided into four groups according to specific signatures in the nucleotidic and amino acid sequences. The mean nucleotide diversity is 4.8 ± 0.1% and reflects mainly the divergence of inactive elements over different periods. Although this probably gives rise to occasional homoplasies between distantly related taxa, the elements of each species remain grouped together. Horizontal transfer, reported previously between D. mauritiana and Zaprionus tuberculatus, can be extended to Z. verruca, while the Mos1-like element of Z. indianus belongs to another group. Interpretation of the phylogeny leads to a comparison of the influence of common ancestral sequences and putative horizontal transfers. Received: 31 May 1999 / Accepted: 28 June 1999     

<Emphasis Type="Italic">Gallus gallus</Emphasis> aggrecan gene-based phylogenetic analysis of selected avian taxonomic groups

Mitochondrial DNA (mtDNA) sequences remain the most widely used for phylogenetic analysis in birds. A major limitation of mtDNA sequences, however, is that mitochondria genes are inherited as a single linkage group. Here we describe the use of a 540-bp DNA sequence corresponding to the G3 domain of Gallus gallus nuclear aggrecan gene (AGC1) for phylogenetic analysis of the main groups of Galliformes including Phasianidae, Numididae, and Odontophoridae. We also included species from Cracidae and Megapodiidae which are considered by some as Craciformes and others, including here as Galliformes. The uncorrected sequence divergence of the G3 fragments ranges from 1 among the grouses to 36% between some of the distant groups within Galliformes. These sequences contain 39–48% AT nucleotides and the ratios of transition versus transversion are above 1.5 in majority of the comparisons. Using G3 sequences from an Anseriform, Oxyura jamaicensis, as out-groups, phylogenetic trees were obtained using maximum parsimony and distance algorithms and bootstrap analyses. These trees were consistent with those described using Avian sarcoma and leucosis virus gag genes and those from amino acid sequences of hemoglobin and lysozyme c. Our data also support relationships among Galliformes which were defined using mtDNA sequences. In addition to the general support of the five main families of Galliformes, our data are also consistent with previous work that showed Francolinus africanus and Gallus gallus are in the same clade and that Tetraoninae is a well-supported monophyletic subfamily within Phasianidae. The results presented here suggest that the AGC1 sequences meet the criterion of novel nuclear DNA sequences that can be used to help resolve the relationships among Galliformes.     

Molecular diversity of chrysoviruses in Korean isolates of a new fungal species, <Emphasis Type="Italic">Cryphonectria nitschkei</Emphasis>

Genetic diversity of the chrysovirus within the four fungal strains was analyzed by comparing the full-length sequences of cloned chrysoviral genes encoding the RNA-dependent RNA polymerase (RdRp) and capsid protein (CP). Because the morphological characteristics of four chrysovirus-infected Cryphonectria spp. strains were different, strain identification was conducted via sequence comparison of the internal transcribed spacers (ITSs) of the fungal rRNA gene. Phylogenic analysis of the ITS regions revealed that the four strains were closely clustered with the reference strain of Cryphonectria nitschkei, while they were more distantly related to other common Cryphonectria species, indicating that they were likely C. nitschkei. Sequence comparison among chrysoviruses from Korean C. nitschkei strains revealed that similarities of the RdRp and CP genes ranged from 98% to 100% and from 95% to 100%, respectively, at the protein level. Their corresponding nucleotide sequences showed 97% to 100% and 84% to 100% identities, respectively. Compared to RdRp, the CP gene had more divergence, suggesting the presence of genes possessing different evolutionary rates within the chrysovirus genome. Sequence comparisons with other known chrysoviruses showed that the four Korean chrysoviruses were clustered together at the next lineage level. Discovering why two strains (bsl31 and bsl32) containing identical ITS sequences and chrysoviruses display different phenotypes should prove interesting.     

Polymorphism of the <Emphasis Type="Italic">cox1</Emphasis> mtDNA gene from cercarial isolates of the avian schistosome <Emphasis Type="Italic">Bilharziella polonica</Emphasis> (Trematoda: Schistosomatidae) from Belarussian lakes

We have studied the phylogeographic structure of avian schistosomes Bilharziella polonica (Trematoda: Schistosomatidae), parasites of 18 molluscs Planorbarius corneus (family Planorbidae) from three Belarussian lakes. Low nucleotide (π < 0.5%) and high haplotype (h = 85.6%) diversity of the gene encoding the cytochrome C oxidase first subunit (cox1) was found on the part of the species range studied. The phylogenetic reconstructions showed that the sample examined consists of at least two lineages of haplotypes, A and C. Haplotype diversity was somewhat higher in lineage C. The genetic divergence between these genealogical lines reached 0.55%, while the time of possible divergence was 180000–270000 years. Possible evolutionary scenarios for differentiation of the B. polonica lines and effectiveness of using cox1 for barcoding trematode populations and finding coevolutionary parasite-host relationships are discussed.     

Molecular phylogenetics of the subtribeGentianinae (Gentianaceae) inferred from the sequences of internal transcribed spacers (ITS) of nuclear ribosomal DNA

The internal transcribed spacer (ITS) regions of 18S–25S nuclear ribosomal DNA from representatives of 23 species of the subtribeGentianinae and one outgroup species (Centaurium capitatum) were analyzed by polymerase chain reaction amplification and direct DNA sequencing. Within the taxa analyzed, the length of the ITS1 region varied from 221 to 233 bp, ITS2 from 226 to 234 bp. Of the aligned sequences of 497 positions, 151 sites involved gaps or nucleotide ambiguity, 133 were invariable and 213 showed divergence. In pairwise comparisons among the taxa of the subtribeGentianinae and the outgroup, sequence divergence ranged from 1.3% to 34.1% in ITS1, from 0 to 28.1% in ITS2 and from 0.6% to 27.5% in combined ITS1 and ITS2. Phylogenetic trees generated from ITS sequences were highly resolutive and principally concordant with morphological classifications for the major phylogenetic divisions in the subtribe. An ancient divergence leading to two evolutionary lines was suggested in the subtribe by both DNA sequence and morphological data. One line encompasses the generaGentiana, Crawfurdia andTripterospermum, morphologically characterized by their glands on the base of ovary and their plicate corolla, while the other line involves all other members of the subcribe surveyed, characterized by their epipetalous glands and simple corolla without plicae.Megacodon, with glands on the base of ovary but without plicae on its corolla, was revealed to be more related to the latter group than to the former.Comastoma, Gentianella andGentianopsis were shown to be well-defined monophyletic genera.Pterygocalyx showed much closer affinity toGentianopsis than to any other genus. Some conflictions were detected in the genusSwertia.