Phylogenetic Relationships of Globodera millefolii,G. artemisiae,and Cactodera salina Based on ITS Region of Ribosomal DNA

Globodera millefolii and G. artemisiae are interesting because their type localities (Estonia and Russia, respectively) are geographically distant from those of the potato cyst nematodes and other Globodera species that seem to have originated in the Western world, and because the type host for each is a member of Compositae rather than Solanaceae. Sequence data for ITS1, ITS2, and 5.8S ribosomal DNA (ITS rDNA) for G. millefolii and G. artemisiae were nearly identical to sequence data for Cactodera salina from the rhizosphere of the estuary plant Salicornia bigelovii in Sonora, Mexico. The ITS rDNA sequences of these three species were all about 94% similar to those of two other Cactodera species for which ITS rDNA data were obtained. Phylogenetic analysis indicated that, based on the ITS rDNA data, G. millefolii and G. artemisiae are more closely related phylogenetically to the Cactodera species than to other nominal Globodera species. The molecular data further suggest that the genus Cactodera may comprise two or more morphologically similar but separate groups.     

Molecular barcoding for central-eastern European <Emphasis Type="Italic">Crioceris</Emphasis> leaf-beetles (Coleoptera: Chrysomelidae)

Among Crioceris leaf-beetles, the two most widespread species (Crioceris asparagi and C. duodecimpunctata) are serious invasive plant pests, while another two (C. quatuordecimpunctata and C. quinquepunctata) are rare species restricted to steppe-like habitats in Eurasia. The aim of the research was to check the genetic distinctiveness of these four species and develop barcodes for their molecular identification using the mitochondrial Cytochrome Oxidase I (COI) gene and two nuclear markers: Elongation Factor 1-α (EF1-α) and Internal Transcribed Spacer 1 (ITS1). The identification of each species was possible and reliable with the use of COI and ITS1 markers. EF1-α was omitted in analyses due to its high level of heterozygosity (presence of multiple PCR products). C. duodecimpunctata and C. quatuordecimpunctata were shown to be sister taxa, but the similar genetic distances between all of the species indicate that these species originated almost simultaneously from a common ancestor. Identification of two separate clades in populations of C. quatuordecimpunctata suggested that the clades are isolated and can be considered as separate conservation units.     

Mitochondrial DNA variation of Gyrodactylus spp (Monogenea, Gyrodactylidae) populations infecting Atlantic salmon, grayling, and rainbow trout in Norway and Sweden

Approximately 800 bp of the mitochondrial cytochrome oxidase I (COI) gene were sequenced from 76 Gyrodactylus specimens of 32 salmonid host populations, i.e. from Salmo salar, Thymallus thymallus, and Oncorhynchus mykiss in Norway, Sweden and Latvia. The COI sequences indicated a substantial intraspecific differentiation of Gyrodactylus salaris and Gyrodactylus thymalli. In total, 12 haplotypes were identified which group into five well supported clades, three clades with parasites from Atlantic salmon and two clades with parasites from grayling. The basal nodes linking the five clades together are only weakly supported. Thus, there is no support for the monophyly of all G. salaris haplotypes and the monophyly of all G. thymalli haplotypes. The lack of monophyly of the mitochondrial haplotypes of G. salaris and G. thymalli may indicate that G. salaris and G. thymalli represent (i). two polytypic species or (ii). one polytypic species, or (iii). refer to a complex of more than two sibling species. The mtDNA data indicate multiple introductions of G. salaris and G. thymalli into Norway. A minimum of three independent introductions of G. salaris and two independent introductions of G. thymalli are supported. This is congruent with earlier hypotheses on the introduction of G. salaris and G. thymalli into Norway.     

Molecular phylogenetic analysis of white prawns species and the existence of two clades in Penaeus merguiensis

Closely related species, Penaeus merguiensis and Penaeus silasi from Thai waters, were genetically examined using variation observed in 558 base pairs (bp) of sequence from cytochrome oxidase subunit I (COI) gene of mtDNA. The sequence divergences of COI between P. merguiensis and other Penaeus species were 5.76-6.15% (P. silasi), 13.17-13.97% (Penaeus indicus), 16.43% (Penaeus vannamei), 16.63% (Penaeus monodon), and 18.37% (Penaeus japonicus). From the alignment reported that there were four clades on phylogenetic tree, the distinction of the two monophyletic clades was referred as P. merguiensis, one monophyletic clade within P. silasi and P. indicus. These results point toward the possibility of P. merguiensis being a complex of two cryptic species or a single species with strong phylogeographic subdivision.     

Two new species of Gyrodactylus von Nordmann, 1832 from Profundulus oaxacae (Pisces: Profundulidae) from Oaxaca,Mexico, studied by morphology and molecular analyses

In the present study, two new species of Gyrodactylus are described from Profundulus oaxacae, a fish endemic to the Pacific slope of Oaxaca State, Mexico. Fishes were collected within their distribution range in 5 localities in the Atoyac-Verde River. Gyrodactylus montealbani n. sp. and G. zapoteco n. sp. were erected and characterized morphologically (sclerites of the attachment apparatus and the male copulatory organ) and molecularly (sequences of the Internal Transcribed Spacer region of rDNA). The haptoral sclerites of the new species are similar to those of Gyrodactylus iunuri and Gyrodactylus tepari, both recently described from the goodeid fish Goodea atripinnis, from the Mexican States of Jalisco and Querétaro, respectively; and to Gyrodactylus xtachuna described from the poeciliid Poeciliopsis gracilis in Veracruz State, Mexico – nonetheless, these species can all be discriminated based on their marginal hook morphology. Specimens of G. montealbani n. sp. and G. zapoteco n. sp. were sequenced, and were aligned with sequences of 25 other Gyrodactylus spp. Both Maximum likelihood and Bayesian inference analyses indicated that the two new species are members of independent, well-supported lineages – these are the first Gyrodactylus species described from Profundulus oaxacae.     

ITS-2 secondary structures and phylogeny of Anopheles culicifacies species

Background: Second internal transcribed spacer (ITS2) has proven to contain useful biological information at higher taxonomic levels. Objectives: This study was carried out to unravel the biological information in the ITS2 region of An. culicifacies and the internal relationships between the five species of Anopheles culicifacies. Methodology: In achieving these objectives, twenty two ITS2 sequences (~370bp) of An. culicifacies species were retrieved from GenBank and secondary structures were generated. For the refinement of the primary structures, i.e. nucleotide sequence of ITS2 sequences, generated secondary structures were used. The improved ITS2 primary structures sequences were then aligned and used for the construction of phylogenetic trees. Results and discussions: ITS2 secondary structures of culicifacies closely resembled near universal eukaryotes secondary structure and had three helices, and the structures of helix II and distal region of helix III of ITS2 of An. culicifacies were strikingly similar to those regions of other organisms strengthening possible involvement of these regions in rRNA biogenesis. Phylogenetic analysis of improved ITS2 sequences revealed two main clades one representing sibling B, C and E and A and D in the other. Conclusions: Near sequence identity of ITS2 regions of the members in a particular clade indicate that this region is undergoing parallel evolution to perform clade specific RNA biogenesis. The divergence of certain isolates of An. culicifacies from main clades in phylogenetic analyses suggests the possible existence of camouflaged sub-species within the complex of culicifacies. Using the fixed nucleotide differences, we estimate that these two clades have diverged nearly 3.3 million years ago, while the sibling species in clade 2 are under less evolutionary pressure, which may have evolved much later than the members in clade 1.     

First report of Gyrodactylus spp. (Platyhelminthes: Monogenea) in the western Mediterranean Sea: molecular and morphological descriptions

The Gyrodactylus spp. fauna on species of gobies, Pomatoschistus, Gobiusculus, and Knipowitschia (Gobiidae: Teleostei), from the western Mediterranean and Adriatic seas is strikingly similar to that found in the Baltic Sea and eastern Atlantic Ocean, both in morphology and in internal transcribed spacer (ITS) rRNA. The fauna consisted of Gyrodactylus branchialis, G. ostendicus, G. gondae, G. rugiensis, G. rugiensoides, and G. arcuatus. No new species have been found. A morphometric comparison between G. branchialis from the Mediterranean and Adriatic seas and its type locality in Ostend (Belgium) showed significant differences in ventral bar and marginal hook features. The morphometric variation was lower in G. rugiensis, whereas no significant differences were found in G. ostendicus. Gyrodactylus branchialis and G. ostendicus collected on P. microps were slightly different in the ITS rDNA (-0.6%) compared with specimens on the closely related P. marmoratus, probably reflecting ongoing speciation. A hybrid zone was identified in the Vaccarès lagoon complex (France) where both host species are sympatric. There was no clear geographic or host-related pattern in the variation found in the ITS2 rDNA in G. arcuatus sampled from P. microps, G. flavescens, Pungitius pungitius, K. panizzae, and its original host Gasterosteus aculeatus (2 polymorphic sites). Of all studied species, only G. arcuatus, G. rugiensis, and G. rugiensoides showed minor intraspecific variation in the ITS rDNA. Hence, the physical separation by a shoreline of more than 10,000 km is hardly reflected in the parasite ITS rDNA.     

Mitochondrial COI and morphological specificity of the mealy aphids (Hyalopterus ssp.) collected from different?hosts in Europe (Hemiptera,Aphididae)

Forty three European population samples of mealy aphids from various winter and summer host plants were attributed to respective species of Hyalopterus by means of their partial sequences of mitochondrial COI gene. Used Hyalopterus samples emerged as monophyletic relative to outgroup and formed three major clades representing three host specific mealy aphid species in the Neighbor joining, Maximum parsimony, Maximum likelihood and Bayesian inference trees. Hyalopterus pruni and Hyalopterus persikonus emerged as a sister species, whilst Hyalopterus amygdali was located basally. Samples representing different clades in the molecular trees were used for canonical discrimination analysis based on twenty two morphological characters. Length of the median dorsal head hair enabled a 97.3 % separation of Hyalopterus amygdali from the remaining two species. No single character enabled satisfactory discrimination between apterous viviparous females of Hyalopterus pruni and Hyalopterus persikonus. A modified key for the morphological identification of Hyalopterus species is suggested and their taxonomic status discussed.     

Species delimitation in the European species of Clavulina (Cantharellales, Basidiomycota) inferred from phylogenetic analyses of ITS region and morphological data

The identification of the conventionally accepted species of Clavulina (Cantharellales, Basidiomycota) in Europe (Clavulina amethystina, Clavulina cinerea, Clavulina cristata, and Clavulina rugosa) is often difficult and many specimens are not straightforwardly assignable to any of those four species, which is why some authors have questioned their identity. In order to assess the status of those species, a morphological examination was combined with the molecular analysis of the ITS region. The same six major clades were obtained in the Bayesian and parsimony phylogenetic analyses, and all six clades were well-supported at least by one of the analyses. Morphological characters, such as the overall branching pattern, the presence and intensity of grey colour, the cristation of the apices, and basidiospore size and shape were to various extents correlated with the phylogenetic signal obtained from the ITS region. The congruence between the molecular analyses and morphology, rather than geographical origin, suggests the existence of several species that can be delimited using a combined phylogenetic and morphological species recognition. The analyses revealed that C. cristata and C. rugosa are well-delimited species. In contrast, more than one taxa could be subsumed under the names C. amethystina and C. cinerea, the taxonomical complexity of which is discussed. The ITS region is proved to be adequate to separate phylogenetic species of Clavulina.     

DNA barcodes and phylogenetic affinities of the terrestrial slugs Arion gilvus and A. ponsi (Gastropoda,?Pulmonata,Arionidae)

The Iberian Peninsula is a region with a high endemicity of species of the terrestrial slug subgenus Mesarion. Many of these species have been described mainly on subtle differences in their proximal genitalia. It therefore remains to be investigated 1) whether these locally diverged taxa also represent different species under a phylogenetic species concept as has been shown for other Mesarion species outside the Iberian Peninsula, and 2) how these taxa are phylogenetically related. Here, we analysed DNA sequence data of two mitochondrial (COI and 16S) genes, and of the nuclear ITS1 region, to explore the phylogenetic affinities of two of these endemic taxa, viz. Arion gilvus Torres Mínguez, 1925 and A. ponsi Quintana Cardona, 2007. We also evaluated the use of these DNA sequence data as DNA barcodes for both species. Our results showed that ITS did not allow to differentiate among most of the Mesarion molecular operational taxonomic units (MOTUs) / morphospecies in Mesarion. Yet, the overall mean p-distance among the Mesarion MOTUs / morphospecies for both mtDNA fragments (16.7% for COI, 13% for 16S) was comparable to that between A. ponsi and its closest relative A. molinae (COI: 14.2%; 16S: 16.2%) and to that between A. gilvus and its closest relative A. urbiae (COI: 14.4%; 16S: 13.4%). Hence, with respect to mtDNA divergence, both A. ponsi and A. gilvus, behave as other Mesarion species or putative species-level MOTUs and thus are confirmed as distinct ‘species’.     

Morphometric comparison of three members of the Gyrodactylus nemachili-like species group (Monogenea: Gyrodactylidae) on Barbatula barbatula L. in the Czech Republic, with a reinstatement of G. papernai Ergens & Bychowsky, 1967

The Gyrodactylus nemachili-like species complex, parasitising stone loach Barbatula barbatula L. in the Czech Republic, was investigated based on the detailed morphometric analysis of its haptoral hard parts. The existence of two species, G. jiroveci Ergens & Bychowsky, 1967 and G. pseudonemachili Ergens & Bychowsky, 1967 was confirmed. A standard discriminant analysis of the morphometric data showed a clear separation between specimens of G. jiroveci and another species or variant. Based on the morphology of the haptoral hard parts, this third species was more similar to G. jiroveci than to G. pseudonemachili. The differences between G. jiroveci and the third species were mainly in the morphology of the marginal hook sickle, but included the total length of the marginal hook, the length of the hamulus point, the length of the marginal hook sickle, the width of the ventral bar, and the width of the dorsal bar. Based on comparison with the original material, the third species was identified as G. papernai Ergens & Bychowsky, 1967, which has recently been considered a synonym of G. jiroveci. These analyses provided sufficient evidence for the restoration of G. papernai as a valid taxon. Partial sequences of the internal transcribed spacer rRNA genes (ITS rDNA) verified the validity of all three Gyrodactylus species studied.     

The Praon dorsale–yomenae s.str. complex (Hymenoptera,Braconidae, Aphidiinae): Species discrimination using geometric morphometrics and molecular markers with description of a new species

In this paper we apply an integrative approach combining morphometric and molecular analyses to explore parasitoids of the Praon dorsale–yomenae s.str. complex. These parasitoids occur in natural and agricultural ecosystems throughout the Palaearctic and parasitize aphid hosts belonging to the tribe Macrosiphini. The P. dorsale–yomenae species complex represents a morphologically cryptic group, consisting of several hidden taxa with specific host adaptations and distributions. For the morphometric analyses we used a large dataset comprising 230 female specimens that emerged from 30 different species of aphid hosts throughout the Palaearctic. The molecular analysis included a reduced dataset of 44 specimens that emerged from 19 aphid hosts. The mitochondrial cytochrome oxidase subunit I (COI) and the nuclear second expansion segment of the 28S rRNA gene (28SD2) were used to estimate a genetic diversity within this complex. Although all Praon species clustered closely together in the 28SD2 tree, confirming their close relatedness, the molecular characterization based on COI identified five clearly separate taxa with sequence divergences in the range of 4.7–8.9%. These taxa also exhibited significant differences in forewing shape as revealed by geometric morphometric analyses. Classical morphometric analyses revealed the length of m-cu vein as a new taxonomic character, but suggested that one commonly used trait, the color pattern of the Rs + M and m-cu veins, cannot be used for species distinction as it was highly variable in one of the taxa. Based on the combined morphometric and genetic data, we confirm the species status of Praon dorsale, P. yomenae, P. longicorne and P. volucre, and describe and illustrate a new parasitoid species of the “dorsale–yomenae” group, i.e. Praon staticobii n.sp. associated with Staticobium limonii on Limonium angustifolium.     

Morphological and Molecular Characterization of Reared Parasitoid Wasps of the Genus Glyptapanteles Ashmead 1904 (Insecta: Hymenoptera: Braconidae: Microgastrinae) Associated with Lepidoptera in India

Glyptapanteles Ashmead (Hymenoptera: Braconidae: Microgastrinae) is a cosmopolitan group of hyperdiverse parasitic wasps. The genus remains taxonomically challenging in India due to its highly speciose nature, morphological similarity amongst species and negligible host records. The Indian fauna is one of the most diverse and also the least studied. The present study is based on 60 populations reared from 35 host species, 100+ individual caterpillar rearings (1100 wasp specimens pinned and 2000 in alcohol) and from 12 different geographical locations of the country (11 states and one Union territory) that represent 26 provisional Glyptapanteles species within 8 species-groups. Out of 60 populations, phylogenetic analyses were performed on 38 based on mitochondrial cytochrome oxidase subunit I (COI) nucleotide sequences. Maximum likelihood and Bayesian inference methods displayed three and four major discrete Glyptapanteles clades, respectively. In clade A very few Indian species were grouped along with Neotropical and Thailand species. The other clades B and C grouped the majority of the Indian species and showed considerable host specificity in both the trees. All parasitic wasp species were gregarious in nature, except for two populations. Three different sets of data (morphology, host records, and COI) were integrated in order to generate accurate boundaries between species/species-groups. Illustrations of all parasitized caterpillars/cocoons and 42 habitus views of Glyptapanteles spp., distributional information, and GenBank accession numbers, are presented. The present study, perhaps the most comprehensive done to date in India, suggests the presence of several additional Glyptapanteles species, which were previously unrecognized.     

Cytochrome oxidase I sequences reveal possible cryptic diversity in the cosmopolitan symbiotic copepod Nesippus orientalis Heller, 1868 (Pandaridae: Siphonostomatoida) on elasmobranch hosts from the KwaZulu-Natal coast of South Africa

Over the past decade, numerous molecular phylogenetic studies uncovered cryptic diversity within the Copepoda, yet very few investigations focused on symbiotic copepods. Here we report mitochondrial DNA cytochrome oxidase I diversity in the cosmopolitan elasmobranch symbiont Nesippus orientalis off the KwaZulu-Natal coast of South Africa. Analysis of partial COI sequences of copepods sampled from a diversity of shark hosts, revealed the presence of two divergent clades. Diversity within the clades does not appear to be structured based on host species, host individual, geographic locality or time of sampling. However, divergence between the two clades seems to be related to host species. Phylogenetic analyses of representatives from the two clades, along with Nesippus spp., Caligus spp. and Lepeophtheirus spp. outgroups, further supports the distinction between the two clades. Future molecular phylogenetic investigations of widespread copepod symbionts most likely will reveal far greater levels of biodiversity than currently recognized.     

Relationships within aphids Cinara (Cupressobium) (Hemiptera) based on mitochondrial and nuclear DNA sequences

The relationships between Cinara (Cupressobium) aphids inhabiting woody parts and leaves of conifers belonging to Cupressaceae have been studied using a mitochondrial gene (COI) and a nuclear gene (EF1-α). Based on the COI sequences, genetic distances between species ranged from 5.6 % between Cinara (C.) tujafilina (del Guercio) and Cinara (C.) juniperi (De Geer) to 10.5 % between C. (C.) tujafilina and Cinara (C.) mordvilkoi (Pa?ek). Genetic distances among EF1-α sequences were lower and showed from 0.1 % between C. cupressi and C. juniperi to 2.3 % between C. tujafilina and C. mordvilkoi. Molecular phylogenetic trees were constructed using the Bayesian inference (BI) phylogenetic analysis and maximum parsimony (MP) criterion. Phylogenetic trees obtained based on COI and EF1-α marker genes created two sister clades. Our results indicate that Cinara (Cupressobium) are a monophyletic group of aphids. Phylogenetic relationships amongst Cupressobium aphids do not result from the association with the host plant, but from the feeding site on the host plant or an ability to change the microhabitat on the plant. As closely related species inhabit similar microhabitats on different host plants, it suggests that the host switching is the main mode of speciation in this subgenus.     

Host-associated speciation in the coral barnacle Wanella milleporae (Cirripedia: Pyrgomatidae) inhabiting the Millepora coral

Speciation by host shift is a common phenomenon observed in many symbiotic animals. The symbiont–host interaction is highly dynamic, but it is poorly documented in the marine realm. In the present study, we examined the genetic and morphological differentiation of the coral barnacle Wanella milleporae (obligate to fire corals) collected from four different Millepora host species in Taiwan to investigate the host specificity of this barnacle. Phylogenetic analysis of mitochondrial COI gene for 241 individuals of Wanella revealed five distinct clades, whose sequence divergences are comparable to values between other cogeneric barnacle species. The five clades also differ in shell and opercular plate morphology and colour. Genetic and morphological differentiations together strongly suggest the presence of cryptic species. Although the five clades do not display species-level host specificity, they showed a significant difference in preference on host growth form. Clades 1 and 2 were predominantly found on encrusting Millepora exaesa and Millepora platyphylla , while clades 3, 4 and 5 live exclusively on branching-form fire corals Millepora dichotoma and Millepora tenella . Phylogeny inferred from the combined mitochondrial COI, 16S and 12S (2182 bp) analysis suggests the division of the five clades into two major lineages congruent with the morphology of the host coral. Multiple independent invasions to the same form of host and subsequent speciation are evident in the Red Sea and Taiwan. Our results indicate that ecological/sympatric speciation could occur in marine symbiotic invertebrates through host shift and specialization. It appears that, as in their terrestrial counterparts, host–symbiont radiations in the marine realm are more prevalent than we expected and thus warrant further investigation.     

Symbiodinium diversity in the soft coral Heteroxenia sp. and its nudibranch predator Phyllodesmium lizardensis

We examined the diversity of the photosynthetic dinoflagellate, Symbiodinium, over a 2-year period in two invertebrates from Australia’s Northern Great Barrier Reef: the nudibranch Phyllodesmium lizardensis and an octocoral of the genus Heteroxenia. In years one and two, we used denaturing gradient gel electrophoresis with internal transcribed spacer 2 (ITS2) region amplicons and identified two nearly identical genotypes of clade C (C64 and a variant) in all samples of each species. We examined the secondary structure of both sequences and found that each had predicted ∆G values within the range of stable free energy values for Symbiodinium ITS2 sequences. In year two, we also used real-time quantitative polymerase chain reaction assays (qPCR) with clade-specific internal transcribed spacer 1 primers to determine whether there were cryptic clades (A, B, and/or D) associated with either host in addition to clade C. qPCR revealed that clades B, C, and D were present in all animals of both species and that all but two nudibranch samples also harbored clade A. These findings suggest that there may be more flexibility in this host/symbiont interaction than has previously been assumed.

     

Genetic compatibility between Anopheles lesteri from Korea and Anopheles paraliae from Thailand

To assess differentiation and relationships between Anopheles lesteri and Anopheles paraliae we established three and five iso-female lines of An. lesteri from Korea and An. paraliae from Thailand, respectively. These isolines were used to investigate the genetic relationships between the two taxa by crossing experiments and by comparing DNA sequences of ribosomal DNA second internal transcribed spacer (ITS2) and mitochondrial DNA cytochrome c oxidase subunit I (COI) and subunit II (COII). Results of reciprocal and F₁-hybrid crosses between An. lesteri and An. paraliae indicated that they were compatible genetically producing viable progenies and complete synaptic salivary gland polytene chromosomes without inversion loops in all chromosome arms. The pairwise genetic distances of ITS2, COI and COII between these morphological species were 0.040, 0.007-0.017 and 0.008-0.011, respectively. The specific species status of An. paraliae in Thailand and/or other parts of the continent are discussed.