D2 Region Of 28S rDNA Research Articles

MOLECULAR IDENTIFICATION AND ANTIBACTERIAL ACTIVITY OF MACROFUNGUS Trametes sanguineus (L.)

Aim. The molecular identification of Pycnoporus sanguineus, a previously morphologically mushroom, was done to see the antibacterial activity against pathogenic bacteria Staphylococcus aureus and Salmonella typhi. Methods. A fragment of the D2 region of 28S rDNA was amplified by PCR, sequenced, and BLAST was performed using the consensus sequence. The maximum identity score was used to build a phylogenetic tree. Agar well diffusion was used to study the antibacterial activity. Results. Sequencing of a 700 base pair PCR amplicon was carried and a 616 base pair of D2 region of large subunit gene was generated. The 100 blast hits on the D2 region of LSU gene showed similarity to Trametes sanguineus voucher PRSC95 (GenBank Accession Number: JN164795.1) based on nucleotide homology and phylogenetic analysis. Antibacterial screening revealed that the crude extract had higher activity on Staphylococcus aureus, with a 3mm to 13mm zone of inhibition and a 100µg minimum inhibitory concentration, compared to Salmonella typhi. Salmonella typhi had a 5 mm to 15 mm zone of inhibition and a 200 µg minimum inhibitory concentration. Conclusion. According to the obtained result, the morphologically identified mushroom Pycnoporus sanguies can be referred to as Trametes sanguine, and it can be used for producinig antibacterial agents.

<strong>Hidden but not enough: DNA barcodes reveal two new species in <em>Hygrobates fluviatilis </em>complex from Iran (Acariformes, Hydrachnidia, Hygrobatidae)</strong>

Water mites of the Hygrobates fluviatilis complex are common in Palaearctic inhabiting mainly running and slowly flowing waters. In this study, we examined the specimens collected in a stream in Golestan Province of Iran which is currently the easternmost distribution limit of the latter species complex. We used a fragment of the mtDNA COI gene and the D2 region of 28S rDNA to test the genetic structure. Molecular analyses revealed presence of two new well-supported species with a mean genetic divergence of 12.80% and 14.66% COI, respectively, from their closest relatives. Phylogenetic analysis based on COI and nuclear DNA data placed H. grabowskii sp. nov. as the sister species of H. persicus, and H. ulii sp. nov. as the sister species of H. turcicus. Discriminant statistical analysis revealed that the two newly discovered species could be separated on the basis of morphometric characters, but robust morphological differences were not found. Yet we were able to show that diversity in water mites is underestimated and that species delimitation requires an integrative approach.

Phylogeny of the subfamilies of Ichneumonidae (Hymenoptera)

A combined morphological and molecular phylogenetic analysis was performed to evaluate the subfamily relationships of the parasitoid wasp family Ichneumonidae (Hymenoptera). Data were obtained by coding 135 morphological and 6 biological characters for 131 exemplar species of ichneumonids and 3 species of Braconidae (the latter as outgroups). The species of ichneumonids represent all of the 42 currently recognized subfamilies. In addition, molecular sequence data (cytochrome oxidase I “DNA barcoding” region, the D2 region of 28S rDNA and part of the F2 copy of elongation factor 1-alpha) were obtained from specimens of the same species that were coded for morphology (1309 base pairs total). The data were analyzed using parsimony and Bayesian analyses. The parsimony analysis using all data recovered previously recognized informal subfamily groupings (Pimpliformes, Ophioniformes, Ichneumoniformes), although the relationships of these three groups to each other differed from previous studies and some of the subfamily relationships within these groupings had not previously been suggested. Specifically, Ophioniformes was the sister group to (Ichneumoniformes + Pimplformes), and Labeninae was placed near Ichneumoniformes, not as sister group to all Ichneumonidae except Xoridinae. The parsimony analysis using only morphological characters was poorly resolved and did not recover any of the three informal subfamily groupings and very few of the relationships were similar to the total-evidence parsimony analysis. The molecular-only parsimony analysis and both Bayesian analyses (total-evidence and molecular-only) recovered Pimpliformes, a restricted Ichneumoniformes grouping and many of the subfamily groupings recovered in the total-evidence parsimony analysis. A comparison and discussion of the results obtained by each phylogenetic method and different data sets is provided. It is concluded that the molecular characters produced results that were relatively consistent with traditional, non-phylogenetic concepts of relationships between the ichneumonid subfamilies, whereas the morphological characters did not (at least not by themselves). The inclusion of both molecular and morphological characters using parsimony produced a topology that was the closest to the traditional subfamily relationships. The method of analysis did not greatly affect the overall topology for the molecular-only analyses, but there were differences between Bayesian and parsimony results for the total-evidence analyses (especially near the root of the tree). The Bayesian results did not seem to be altered very much by the inclusion of morphological characters, unlike in the parsimony analysis. In summary, the following groups were supported in multiple analyses regardless of the characters used or method of tree-building: Pimpliformes, higher Ophioniformes, higher Pimpliformes, (Claseinae + Pedunculinae), (Banchinae + Stilbopinae), Campopleginae, Cremastinae, Diplazontinae, Ichneumoninae (includingAlomya), Labeninae, Ophioninae, Poemeniinae, Rhyssinae, and Tersilochinae sensu stricto. Conversely, Ctenopelmatinae and Tryphoninae were never recovered without inclusion of other taxa. Based on the hypothesis of relationships obtained by the total-evidence parsimony analysis, the following formal taxonomic changes are proposed: Alomyinae Förster (=AlomyaPanzer andMegalomyaUchida) is once again synonymized with Ichneumoninae and is now considered a tribe (Alomyinirev. stat.); andNotostilbopsTownes is transferred from Stilbopinae to Banchinae, tribe Atrophini.

Efficacy of using DNA barcoding to identify parasitoid wasps of the melon-cotton aphid (Aphis gossypii) in watermelon cropping system

Parasitoid wasps have received a great deal of attention in the biological control of melon-cotton aphid (Aphis gossypii Glover). The species of parasitoids are often difficult to identify because of their small body size and profound diversity. DNA barcoding offers scientists who are not expert taxonomists a powerful tool to render their field studies more accurate. Using DNA barcodes to identify aphid parasitoid wasps in specific cropping systems may provide valuable information for biological control. Here, we report the use of DNA barcoding to confirm the morphological identification of 14 species (belonging to 13 genera of 7 families) of parasitoid wasps from two-year field samples in a watermelon cropping system. We generated DNA sequences from the mitochondrial COI gene and the nuclear D2 region of 28S rDNA to assess the genetic variation within and between parasitoid species. Automatic Barcode Gap Discovery (ABGD) supported the presence of 14 genetically distinct groups in the dataset. Among the COI sequences, we found no overlap between the maximum K2P distance within species (0.49%) and minimum distance between species (6.85%). The 28S sequences also showed greater interspecific distance than intraspecific distance. DNA barcoding confirmed the morphological identification. However, inconsistency and ambiguity of taxonomic information available in the online databases has limited the successful use of DNA barcoding. Only five species matched those in the BOLD and GenBank. Four species did not match the entries in GenBank and five species showed ambiguous results in BOLD due to confusing nomenclature. We suggested that species identification based on DNA barcodes should be performed using both COI and other genes. Nonetheless, we demonstrate the potential of the DNA barcoding approach to confirm field identifications and to provide a foundation for studies aimed at improving the understanding of the biocontrol services provided by parasitoids in the melon ecosystem.

Phytophagous mites (Acari: Eriophyoidea) recorded from Svalbard, including the description of a new species

Eriophyoid mites (Eriophyoidea) are minute phytophagous mites with great economic importance and great invasive potential. In spite of their demonstrated impact on ecosystem functions, knowledge of eriophyoid mite fauna in the Arctic is lacking. To date, only eight eriophyoid mite species have been recorded from the entire region north of the Arctic Circle. The Svalbard archipelago is one of the most biologically investigated Arctic areas. Despite the fact that studies on invertebrates on Svalbard have been conducted for more than one hundred years, eriophyoids have never been recorded before from this place, except for one likely accidental record of a single specimen belonging to the genus Eriophyes. Thus, each new study of eriophyoid mite fauna in this region is important. In this paper, a new species of eriophyoid mite, Cecidophyes siedleckii n. sp., is described and illustrated. Nucleotide sequence data (D2 region of 28S rDNA) were employed to complement traditional morphological taxonomy. The first record of Aceria saxifragae (Rostrup 1900) from Svalbard is also provided, with supplementary morphological descriptions and illustrations. Eriophyoid mites represent an important and underutilized taxon that is available to ecologists studying the effects of changing climatic conditions on Svalbard.

Exceptionally High Levels of Genetic Diversity in Wheat Curl Mite (Acari: Eriophyidae) Populations from Turkey.

Recent research on the wheat curl mite species complex has revealed extensive genetic diversity that has distinguished several genetic lineages infesting bread wheat (Triticum aestivum L.) and other cereals worldwide. Turkey is the historical region of wheat and barley (Hordeum vulgare L.) domestication and diversification. The close relationship between these grasses and the wheat curl mite provoked the question of the genetic diversity of the wheat curl mite in this region. The scope of the study was to investigate genetic differentiation within the wheat curl mite species complex on grasses in Turkey. Twenty-one wheat curl mite populations from 16 grass species from nine genera (Agropyron sp., Aegilops sp., Bromus sp., Elymus sp., Eremopyrum sp., Hordeum sp., Poa sp., Secale sp., and Triticum sp.) were sampled in eastern and southeastern Turkey for genetic analyses. Two molecular markers were amplified: the cytochrome oxidase subunit I coding region of mtDNA (COI) and the D2 region of 28S rDNA. Phylogenetic analyses revealed high genetic variation of the wheat curl mite in Turkey, primarily on Bromus and Hordeum spp., and exceptionally high diversity of populations associated with bread wheat. Three wheat-infesting wheat curl mite lineages known to occur on other continents of the world, including North and South America, Australia and Europe, were found in Turkey, and at least two new genetic lineages were discovered. These regions of Turkey exhibit rich wheat curl mite diversity on native grass species. The possible implications for further studies on the wheat curl mite are discussed.

Genetic and morphological diversity of Trisetacus species (Eriophyoidea: Phytoptidae) associated with coniferous trees in Poland: phylogeny, barcoding, host and habitat specialization

Eriophyoid species belonging to the genus Trisetacus are economically important as pests of conifers. A narrow host specialization to conifers and some unique morphological characteristics have made these mites interesting subjects for scientific inquiry. In this study, we assessed morphological and genetic variation of seven Trisetacus species originating from six coniferous hosts in Poland by morphometric analysis and molecular sequencing of the mitochondrial cytochrome oxidase subunit I gene and the nuclear D2 region of 28S rDNA. The results confirmed the monophyly of the genus Trisetacus as well as the monophyly of five of the seven species studied. Both DNA sequences were effective in discriminating between six of the seven species tested. Host-dependent genetic and morphological variation in T. silvestris and T. relocatus, and habitat-dependent genetic and morphological variation in T. juniperinus were detected, suggesting the existence of races or even distinct species within these Trisetacus taxa. This is the first molecular phylogenetic analysis of the Trisetacus species. The findings presented here will stimulate further investigations on the evolutionary relationships of Trisetacus as well as the entire Phytoptidae family.

Application of DNA barcoding to the identification of Hymenoptera parasitoids from the soybean aphid (Aphis glycines) in China

Aphis glycines Matsumura is an important pest of soybean in Asia and North America. Hymenoptera parasitoids play a key role in the control of the soybean aphid. The correct identification of parasitoids is a critical step that precedes the assessment of their potential biological control agents. Accurate identification of the majority of the species attacking the soybean aphid often requires elaborate specimen preparation and expert taxonomic knowledge. In this study, we facilitated the identification of soybean aphid parasitoids by applying a DNA barcoding approach following a preliminary morphological identification. We generated DNA sequence data from the mitochondrial COI gene and the D2 region of 28S rDNA to assess the genetic variation within and between parasitoid species emerging from the soybean aphid in China. Fifteen Hymenoptera parasitoid species belonging to 10 genera of five families were identified with little intra-specific variation (0.09%±0.06% for 28S and 0.36%±0.18% for COI) and large inter-specific divergence (30.46%±3.42% for 28S and 20.4%±1.20% for COI).

The wheat curl miteAceria tosichella(Acari: Eriophyoidea) is a complex of cryptic lineages with divergent host ranges: evidence from molecular and plant bioassay data

Aceria tosichella (the wheat curl mite, WCM) is a global pest of wheat and other cereals, causing losses by direct damage, as well as the transmission of plant viruses. The mite is considered to have an unusually wide host range for an eriophyoid species. The present study tested the commonly held assumption that WCM is a single, highly polyphagous species by assessing the host range of genetically distinct lineages of WCM occurring in Poland on different host plants. Genotyping was performed by analyzing nucleotide sequence data from fragments of the mitochondrial cytochrome c oxidase subunit I (COI) and the nuclear D2 region of 28S rDNA. Mean between-lineage distance estimated using COI data was found to be one order of magnitude greater than the within-clade lineage and, in some cases, comparable to distances between WCM lineages and a congeneric outgroup species. Host acceptance was tested by quantifying population growth for different WCM mitochondrial (mt)DNA lineages when transferred from source host plants to test plants. These experiments revealed significant differences in host colonization ability between mtDNA lineages, ranging from highly polyphagous to more host-specific. The present study reveals that WCM is composed of several discrete genetic lineages with divergent host-acceptance and specificity traits. Genetic variation for host acceptance within A. tosichella s.l. may act as a reproductive barrier between these lineages, most of which had narrow host ranges. Two lineages appear to have high pest potential on cereals, whereas several others appear to specialize on wild grass species. We conclude that WCM is not a homogeneous species comprising polyphagous panmictic populations rather it is a complex of genetically distinct lineages with variable host ranges and therefore variable pest potential. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 109, 165–180. ADDITIONAL KEYWORDS: 28S rDNA D2 – DNA barcoding – acceptance – specificity – mtDNA COI – species complex.

A new feather mite species of the genus Proctophyllodes Robin, 1877 (Astigmata: Proctophyllodidae) from the Long-tailed Tit Aegithalos caudatus (Passeriformes: Aegithalidae)—morphological description with DNA barcode data

A new feather mites species, Proctophyllodes valchukae sp. n., is described from the Long-tailed Tit, Aegithalos caudatus(Linnaeus, 1758) (Passeriformes: Aegithalidae), captured in the Primoriye (Russian Far East). The new species belongsto the tricetratus species group and is most closely related to P. stachyris Atyeo et Braasch, 1966. For the first time forfeather mites the standard morphological description is supplemented by sequence data of the mitochondrial cytochrome c oxidase subunit I gene fragment (COI) and nuclear D2 region of 28S rDNA.

Cryptic species within the wheat curl mite Aceria tosichella (Keifer) (Acari : Eriophyoidea), revealed by mitochondrial, nuclear and morphometric data

The wheat curl mite (WCM), Aceria tosichella (Keifer, 1969), is one of the primary pests of wheat and other cereals throughout the world. Traditional taxonomy recognises WCM as a single eriophyoid species; however, a recent study suggested that two genetic lineages of WCM in Australia might represent putative species. Here, we investigate WCM populations from different host plants in Australia, South America and Europe and test the hypothesis that WCM is, in fact, a complex of cryptic species. We used morphological data in combination with nucleotide sequences of the mitochondrial cytochromec oxidase subunitI (COI) and nuclear D2 region of 28S rDNA and internal transcribed spacer region (ITS1, ITS2) sequences. The molecular analyses did not support the monophyly of A. tosichella because the outgroup A. tulipae (Keifer, 1938) is grouped within WCM. The molecular datasets indicated the existence of distinct lineages within WCM, with the distances between lineages corresponding to interspecific divergence. Morphological analyses failed to clearly separate WCM populations and lineages, but completely separated A. tulipae from A. tosichella. The results suggest that what has been recognised historically as a single species is, in fact, a complex of several genetically isolated evolutionary lineages that demonstrate potential as cryptic species. Hence, their discrimination using solely morphological criteria may be misleading. These findings are particularly significant because of the economic importance of WCM as a direct pest and vector of plant viruses.

DNA barcoding reveals andropolymorphism in Aclerogamasus species (Acari: Parasitidae) MIROSŁAWA DABERT, AGATA BIGOŚ & WOJCIECH WITALIŃSKI (Poland)

Andropolymorphism, defined as discontinuous morphological variability in males, can lead to taxonomic confusion when different male morphs are determined and described as separate species. This study addresses this issue in two occasionally sympatric mite species Aclerogamasus similis (Willmann, 1953) and A. holzmannae (Micherdziński, 1969) collected in Poland. The females of these two taxa are morphologically indistinguishable but males are quite different, and could be either separate species or one species with two male morphs. We address this question by performing molecular assays, testing variation in a fragment of the mtDNA COI gene and the D2 region of 28S rDNA. Molecular analysis of populations revealed very low variation in the studied gene fragments. All sequences of the D2 region of 28S rDNA (size 375 bp) were identical. Only two COI haplotypes were found, differing by two out of 644 nucleotide positions (0.3% K2P distance). The variant haplotype was found in one A. similis male, and probably represents intraspecific variability. The results strongly suggest that all studied females and males belong to only one species with dimorphic males. This finding confirms some earlier opinions on synonymy of both species. Therefore, Aclerogamasus holzmannae (Micherdziński, 1969) should be regarded as a junior synonym of Aclerogamasus similis (Willmann, 1953). As andropolymorphism has been rarely observed in gamasid mites, we briefly discuss its possible origins and consequences.

The cereal rust miteAbacarus hystrix(Acari: Eriophyoidea) is a complex of species: evidence from mitochondrial and nuclear DNA sequences

The cereal rust mite Abacarus hystrix (Nalepa), a significant pest of grasses, has been regarded as one of a few exceptions among eriophyoid mites with reference to the pattern of host plant utilization. At least 60 grass species have been recorded as its hosts. Thus, the mite has long been considered as a host generalist in which host specialization would not be likely to evolve. However, recent studies have revealed that host-associated specialization is possible in A. hystrix. Here, we aimed to discriminate between the three populations of A. hystrix associated with the different hosts (namely quackgrass, ryegrass and smooth brome) on the basis of mitochondrial (COI) and nuclear (D2 region of 28S rDNA) DNA sequences. The phylogenetic trees obtained with the maximum likelihood analysis of both COI and D2 region data sets showed that host-adapted strains of A. hystrix form distinct clades. Furthermore, on the COI nucleotide tree, the quackgrass- and brome-associated strains were internally divided each into two well-supported monophyletic clusters. The nucleotide D2 region data set tree showed that brome-associated strain is polyphyletic in origin. There is clear co-variation of DNA results with earlier morphological and ecological traits, as well as the results of crossing experiments. We showed that reproductively incompatible strains of A. hystrix exhibit more than 20% sequence divergence in the COI gene and 0.2% sequence divergence in the D2 28S rDNA. Our results did not confirm the placement of three host-associated strains of A. hystrix within one, ostensibly generalist, species.

Phylogeny of the Nasonia species complex (Hymenoptera: Pteromalidae) inferred from an internal transcribed spacer (ITS2) and 28S rDNA sequences.

The phylogeny of the cryptic species complex of wasps in the genus Nasonia was inferred by analysis of nucleotide sequences of an rDNA internal transcribed spacer (ITS2) and the D2 region of 28S rDNA. Phylogenetic analysis showed that N. vitripennis descended from a theoretical common ancestor with that of a lineage that diverged into N. longicornis and N. giraulti. Differences in the ITS2 regions clearly distinguished two strains of N. giraulti. Another member of the Dibrachys Group, Trichomalopsis dubius, was placed outside of the Nasonia complex. The D2 region had a base substitution rate approximately 2 times slower than the ITS2 region and was used to resolve the phylogenetic affiliation of an eulophid, Melittobia digitata, to the pteromalids. Tree topology of the Nasonia complex was congruent with the phylogeny of a cluster of Wolbachia bacteria which infect these insects. The possible role of these bacteria in driving speciation is discussed.