Developing a new molecular marker for aphid species identification: Evaluation of eleven candidate genes with species-level sampling

The mitochondrial cytochrome c oxidase subunit I (COI) gene has been utilized as a molecular marker for aphid species identification. However, this gene has sometimes resulted in misidentification because of low interspecific genetic divergences between some species pairs. In this study, to propose new molecular markers for the family Aphididae, we first screened 2289 sequences of 11 genes (COI, COII, CytB, ATP6, lrRNA, srRNA, ITS1, ITS2, EF1a, 18S, and 28S) collected from the GenBank. Among the 11 genes, ATP6 gene revealed the largest genetic divergence among congeneric species with the smallest divergence among conspecific individuals; in contrast, species pairs with low genetic divergences (< 1%) were not observed. Secondly, for statistically testing the usefulness of ATP6 gene in species identification, we analyzed genetic distances between all of the combinations of 32 individuals of 20 species for both COI and ATP6 genes. The ATP6 gene showed lower intraspecific (on average 0.08%) and higher interspecific (on average 8.28%) genetic distances than the COI gene (on average 0.19% and 6.24%, respectively) for the same pairs of individuals. This study corroborates the usefulness of the ATP6 gene as a new molecular marker that could improve the misidentification problems that are inherent with the COI gene.     

Genetic Differentiation of the Mitochondrial Cytochrome Oxidase c Subunit I Gene in Genus Paramecium (Protista,Ciliophora)

Background

The mitochondrial cytochrome c oxidase subunit I (COI) gene is being used increasingly for evaluating inter- and intra-specific genetic diversity of ciliated protists. However, very few studies focus on assessing genetic divergence of the COI gene within individuals and how its presence might affect species identification and population structure analyses.

Methodology/Principal findings

We evaluated the genetic variation of the COI gene in five Paramecium species for a total of 147 clones derived from 21 individuals and 7 populations. We identified a total of 90 haplotypes with several individuals carrying more than one haplotype. Parsimony network and phylogenetic tree analyses revealed that intra-individual diversity had no effect in species identification and only a minor effect on population structure.

Conclusions

Our results suggest that the COI gene is a suitable marker for resolving inter- and intra-specific relationships of Paramecium spp.     

Molecular Identification of Sibling Species of Sclerodermus (Hymenoptera: Bethylidae) That Parasitize Buprestid and Cerambycid Beetles by Using Partial Sequences of Mitochondrial DNA Cytochrome Oxidase Subunit 1 and 28S Ribosomal RNA Gene

The species belonging to Sclerodermus (Hymenoptera: Bethylidae) are currently the most important insect natural enemies of wood borer pests, mainly buprestid and cerambycid beetles, in China. However, some sibling species of this genus are very difficult to distinguish because of their similar morphological features. To address this issue, we conducted phylogenetic and genetic analyses of cytochrome oxidase subunit I (COI) and 28S RNA gene sequences from eight species of Sclerodermus reared from different wood borer pests. The eight sibling species were as follows: S. guani Xiao et Wu, S. sichuanensis Xiao, S. pupariae Yang et Yao, and Sclerodermus spp. (Nos. 1–5). A 594-bp fragment of COI and 750-bp fragment of 28S were subsequently sequenced. For COI, the G-C content was found to be low in all the species, averaging to about 30.0%. Sequence divergences (Kimura-2-parameter distances) between congeneric species averaged to 4.5%, and intraspecific divergences averaged to about 0.09%. Further, the maximum sequence divergences between congeneric species and Sclerodermus sp. (No. 5) averaged to about 16.5%. All 136 samples analyzed were included in six reciprocally monophyletic clades in the COI neighbor-joining (NJ) tree. The NJ tree inferred from the 28S rRNA sequence yielded almost identical results, but the samples from S. guani, S. sichuanensis, S. pupariae, and Sclerodermus spp. (Nos. 1–4) clustered together and only Sclerodermus sp. (No. 5) clustered separately. Our findings indicate that the standard barcode region of COI can be efficiently used to distinguish morphologically similar Sclerodermus species. Further, we speculate that Sclerodermus sp. (No. 5) might be a new species of Sclerodermus.     

DNA barcoding of sunn pest adult parasitoids using cytochrome c oxidase subunit I (COI)

In this study, DNA barcoding was used in the identification of potential biological control agents of sunn pest adult parasitoid species, including Eliozeta helluo (F.), Phasia subcoleoptrata (L.), Ectophasia crassipennis (F.) and Elomyia lateralis (Meig). DNA analyses were assessed by sequencing cytochrome c oxidase subunit I (COI) gene. The obtained sequences were analyzed in terms of nucleotide composition, nucleotide pair frequency and haplotype diversity. Genetic divergence among haplotypes was estimated by constructing genetic distance matrix using DNA sequence variations, by Kimura 2-parameter model. Variable sites and average variations of the sequenced 603 base pair long DNA fragment were calculated. All COI barcodes were matched with reference sequences of expected species according to morphological identification. Neighbor-joining tree was drawn based on DNA barcodes and all the specimens clustered in agreement with their taxonomic classification at species level. The evolutionary history inferred using the UPGMA method indicated two distinct mitochondrial haplotype lineages. The genetic variation between sunn pest adult parasitoids will be useful in sunn pest management, regulatory and environmental applications.     

Position of neck banded blenny Leptostichaeus pumilus (Perciformes: Zoarcoidei) in the system of the suborder Zoarcoidei as inferred from molecular genetic data

Molecular genetic study of genetic variations within COI, cytochrome b, and 16S rRNA of mitochondrial DNA in neck banded blenny Leptostichaeus pumilus, which was earlier assigned to the family Stichaeidae, indicates that the species is most close to a group combining the families Zoarcidae, Neozoarcidae, and Anarhichadidae. Significant genetic differences between neck banded blenny and the family Stichaeidae correspond to the level of divergence between other families of the suborder Zoarcoidei (Zaproridae, Ptilichthyidae, Pholidae, Cryptacanthodidae, and Bathymasteridae).     

Molecular identification and genetic structure of Aedes phoeniciae (Diptera: Culicidae) in Northern Cyprus and Turkey

Aedes mariae, Aedes phoeniciae and Aedes zammitii are three sibling species of the Mariae complex with a limited distribution. All these three species use semi-saline/saline rock pool habitats on the coastal areas of Mediterranean countries. The Eastern coast of the Mediterranean basin, including Turkey and Cyprus coasts, is a unique distribution area for Ae. phoeniciae. This study was designed to identify three sibling species of the Mariae complex based on molecular markers and also to determine the distribution pattern and genetic structure of the Ae. phoeniciae along the eastern Mediterranean basin. Larval and adult samples of Ae. zammitii and Ae. phoeniciae were collected from different rock pool habitats along the coastal regions of Turkey and Northern Cyprus. Species identifications were done primarily based on morphological identification key. Identifications were supported by using mitochondrial DNA cytochrome oxidase I (COI) and nuclear DNA Internal transcribed spacer 2 (ITS2) sequences. Population genetic analyses of Ae. phoeniciae were implemented based on NADH dehydrogenase 4 (ND4) sequence and 17 haplotypes were detected in the study area. Analyses of molecular variance (AMOVA) determined that the majority of the variation was within the populations (60.15 %) while variation between groups was 30.68 % and the variation within groups was 9.17 %, indicating low genetic structuring among groups.     

Reassessment of Species Diversity of the Subfamily Denticollinae (Coleoptera: Elateridae) through DNA Barcoding

The subfamily Denticollinae is a taxonomically diverse group in the family Elateridae. Denticollinae includes many morphologically similar species and crop pests, as well as many undescribed species at each local fauna. To construct a rapid and reliable identification system for this subfamily, the effectiveness of molecular species identification was assessed based on 421 cytochrome c oxidase subunit I (COI) sequences of 84 morphologically identified species. Among the 84 morphospecies, molecular species identification of 60 species (71.4%) was consistent with their morphological identifications. Six cryptic and/or pseudocryptic species with large genetic divergence (>5%) were confirmed by their sympatric or allopatric distributions. However, 18 species, including a subspecies, had ambiguous genetic distances and shared overlapping intra- and interspecific genetic distances (range: 2.12%–3.67%) suggesting incomplete lineage sorting, introgression of mitochondrial genome, or affection by endosymbionts, such as Wolbachia infection, between species and simple genetic variation within species. In this study, we propose a conservative threshold of 3.6% for convenient molecular operational taxonomic unit (MOTU) identification in the subfamily Denticollinae based on the results of pairwise genetic distances analyses using neighbor-joining, mothur, Automatic Barcode Gap Discovery analysis, and tree-based species delimitation by Poisson Tree Processes analysis. Using the 3.6% threshold, we identified 87 MOTUs and found 8 MOTUs in the interval between 2.5% to 3.5%. Evaluation of MOTUs identified in this range requires integrative species delimitation, including review of morphological and ecological differences as well as sensitive genetic markers. From this study, we confirmed that COI sequence is useful for reassessing species diversity for polymorphic and polytypic species occurring in sympatric and allopatric distributions, and for a single species having an extensively large habitat.     

Dissimilarity of Species and Forms of Planktonic Neocalanus Copepods Using Mitochondrial COI, 12S,Nuclear ITS,and 28S Gene Sequences

Background

A total of six Neocalanus species inhabit the oceans of the world. Of these, three species plus form variants (N. cristatus, N. plumchrus, N. flemingeri large form, and N. flemingeri small form), which constitute a monophyletic group among Neocalanus copepods, occur in the Northwestern Pacific off Japan. In the present study, we have tried to discriminate the three species plus form variants of Neocalanus copepods based on sequences of four DNA marker regions.

Methodology/Principal Findings

Discrimination was performed based on the DNA sequence information from four genetic markers, including the mitochondrial COI, 12S, nuclear ITS, and 28S gene regions. Sequence dissimilarity was compared using both distance- and character-based approaches. As a result, all three species were confirmed to be distinct based on the four genetic marker regions. On the contrary, distinction of the form variants was only confirmed based on DNA sequence of the mitochondrial COI gene region.

Conclusions/Significance

Although discrimination was not successful for the form variants based on the mitochondrial 12S, nuclear ITS, and 28S genes, diagnostic nucleotide sequence characters were observed in their mitochondrial COI gene sequences. Therefore, these form variants are considered to be an important unit of evolution below the species level, and constitute a part of the Neocalanus biodiversity.     

DNA barcoding as a tool for coral reef conservation

DNA Barcoding (DBC) is a method for taxonomic identification of animals that is based entirely on the 5′ portion of the mitochondrial gene, cytochrome oxidase subunit I (COI-5). It can be especially useful for identification of larval forms or incomplete specimens lacking diagnostic morphological characters. DBC can also facilitate the discovery of species and in defining “molecular taxonomic units” in problematic groups. However, DBC is not a panacea for coral reef taxonomy. In two of the most ecologically important groups on coral reefs, the Anthozoa and Porifera, COI-5 sequences have diverged too little to be diagnostic for all species. Other problems for DBC include paraphyly in mitochondrial gene trees and lack of differentiation between hybrids and their maternal ancestors. DBC also depends on the availability of databases of COI-5 sequences, which are still in early stages of development. A global effort to barcode all fish species has demonstrated the importance of large-scale coordination and is yielding promising results. Whether or not COI-5 by itself is sufficient for species assignments has become a contentious question; it is generally advantageous to use sequences from multiple loci.     

Host Chemical Footprints Induce Host Sex Discrimination Ability in Egg Parasitoids

Trissolcus egg parasitoids, when perceiving the chemical footprints left on a substrate by pentatomid host bugs, adopt a motivated searching behaviour characterized by longer searching time on patches were signals are present. Once in contact with host chemical footprints, Trissolcus wasps search longer on traces left by associated hosts rather than non-associated species, and, in the former case, they search longer on traces left by females than males. Based on these evidences, we hypothesized that only associated hosts induce the ability to discriminate host sex in wasps. To test this hypothesis we investigated the ability of Trissolcus basalis, T. brochymenae, and Trissolcus sp. to distinguish female from male Nezara viridula, Murgantia histrionica, and Graphosoma semipunctatum footprints. These three pentatomid bugs were selected according to variable association levels. Bioassays were conducted on filter paper sheets, and on Brassica oleracea (broccoli) leaves. The results confirmed our hypothesis showing that wasps spent significantly more time on female rather than male traces left by associated hosts on both substrates. No differences were observed in the presence of traces left by non-associated hosts. The ecological consequences for parasitoid host location behaviour are discussed.     

Molecular diversity and phylogeography of Andricus curtisii (Hymenoptera,Cynipidae) in Turkey

A 433-bp fragment of the mitochondrial cytochrome b gene and the entire nuclear ITS2 region were sequenced in an oak gall wasp species, Andricus curtisii, which represents 21 populations across its range in Turkey. Forty cytochrome b haplotypes and 63 ITS2 alleles were discovered among the 161 individuals studied. Remarkable genetic variation was detected in the species, with an average haplotype diversity (h) of 0.72 and 0.76 and an average nucleotide diversity (π) of 0.06 and 0.01 for the cyt b gene and the ITS region, respectively. Most populations were genetically distinct, possessing unique cytochrome b haplotypes. Maximum likelihood and Bayesian phylogenetic analyses and application of the haplotype network to both datasets revealed that the most basally located haplotypes/alleles were from the eastern part of Turkey. The detection of older sequences in the phylogenetic trees of the easterly located populations implied an eastern origin of the species. The application of a conventional insect mitochondrial DNA clock to the dataset suggested a splitting of ingroup haplotypes from the outgroup lineages predating the Pleistocene epoch and the formation of two major haplogroups that coincided with the oscillation of the early Pleistocene glaciations.     

Phylogeography of the cold‐water barnacle Chthamalus challengeri in the north‐western Pacific: effect of past population expansion and contemporary gene flow

Aim Phylogeographical patterns of marine organisms in the north‐western Pacific are shaped by the interaction of past sea‐level fluctuations during glacial maxima and present‐day gene flow. This study examines whether observed population differentiation in the barnacle Chthamalus challengeri, which is endemic to the north‐western Pacific, can be explained by the interactions between historical glacial events and patterns of contemporary gene flow. Location Eleven locations in the north‐western Pacific. Methods Partial sequences of mitochondrial cytochrome c oxidase subunit I (COI), 12S, 16S and nuclear internal transcribed spacer 1 (ITS1) were obtained from 312 individuals. Parsimony haplotype networks and analysis of molecular variance (AMOVA) were used to determine whether the observed genetic structure corresponds to marine provinces (Kuroshio Current and China Sea Coastal Provinces), zoogeographical zones (oriental and Japan warm‐temperate zones) and/or potential refugial areas (Sea of Japan and East China Sea) in the north‐western Pacific. Neutrality tests, mismatch distribution analysis and Bayesian skyline plots were used to infer the demographic history of C. challengeri. Results In total, 312, 117, 182 and 250 sequences were obtained for COI, 12S, 16S and ITS1, respectively. A panmictic population was revealed, which did not conform to the ‘isolation by distance’ model. None of the a priori population groupings based on marine provinces, zoogeographical zones or potential refugial areas was associated with observed genetic patterns. Significant negative values from neutrality tests and the unimodal mismatch distribution and expansion patterns in Bayesian skyline plots for the COI and 16S data sets indicate a population expansion in the mid‐Pleistocene (c. 200 ka). Information from the fossil record suggests that there has been a northward range shift of this species from the East China Sea or the Palaeo‐Pacific coast of Japan to the Sea of Japan since the mid‐Pleistocene. Main conclusions Chthamalus challengeri has experienced a population expansion and range shift since the mid‐Pleistocene. The observed lack of population differentiation can be explained by this past population expansion and present‐day wide‐scale larval dispersal (owing to the long planktonic larval duration) across marine provinces, which have led to the successful establishment of the species in different zoogeographical zones and habitats.     

The Identity of Specimens of the Anastrepha fraterculus Complex (Diptera, Tephritidae) with Atypical Aculeus Tip

Several specimens collected in Paraguay along with Anastrepha fraterculus (sensu lato) have an aculeus tip similar to species from the fraterculus complex, but the teeth of the aculeus of these specimens are poorly defined. As Anastrepha species identification is based mostly on subtle differences in the aculeus tip, we studied these specimens with atypical aculeus tips (with poorly defined teeth) that slightly differs from the aculeus tip of species of the fraterculus complex (with well-developed blunt teeth), to determine if this is due to intraspecific variation or if it can characterize a full species. The Paraguayan specimens were separated in six groups under stereomicroscope according to variation in their aculeus tip. Specimens within each group were studied by means of morphometrics (traditional and geometric) and gene sequence analysis (COI and ITS1). Morphometric analyses were significant, but no clear groups were formed by the discriminant analyses of the aculeus and wing, and the COI and ITS1 sequence analysis clustered specimens with all six aculeus variations. Therefore, the subtle morphological differences observed in the aculeus tip of Paraguayan specimens are intraspecific variations and the Paraguayan specimens were more genetically closely related to Anastrepha sp. 3 from the fraterculus complex.     

Exploring phylogenetic informativeness and nuclear copies of mitochondrial DNA (numts) in three commonly used mitochondrial genes: mitochondrial phylogeny of peppermint,cleaner, and semi‐terrestrial shrimps (Caridea: Lysmata,Exhippolysmata, and Merguia)

Of paramount importance to studies that profit from molecular trees is the accuracy and robustness of the reconstructed phylogenies. Causes of systematic error that can mislead phylogenetic methods include nuclear copies of mitochondrial DNA (numts) and low phylogenetic informativeness (PI). Herein, numts and PI were explored in three mitochondrial genes commonly used for phylogenetic reconstruction: 16S, 12S, and cytochrome c oxidase I (COI). Shrimps from the genera Lysmata, Exhippolysmata, and Merguia were used as a model system. The existence of: (1) multiple bands on gels of COI and 12S polymerase chain reaction (PCR) products from various species; (2) double peaks, background noise, and ambiguity in sequence chromatograms of COI and 12S PCR products that produced a single clear band in other species; and (3) indels, stop codons, and considerable composition bias in COI‐like cloned sequences of one problematic species (Lysmata seticaudata), was interpreted as evidence of pervasive non‐functional nuclear copies of mitochondrial DNA (numts) of the targeted COI (and probably 12S) mtDNA fragment. The information content of the three mtDNA markers studied was investigated using PI profiling, spectral analysis, and neighbour‐nets. Marker‐specific PI profiles suggested that the COI marker has the highest information content and greatest power for resolving both shallow and deep nodes in trees depicting the phylogenetic relationship among the species studied. Nonetheless, spectral analysis of splits and neighbour‐nets suggested that the 16S and 12S markers were equally or even more powerful than the COI marker for resolving nodes at all phylogenetic levels. Altogether, these analyses suggest that all three mtDNA markers are equally useful for resolving phylogenetic relationships in the shrimps studied, and that PI profiling is not necessarily useful to estimate overall gene utility. A ‘total‐evidence’ phylogenetic analysis that included 34 species and used a concatenated data set of 1403 characters (from reliable 16S, 12S and COI sequences), demonstrated that the genus Lysmata is paraphyletic, and that the monophyletic clade comprising species of Lysmata and Exhippolysmata can be divided into four well‐supported subclades (Neotropical, Cleaner, Cosmopolitan, and Morphovariable). © 2013 The Linnean Society of London     

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

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

Host preference: parasitism, emergence and development of Trissolcus semistriatus (Hym., Scelonidae) in various host eggs

Abstract: Trissolcus semistriatus is an important egg parasitoid of the sunn bug, Eurygaster integriceps. Conservation and the supportive augmentation of populations of egg parasitoids appears to offer high potential for the biological control of sunn bug in an integrated pest management system. The present study was conducted to determine the host preference of T. semistriatus on various heteropteran host eggs. When each host species was examined separately, the eggs of E. integriceps, Dolycoris baccarum, Graphosoma lineatum, Carpocoris pudicus and Holcostethus vernalis, were parasitized at high levels, namely 88.0, 83.6, 94.8, 87.3 and 80.8%, respectively, whereas a smaller portion of Eurydema ornatum eggs were parasitized (24.0%). There was no parasitism in Nezara viridula eggs by T. semistriatus. Similar high rates were recorded for the percentage of adult emergence in each host species, with the exception of E. ornatum. The average development time was shorter for males than for females. The average development period for both male and female was significantly longer in eggs of E. ornatum than in those of the other host species. These results and observations recorded during the experiments showed that G. lineatum and D. baccarum were available, cheap hosts and it was demonstrated that these hosts could be used for the mass production of egg parasitoids.