Existence of two strains of <Emphasis Type="Italic">Habrobracon hebetor</Emphasis> (Hymenoptera: Braconidae): a complex in Thailand and Japan

Habrobracon hebetor Say (Hymenoptera: Braconidae) is a cosmopolitan gregarious ectoparasitoid that attacks larvae of several species of Lepidoptera. Although there are two genetically different strains within H. hebetor, distribution of the strains has been poorly understood. In 2010, in Thailand, where H. hebetor has been known as a parasitoid of stored grain pests, it was found that H. hebetor attacked Opisina arenosella Walker (Lepidoptera: Oecophoridae), which is an invasive pest of coconut palm. For correct identification of this H. hebetor, we conducted DNA analysis and cross tests using populations collected from O. arenosella and stored grain pests in Thailand and populations in Japan known as H. hebetor. We obtained 413 bp of mitochondrial cytochrome oxidase I (COI) sequences and 414 bp of 16S rRNA gene sequences, and both indicated that there are two distinct clades within H. hebetor: one contains insects from Thailand, Spain, India, and Barbados; the other contains insects from Japan and the USA. There were no genetic differences or sexual isolation between Thai populations from different hosts. Our results also showed that populations in Thailand were sexually isolated from a H. hebetor population in Japan.     

Identification of Japanese <Emphasis Type="Italic">Lymantria</Emphasis> species (Lepidoptera: Lymantriidae) based on DNA sequences

We examined the utility of the cytochrome c oxidase subunit I (COI) and II (COII) genes of mitochondrial DNA (mtDNA) as a tool to identify nine Japanese Lymantria species, including four Asian gypsy moth species [Lymantria dispar japonica (Motschulsky), Lymantria umbrosa (Butler), Lymantria albescens Hori and Umeno, and Lymantria postalba Inoue]. In phylogenetic trees constructed for the COI and COII genes using the maximum likelihood methods, we could identify seven out of the nine Lymantria species (L. albescens and L. postalba could not be identified). These results suggest that the DNA sequences of the COI and COII genes are useful for identifying Japanese Lymantria species.     

Analysis of SSH library of rice variety Aganni reveals candidate gall midge resistance genes

The Asian rice gall midge, Orseolia oryzae, is a serious insect pest causing extensive yield loss. Interaction between the gall midge and rice genotypes is known to be on a gene-for-gene basis. Here, we report molecular basis of HR? (hypersensitive reaction—negative) type of resistance in Aganni (an indica rice variety possessing gall midge resistance gene Gm8) through the construction and analysis of a suppressive subtraction hybridization (SSH) cDNA library. In all, 2,800 positive clones were sequenced and analyzed. The high-quality ESTs were assembled into 448 non-redundant gene sequences. Homology search with the NCBI databases, using BlastX and BlastN, revealed that 73% of the clones showed homology to genes with known function and majority of ESTs belonged to the gene ontology category ‘biological process’. Validation of 27 putative candidate gall midge resistance genes through real-time PCR, following gall midge infestation, in contrasting parents and their derived pre-NILs (near isogenic lines) revealed induction of specific genes related to defense and metabolism. Interestingly, four genes, belonging to families of leucine-rich repeat (LRR), heat shock protein (HSP), pathogenesis related protein (PR), and NAC domain-containing protein, implicated in conferring HR+ type of resistance, were found to be up-regulated in Aganni. Two of the reactive oxygen intermediates (ROI)–scavenging-enzyme-coding genes Cytosolic Ascorbate Peroxidase1, 2 (OsAPx1 and OsAPx2) were found up-regulated in Aganni in incompatible interaction possibly suppressing HR. We suggest that Aganni has a deviant form of inducible, salicylic acid (SA)-mediated resistance but without HR.     

Parasitism rates and parasitoid complexes of the wheat midges, <Emphasis Type="Italic">Sitodiplosis mosellana</Emphasis>, <Emphasis Type="Italic">Contarinia tritici</Emphasis> and <Emphasis Type="Italic">Haplodiplosis marginata</Emphasis>

Three species of cecidomyiid midges (Diptera: Cecidomyiidae), whose larvae overwinter in the soil, can cause significant yield losses on wheat in Europe: the orange wheat blossom midge, Sitodiplosis mosellana (Géhin), the yellow wheat blossom midge, Contarinia tritici (Kirby), and the saddle gall midge, Haplodiplosis marginata (von Roser). The biological control of wheat midges by their parasitoids can contribute to reduce the midge populations. Soil samples were collected in several fields in Belgium in 2012–2014 in order to characterize the parasitism rates and parasitoid complexes in overwintering larvae. The parasitism rates varied greatly between the sampled fields: 3–100, 0–100 and 2% for S. mosellana, H. marginata and C. tritici, respectively. The parasitism rate was not related to the larval density of wheat midge. The three wheat midges have totally distinct parasitoid complexes in Belgium. Eight species (Hymenoptera: Pteromalidae and Platygastridae) were found as parasitoid of S. mosellana: Macroglenes penetrans (Kirby), Amblypasis tritici (Walker), Euxestonotus error (Fitch), Euxestonutus sp. Fouts, Leptacis sp. Foerster, Platygaster gracilipes (Huggert), Platygaster nisus Walker, and Platygaster tuberosula (Kieffer). According to their abundance, M. penetrans, E. error and P. tuberosula appeared as the main parasitoids of S. mosellana in Belgium. For the two other wheat midges, only one species of the family Platygastridae was found for each midge: Platygaster equestris (Spittler) for H. marginata and Synopeas myles (Walker) for C. tritici.     

Genetic diversity of <Emphasis Type="Italic">Haemaphysalis qinghaiensis</Emphasis> (Acari: Ixodidae) in western China

Haemaphysalis qinghaiensis as an endemic species in China mainly infests domestic animals and causes great harm to animals and humans in Northwestern plateau. However, there is no information about genetic diversity within the recently established populations of this tick species. Therefore, the present study analyzed the fragments of mitochondrial 16S rDNA, COI and the nuclear gene ITS1 of 56 H. qinghaiensis ticks across four regions of China which are main endemic areas of this species. Analysis showed 98.1–100% (16S rDNA), 97.9–100% (COI), 99.7–100% (ITS1) identity within individuals. For these sequences, 9, 15 and 8 haplotypes were found for 16S rDNA, COI and ITS1, respectively. Ticks from Yongjing were the most variable group, followed by Lintan, Huangyuan, and Tianzhu. Based on parallel analysis of the mitochondrial and nuclear genetic diversity of H. qinghaiensis, our results indicated that mitochondrial markers (especially COI) were much more useful than nuclear ITS for intraspecific genetic variability analysis.     

Biogeography and designatable units of <Emphasis Type="Italic">Bombus occidentalis</Emphasis> Greene and <Emphasis Type="Italic">B. terricola</Emphasis> Kirby (Hymenoptera: Apidae) with implications for conservation status assessments

Conservation action for species of concern requires that “designatable units” (e.g., species, subspecies, geographic races, genetically distinct forms) are clearly defined, or that the species complex is treated as a whole. Several species of bumble bee are currently threatened, and some of these have cryptic colouration (resembling other species), or form complexes that vary considerably in colour patterning. Here we address the taxonomy and distribution of Bombus occidentalis Greene and B. terricola Kirby, both of which are currently of conservation concern in North America. Bombus occidentalis includes two apparently monophyletic groups of COI barcode haplotypes (recently considered as subspecies) with ranges mostly separated by that of their sister species, B. terricola. The southern B. o. occidentalis ranges throughout the western United States and into western Canada from southern Saskatchewan and Alberta, and throughout British Columbia north to ca. 55°N; the northern B. o. mckayi Ashmead, is restricted to north of this in British Columbia, westernmost Northwest Territories, Yukon Territory and Alaska. Bombus o. mckayi exists, as far as is known, only with a “banded” colour pattern. By contrast, B. o. occidentalis occurs in both banded and non-banded colour patterns, although the southern banded colour pattern is geographically isolated from the northern subspecies. Bombus o. occidentalis has declined throughout its range, perhaps due in part to exposure to novel parasites. Despite having similar levels of parasitism (ca. 40 %) as the southern subspecies, B. o. mckayi appears to have stable populations at present. There is therefore compelling evidence that the two subspecies should be distinguished for conservation and management purposes. We present the evidence for their distinction and provide tools for subspecies recognition.     

Temperature-related activity of <Emphasis Type="Italic">Gomphiocephalus hodgsoni</Emphasis> (Collembola) mitochondrial DNA (COI) haplotypes in Taylor Valley,Antarctica

Gomphiocephalus hodgsoni (Collembola) is the most common and widely distributed arthropod in the Dry Valleys of southern Victoria Land, and is genetically diverse with over 70 mitochondrial cytochrome c oxidase subunit I (COI) haplotypes. There is also considerable physiological variation among G. hodgsoni individuals in their cold tolerance and metabolic activity. Here, we assessed genetic haplotypes of G. hodgsoni relative to the environmental conditions during which individuals were active. We sequenced the COI region of 151 individuals collected in pitfall traps from three sites within Taylor Valley and found 19 unique haplotypes that separated into two distinct lineages (1.6 % divergence), with one lineage comprising 80 % of the sequenced population. During two-hourly sampling, air temperature was the strongest predictor of activity between the two lineages (R ² = 0.56), and when combined with subsurface soil temperature, relative humidity and photosynthetically active radiation, explanatory power increased to R ² = 0.71. With steadily increasing air temperatures predicted for much of Antarctica, it is likely that some haplotypes will have a selective advantage and potentially result in decreased genetic variability within populations. We suggest that temporal monitoring of the relative proportions of COI haplotypes or other appropriate genetic markers may provide a subtle measure of biological responses to environmental changes within Antarctic terrestrial ecosystems.     

Worldwide sampling reveals low genetic variability in populations of the freshwater ciliate <Emphasis Type="Italic">Paramecium biaurelia</Emphasis> (<Emphasis Type="Italic">P. aurelia</Emphasis> species complex,Ciliophora, Protozoa)

Species (or cryptic species) identification in microbial eukaryotes often requires a combined morphological and molecular approach, and if possible, mating reaction tests that confirm, for example, that distant populations are in fact one species. We used P. biaurelia (one of the 15 cryptic species of the P. aurelia complex) collected worldwide from 92 sampling points over 62 years and analyzed with the three above mentioned approaches as a model for testing protistan biogeography hypotheses. Our results indicated that despite the large distance between them, most of the studied populations of P. biaurelia do not differ from each other (rDNA fragment), or differ only slightly (COI mtDNA fragment). These results could suggest that in the past, the predecessors of the present P. biaurelia population experienced a bottleneck event, and that its current distribution is the result of recent dispersal by natural or anthropogenic factors. Another possible explanation for the low level of genetic diversity despite the huge distances between the collecting sites could be a slow rate of mutation of the studied DNA fragments, as has been found in some other species of the P. aurelia complex. COI haplotypes determined from samples obtained during field research conducted in 2015–2016 in 28 locations/374 sampling points in southern Poland were shared with other, often distant P. biaurelia populations. In the Kraków area, we found 5 of the 11 currently known COI P. biaurelia haplotypes. In 5 of 7 reservoirs from which P. biaurelia was obtained, two different COI haplotypes were identified.     

Genetic population structure and low genetic diversity in the over-exploited sea cucumber <Emphasis Type="Italic">Holothuria edulis</Emphasis> Lesson, 1830 (Echinodermata: Holothuroidea) in Okinawa Island

Understanding genetic connectivity is fundamental for ecosystem-based management of marine resources. Here we investigate the metapopulation structure of the edible sea cucumber Holothuria edulis Lesson, 1830 across Okinawa Island, Japan. This species is of economic and ecological importance and is distributed from the Red Sea to Hawai‘i. We examined sequence variation in fragments of mitochondrial cytochrome oxidase subunit I (COI) and 16S ribosomal RNA (16S), and nuclear histone (H3) at six locations across Okinawa Island. We found higher haplotype diversity for mtDNA (COI: Hd = 0.69 and 16S: Hd = 0.67) and higher heterozygosity of nDNA (H3: H _E = 0.39) in populations from the west coast of Okinawa compared to individuals from populations on the east coast (COI: Hd = 0.40; 16S: Hd = 0.21; H3: H _E = 0.14). Overall population structure was significant (AMOVA results for COI: Φ _ST = 0.49, P < 0.0001; 16S: Φ _ST = 0.34, P < 0.0001; H3: Φ _ST = 0.12, P < 0.0001). One population in the east, Uruma, showed elevated pairwise Φ _ST values in comparisons with all other sites and a marked reduction of genetic diversity (COI: Hd = 0.25 and 16S: Hd = 0.24), possibly as a consequence of a shift to a more dominant asexual reproduction mode. Recent reports have indicated that coastal development in this area influences many marine organisms, and ecosystem degradation in this location could cause the observed decrease of genetic diversity and isolation of H. edulis in Uruma. Our study should provide valuable data to help with the urgently needed management of sea cucumber populations in Okinawa, and indicates particular attention needs to be paid to vulnerable locations.     

Molecular genetic analysis of five extant reserves of black honeybee <Emphasis Type="Italic">Apis melifera melifera</Emphasis> in the Urals and the Volga region

Local populations of the black honeybee Apis mellifera mellifera from the Urals and the Volga region were examined in comparison with local populations of southern honeybee subspecies A. m. caucasica and A. m. carpatica from the Caucasus and the Carpathians. Genetic analysis was performed on the basis of the polymorphism of nine microsatellite loci of nuclear DNA and the mtDNA COI–COII locus. On the territory of the Urals and the Volga region, five extant populations (reserves) of the black honeybee A. m. mellifera were identified, including the Burzyanskaya, Tatyshlinskaya, Yuzhno-Prikamskaya, Visherskaya, and Kambarskaya populations. These five populations are the basis of the modern gene pool of the black honeybee A. m. mellifera from the Urals and the Volga region. The greatest proportion of the remaining indigenous gene pool of A. m. mellifera (the core of the gene pool of the population of A. m. mellifera) is distributed over the entire territory of Perm krai and the north of the Republic of Bashkortostan. For the population of A. m. mellifera from the Urals and the Volga region, the genetic standards were calculated, which will be useful for future population studies of honeybees.     

A new <Emphasis Type="Italic">Gephyraulus</Emphasis> species (Diptera: Cecidomyiidae) inducing flower bud galls on the European sea rocket <Emphasis Type="Italic">Cakile maritima</Emphasis> Scop. (Brassicaceae)

The European sea rocket Cakile maritima Scop. (Brassicaceae) is a common herb growing on sandy coastlines worldwide and is considered a useful plant because of its medicinal importance, its edibility, and potential as an oilseed crop. However, C. maritima is an invasive plant over a wide range, e.g., eastern South America, North America, northern Iran, Australia and New Zealand, and has a limited number of associated herbivorous insects. During investigations on gall midges (Diptera: Cecidomyiidae) in Egypt, we found a gall midge inducing flower bud galls on C. maritima and preventing fruit production, which suggested that this gall midge is a potential pest of this plant. In this paper, we describe this gall midge species, Gephyraulus zewaili Elsayed and Tokuda sp. nov., as new to science by comparing its morphology with that of close congeners. Partial sequence data of the mitochondrial DNA cytochrome oxidase subunit I gene are also provided.     

A deep sequencing approach to estimate <Emphasis Type="Italic">Plasmodium falciparum</Emphasis> complexity of infection (COI) and explore apical membrane antigen 1 diversity

Background

Humans living in regions with high falciparum malaria transmission intensity harbour multi-strain infections comprised of several genetically distinct malaria haplotypes. The number of distinct malaria parasite haplotypes identified from an infected human host at a given time is referred to as the complexity of infection (COI). In this study, an amplicon-based deep sequencing method targeting the Plasmodium falciparum apical membrane antigen 1 (pfama1) was utilized to (1) investigate the relationship between P. falciparum prevalence and COI, (2) to explore the population genetic structure of P. falciparum parasites from malaria asymptomatic individuals participating in the 2007 Demographic and Health Survey (DHS) in the Democratic Republic of Congo (DRC), and (3) to explore selection pressures on geospatially divergent parasite populations by comparing AMA1 amino acid frequencies in the DRC and Mali.

Results

A total of 900 P. falciparum infections across 11 DRC provinces were examined. Deep sequencing of both individuals, for COI analysis, and pools of individuals, to examine population structure, identified 77 unique pfama1 haplotypes. The majority of individual infections (64.5%) contained polyclonal (COI > 1) malaria infections based on the presence of genetically distinct pfama1 haplotypes. A minimal correlation between COI and malaria prevalence as determined by sensitive real-time PCR was identified. Population genetic analyses revealed extensive haplotype diversity, the vast majority of which was shared across the sites. AMA1 amino acid frequencies were similar between parasite populations in the DRC and Mali.

Conclusions

Amplicon-based deep sequencing is a useful tool for the detection of multi-strain infections that can aid in the understanding of antigen heterogeneity of potential malaria vaccine candidates, population genetics of malaria parasites, and factors that influence complex, polyclonal malaria infections. While AMA1 and other diverse markers under balancing selection may perform well for understanding COI, they may offer little geographic or temporal discrimination between parasite populations.

     

Genetic diversity of a hitchhiker and prized food source in the Anthropocene: the Asian green mussel <Emphasis Type="Italic">Perna viridis</Emphasis> (Mollusca,Mytilidae)

Insight into a species’ native and introduced range is essential in understanding the invasion process. Genetic diversity, propagule pressure and environmental conditions all have been recognised as playing a determinant role in invasion success. Here, we aimed to investigate the genetic diversity and population genetic structure (using the COI mtDNA gene region and 22 nDNA microsatellite markers) of the Asian green mussel Perna viridis within its potential native range in Asia and at introduced locations in the USA and the Caribbean. We also analyse genetic data from vessel intercepts and an incursion. By doing so, we aimed to identify genetic signatures that could allow to track vessel samples to their source and provide further insight into potential high-risk invasive populations or areas. Three top hierarchical clusters were identified using the individual-based Bayesian clustering method in STRUCTURE, corresponding to populations in three world regions: (1) USA and Caribbean, (2) India and (3) Southeast Asia. Within Southeast Asia, additional analysis indicate a shallow genetic differentiation of three subgroups consisting of (3a) Thailand, (3b) Taiwan and Hong-Kong, and (3c) a cluster of Singapore–Indonesia samples. Overall, the population structure found in this study suggests that the markers used could be useful in identifying source populations, particularly between the three mains world regions. Most surprisingly however, this study shows that the genetic diversity of samples collected from vessel intercepts and incursions did not differ significantly from established populations in Southeast Asia. In this region, in addition to the high vessel connectivity and number of P. viridis transported, all sampled populations are likely to pose a comparable risk in terms of genetic diversity. The present work represents the most comprehensive population genetic study of P. viridis, and the first to address the potential genetic introduction risk posed by populations of this species. The information and genetic markers in this study constitute a valuable addition to the tools already used to infer on potential high-risk source populations of P. viridis. They should therefore prove useful for biosecurity surveillance and management actions directed at this species.     

Natural and human‐mediated selection in a landrace of Thai rice (Oryza sativa)

Landrace rice in Thailand consists of managed populations grown under traditional and long‐standing agricultural practices. These populations evolve both in response to environmental conditions within the local agro‐ecosystem and in response to human activities. Single landraces are grown across varying environments and recently have experienced temporal changes in local environments due to climate change. Here we assess the interplay between natural selection in a changing climate and human‐mediated selection on the population genetic structure of Muey Nawng, a local landrace of Thai rice. Genetic diversity and population structure of landrace rice were assessed by a STRUCTURE analysis of 20 microsatellite loci. The first exon–intron junction of the waxy gene was sequenced to determine genotypes for glutinous or non‐glutinous grain starch. Muey Nawng rice is genetically variable and is structured based on starch grain types and the level of resistance to gall midge pest. A strong positive correlation was found between genetic diversity and the percentage of gall midge infestation. Variation in the waxy locus is correlated with starch quality; selection for non‐glutinous rice appears to involve additional genes. The dynamics of genetic diversity within Muey Nawng rice depends on three factors: (a) a genetic bottleneck caused by strong selection associated with gall midge infestation, (b) selection by local farmers for starch quality and (c) variation introduced by farmer practices for cultivation and seed exchange. These results, when taken in total, document the ability of landrace rice to quickly evolve in response to both natural and human‐mediated selection.     

Molecular and morphological characterization of the predatory mite <Emphasis Type="Italic">Amblyseius largoensis</Emphasis> (Acari: Phytoseiidae): surprising similarity between an Asian and American populations

The accurate characterization of biological control agents is a key step in control programs. Recently, Amblyseius largoensis from Thailand were introduced in Brazil to evaluate their efficiency for the control of the red palm mite, Raoiella indica. The aim of this study was to confirm their identification and to characterize the population from Thailand, comparing it to populations of the Americas and Indian Ocean islands. In addition, a population of A. largoensis from New Caledonia, Oceania, of which DNA sequences were available, was included in phylogenetic analyses. Morphometric data obtained for the population of A. largoensis from Thailand were compared to those of populations from Reunion Island and the Americas through univariate and multivariate analyses. Two DNA fragments were amplified and sequenced: the nuclear ribosomal region ITSS and the mitochondrial 12S rRNA. Haplotypes (12S rRNA) and genotypes (ITSS) were identified and phylogenetic analyses using both fragments were conducted separately and combined using maximum likelihood and the Bayesian information criterion. The integrative approach reveals morphometric and molecular variabilities among populations of A. largoensis and shows that the population identified as A. largoensis collected in Thailand, as well as that from New Caledonia, are conspecific to the populations of the Americas and Indian Ocean islands. Populations from the Americas and Asia are more related to each other than with that from the Indian Ocean islands. Hypotheses to explain this clustering are proposed. Data on the molecular intraspecific variability of this predatory mite from remote areas will be helpful for the development of molecular diagnosis.     

Genetic and ecological differences between <Emphasis Type="Italic">Asphondylia yushimai</Emphasis> and the ivy gall midge, <Emphasis Type="Italic">Asphondylia</Emphasis> sp. (Diptera: Cecidomyiidae), with a new distribution record of the former from Hokkaido and South Korea

The soybean pod gall midge, Asphondylia yushimai, is known to utilize Laurocerasus zippeliana (Rosaceae) and Osmanthus heterophyllus (Oleaceae) as autumn–spring hosts. In addition, ivy, Hedera rhombea (Araliaceae), was thought to be a candidate for an additional autumn–spring host. However, our genetic analysis indicated that no haplotypes of the ivy fruit gall midge, Asphondylia sp., were identical to any of the haplotypes of A. yushimai. Furthermore, the life-history traits of the ivy fruit gall midge, such as voltinism, host-plant range, lower development threshold temperature (LDT), and developmental speed, were clearly different from those of A. yushimai. Thus, the results from genetic analysis and life-history traits revealed that the ivy fruit gall midge was not identical to A. yushimai and that H. rhombea is not an additional autumn–spring host plant for A. yushimai. We also discovered through morphological observation and genetic analysis that A. yushimai is distributed in Hokkaido and South Korea, and that the ivy fruit gall midge exhibits host plant alternation, utilizing both the fruit of Phytolacca americana (Phytolaccaceae) and the flower buds of Paederia foetida (Rubiaceae) as spring–autumn hosts.     

Evidence of multiple introductions and genetic admixture of the Asian brush-clawed shore crab <Emphasis Type="Italic">Hemigrapsus takanoi</Emphasis> (Decapoda: Brachyura: Varunidae) along the Northern European coast

The Asian brush-clawed shore crab Hemigrapsus takanoi is a non-indigenous species along the Northern European coast. Although the history of range expansion of European H. takanoi has been well-documented, little is known about the genetic compositions of either the introduced European populations or the native Asian ones. We therefore collected H. takanoi broadly from their native Asian sites and introduced European ranges, and genotyped them by sequencing the mitochondrial 16S RNA gene and by analyzing nuclear microsatellite loci. Our results revealed that the H. takanoi Bay of Seine (France) populations consisted of a genetic admixture between populations in Japan and those in the Yellow Sea region. These French populations should be carefully monitored in the future, since the genetic admixture of multiple source populations may accelerate range expansion in non-indigenous organisms. Our results also suggested that shipping lines from East Asia were more probable vectors than historical juvenile oyster transportations from Japan for the foundation of present European H. takanoi populations. Interestingly, gene flow between populations in Japan and those in the Yellow Sea region (i.e., domestic invasion) was not observed despite the higher potential for artificial translocations via shipping lines in the native Asian range compared with those from Asia to Europe. The lack of domestic invasions implied that intra-specific priority effects of the resident H. takanoi populations played an important role in preventing the successful colonization of artificially-transferred individuals.     

Identification and genetic diversity of two invasive <Emphasis Type="Italic">Pissodes</Emphasis> spp. Germar (Coleoptera: Curculionidae) in their introduced range in the southern hemisphere

During the first half of the twentieth century, two accidental cases of introduction of Pissodes weevils were recorded from the southern hemisphere. The weevils in South Africa were identified as the deodar weevil (Pissodes nemorensis) and those in South America as the small banded pine weevil (Pissodes castaneus). Wide distribution of the two species in their invasive range, general difficulty in identifying some Pissodes spp., and the varying feeding and breeding behaviours of the species in South Africa has necessitated better evidence of species identity and genetic diversity of both species and population structure of the species in South Africa. Barcoding and the Jerry-to-Pat region of the COI gene were investigated. Morphometric data of the South African species was analysed. Our results confirmed the introduction of only one Pissodes species of North American origin to South Africa. However, this species is not P. nemorensis, but an unrecognized species of the P. strobi complex or a hybrid between P. strobi and P. nemorensis. Only P. castaneus, of European origin, was identified from South America. We identified ten mitochondrial DNA haplotypes from South Africa with evidence of moderate genetic structure among geographic populations. Terminal leader and bole-feeding weevils did not differ at the COI locus. A single haplotype was identified from populations of P. castaneus in South America. Results of the present study will have implications on quarantine, research and management of these insect species.     

Two new complete mitochondrial genomes of <Emphasis Type="Italic">Dorcus</Emphasis> stag beetles (Coleoptera,Lucanidae)

The systematics of Dorcus MacLeay has been a long-standing debate. Mitochondrial genomes were widely used to deeply understand the phylogeny of problematic taxa in virtue of their genetic importance and comprehensiveness. To provide more useful genetic data for resolving the systematic disputation of Dorcus stag beetles. The complete mitochondrial genomes of Dorcus hopei and Dorcus seguyi were obtained using the next generation sequencing. Characteristics of the two genomes are explicated through comparing their genome organization and base composition, protein-coding genes and codon usage, intergenic spacers and non-coding region, transfer and ribosomal RNA genes and control region. Phylogenetic relationships were reconstructed using Maximum likelihood and Bayesian inference analyses based on the concatenated nucleotide sequences of 13 PCGs from 9 stag beetles and 3 scarab beetles. The complete mitogenomes of D. hopei and D. seguyi was 16,026 bp/17,955 bp long, respectively. A tandem repeat with the length of 940 bp was presented in the A+T-rich region in D. hopei. An unexpected non-coding region of 332 bp was located between nad2 and trnW in D. seguyi. The phylogenetic analyses robustly supported that D. hopei formed a branch with the generic type of D. parallelipipedus. Whereas D. seguyi was not covered in the branch of (D. hopei?+?D. parallelipipedus), but was sister to them. The results indicated that D. hopei should be a good member of Dorcus MacLeay. The taxonomic status of D. seguyi remained to be studied furtherly.     

Complete mitochondrial genome sequence of <Emphasis Type="Italic">Cucullaea labiata</Emphasis> (Arcoida: Cucullaeidae) and phylogenetic implications

The complete mitochondrial genome of Cucullaea labiata (Arcoida: Cucullaeidae) was firstly determined in this study in order to better understand the phylogenetic relationship between Cucullaeidae and Arcidae. The C. labiata mitochondrial genome was 25,845 bp in size and contained 12 protein-coding genes, 2 rRNA and 22 tRNA genes. The number and the location of the tRNA genes were different from three Arcidae species (Scapharca broughtonii, Scapharca kagoshimensis and Tegillarca granosa). Gene arrangement also differed dramatically. The length of the non-coding regions was 10,559 bp, in which the largest one (6057 bp) included eight point nine copies of a 659 bp repeat motif. The number of repeated sequences was different in different individuals, similar to the findings from the mitochondrial genome of S. broughtonii and Placopecten magellanicus. One intron was found in cox1 gene both in CL_98 and in CL_99 individuals of C. labiata. The reason why mitochondrial introns are retained so scarcely in bivalve taxa needs further research. Phylogenetic analyses based on 12 concatenated amino acid sequences of protein-coding genes supported Cucullaeidae was the sister group of Arcidae.