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Precise mosquito identification is integral to effective arbovirus surveillance. Nonetheless, the conventional morphological approach to identifying mosquito species is laborious, demands expertise and presents challenges when specimens are damaged. DNA barcoding offers a promising alternative, surmounting challenges inherent in morphological identification. To integrate DNA barcoding into arbovirus surveillance effectively, a robust dataset of mosquito barcode sequences is required. This study established a comprehensive repository of Cytochrome Oxidase I (COI) barcodes, encompassing 177 samples representing 45 mosquito species from southern and northern Western Australia (WA), including 16 species which have not been previously barcoded. The average intraspecific and interspecific genetic distances were 1% and 6.8%, respectively. Anopheles annulipes sensu lato had the highest intraspecific distance at 9.1%, signifying a genetically diverse species. While validating the potential of COI barcodes to accurately differentiate mosquito species, we identified that some species pairs have low COI divergence. This includes Aedes clelandi and Ae. hesperonotius, Tripteroides atripes and Tp. punctolaeralis and Ae. turneri and Ae. stricklandi. In addition, we observed ambiguity in identification of the members of Culex sitiens subgroup (Cx. annulirostris, Cx. palpalis and Cx. sitiens) and three members of Cx. pipiens complex (Cx. australicus, Cx. globocoxitus, Cx. quinquefasciatus). In summary, despite presenting challenges in the identification of some mosquito species, the COI barcode accurately identified most of the species and generated a valuable resource that will support the WA arbovirus surveillance program and enhance public health intervention strategies for mosquito‐borne disease control. [ABSTRACT FROM AUTHOR]

Tephritis angustipennis (Diptera: Tephritidae) and Campiglossa loewiana (Diptera: Tephritidae) are phytophagous pests in China. Their damage has significantly impacted the collection and cultivation of germplasm resources of native Asteraceae plants. However, the genetic characteristics and structure of their population are unclear. This study focused on the highly damaging species of T. angustipennis and C. loewiana collected from the three-river source region (TRSR). We amplified the mitochondrial cytochrome C oxidase subunit I (mt COI) gene sequences of these pests collected from this area and compared them with COI sequences from GenBank. We also analyzed their genetic diversity and structure. In T. angustipennis , 5 haplotypes were identified from 5 geographic locations; the genetic differentiation between France population FRPY (from Nylandia, Uusimaa) and China populations GLJZ (from Dehe Longwa Village, Maqin County), GLDR (from Zhique Village, Dari County), and GLMQ (from Rijin Village, Maqin County) was the strongest. GLJZ exhibited strong genetic differentiation from GLDR and GLMQ, with relatively low gene flow. For C. loewiana , 11 haplotypes were identified from 5 geographic locations; the genetic differentiation between the Chinese population GLMQ-YY (from Yangyu Forest Farm, Maqin County) and Finnish population FDNL (from Nylandia, Uusimaa) was the strongest, with relatively low gene flow, possibly due to geographical barriers in the Qinghai–Tibet plateau. Only 1 haplotype was identified across GLDR, GLMQ, and GLBM. High gene flow between distant locations indicates that human activities or wind dispersal may facilitate the dispersal of fruit flies and across different geographic. Geostatistical analysis suggested a recent population expansion of these 2 species in TRSR. Our findings provide technical references for identifying pests in the TRSR region and theoretical support for managing resistance, monitoring pest occurrences, analyzing environmental adaptability, and formulating biological control strategies for Tephritidae pests on Asteraceae plants. [ABSTRACT FROM AUTHOR]

Morphologically cryptic and pseudo-cryptic species pose a challenge to taxonomic identification and assessments of species diversity and distributions. Such is the case for the sea cucumber Stichopus horrens, commonly confused with Stichopus monotuberculatus. Here, we used mitochondrial cytochrome oxidase subunit I (COI) and microsatellite markers to examine genetic diversity in Stichopus cf. horrens throughout the Philippine archipelago, to aid species identification and clarify species boundaries. Phylogenetic analysis reveals two recently diverged COI lineages (Clade A and Clade B; c. 1.35–2.54 Mya) corresponding to sequence records for specimens identified as S. monotuberculatus and S. horrens, respectively. Microsatellite markers reveal two significantly differentiated genotype clusters broadly concordant with COI lineages (Cluster 1, Cluster 2). A small proportion of individuals were identified as later-generation hybrids indicating limited contemporary gene flow between genotype clusters, thus confirming species boundaries. Morphological differences in papillae distribution and form are observed for the two species, however tack-like spicules from the dorsal papillae are not a reliable diagnostic character. An additional putative cryptic species was detected within Clade B-Cluster 2 specimens warranting further examination. We propose that these lineages revealed by COI and genotype data be referred to as Stichopus cf. horrens species complex. [ABSTRACT FROM AUTHOR]

Simple Summary: Thrips, the notorious sap-sucking insects, serve as the vector of plant viruses. Their accurate species identification is essential for determining the vector species and implementing successful pest management techniques. Mitochondrial COI (DNA barcode) sequence variation has proven to be effective in identifying species in many insect pest groups. In this study, we identified 19 thrips species that were found on different host plants in Bangladesh. Among the 19 species, we identified four prominent vector species (Frankliniella intonsa, Thrips tabaci, Scirtothrips dorsalis and T. palmi) and one significant pollinator, Microcephalothrips abdominalis. The findings presented here emphasize the importance of conducting DNA barcoding, analyzing population structure, and assessing the genetic diversity of thrips species in this region. This research is crucial for comprehending their host preferences, potential for adaptation, and genetic variation at both local and regional levels. Accurate identification of pests and invasive species is essential for implementing effective control and quarantine measures. Misidentifications can lead to the application of ineffective control strategies. Thrips are economically important pests, and some species transmit plant viruses that are widely distributed and can damage vegetables and cash crops. Although few studies on thrips species have been conducted in Bangladesh, the variation and genetic diversity of thrips species remain unknown. In this study, we collected thrips samples from 16 geographical locations throughout the country and determined the nucleotide sequences of the mitochondrial cytochrome c oxidase subunit 1 (mtCOI) gene in 207 thrips individuals. Phylogenetic analysis revealed ten genera (Thrips, Haplothrips, Megalothrips, Scirtothrips, Frankliniella, Dendrothripoides, Astrothrips, Microcephalothrips, Ayyaria, and Bathrips) and 19 species of thrips to inhabit Bangladesh. Among these, ten species had not been previously reported in Bangladesh. Intraspecific genetic variation was diverse for each species. Notably, Thrips palmi was the most genetically diverse species, containing 14 haplotypes. The Mantel test revealed no correlation between genetic and geographical distances. This study revealed that thrips species are expanding their host ranges and geographical distributions, which provides valuable insights into monitoring the diversity of and control strategies for these pests. [ABSTRACT FROM AUTHOR]

The Asian citrus psyllid (ACP) is the main vector of Citrus Huanglongbing, the most damaging citrus disease, causing significant financial losses in the citrus industry. Global warming has expanded the habitat of this pest, allowing it to continue its northward migration to China. Population genetic information of ACP is fundamentally essential for species management. This study investigated the genetic diversity and population structure of Chinese ACP using the mitochondrial cytochrome oxidase subunit I gene by dataset comprised 721 sequences from 27 geographic sites in China. Low haplotype diversity (0.323 ± 0.022) and low nucleotide diversity (0.00071 ± 0.00007) were observed in the entire population, which may indicate recent founder events. Twenty-three haplotypes were identified and clustered into 2 haplogroups: haplogroup I and haplogroup II. Haplogroup II included only 2 unique haplotypes, which occurred exclusively in the Southwest China ACP population. Genetic differentiation analyses were also indicative of Southwest China population was significantly differentiated from the remaining populations. Demographic history analysis showed that ACP population in China has experienced demographic expansion. Our results provided a better understanding of the genetic distribution patterns and structures of ACP populations in China. [ABSTRACT FROM AUTHOR]

Aim: To investigate the drivers of intra‐specific genetic diversity in centipedes, a group of ancient predatory soil arthropods. Location: Asia, Australasia and Europe. Time Period: Present. Major Taxa Studied: Centipedes (Class: Chilopoda). Methods: We assembled a database of 1245 mitochondrial cytochrome c oxidase subunit I sequences representing 128 centipede species from all five orders of Chilopoda. This sequence dataset was used to estimate genetic diversity for centipede species and compare its distribution with estimates from other arthropod groups. We studied the variation in centipede genetic diversity with species traits and biogeography using a beta regression framework, controlling for the effect of shared evolutionary history within a family. Results: A wide variation in genetic diversity across centipede species (0–0.1713) falls towards the higher end of values among arthropods. Overall, 27.57% of the variation in mitochondrial COI genetic diversity in centipedes was explained by a combination of predictors related to life history and biogeography. Genetic diversity decreased with body size and latitudinal position of sampled localities, was greater in species showing maternal care and increased with geographic distance among conspecifics. Main Conclusions: Centipedes fall towards the higher end of genetic diversity among arthropods, which may be related to their long evolutionary history and low dispersal ability. In centipedes, the negative association of body size with genetic diversity may be mediated by its influence on local abundance or the influence of ecological strategy on long‐term population history. Species with maternal care had higher genetic diversity, which goes against expectations and needs further scrutiny. Hemispheric differences in genetic diversity can be due to historic climatic stability and lower seasonality in the southern hemisphere. Overall, we find that despite the differences in mean genetic diversity among animals, similar processes related to life‐history strategy and biogeography are associated with the variation within them. [ABSTRACT FROM AUTHOR]

Simple Summary: Blue oat mite species mainly include Penthaleus major, P. falcatus, P. tectus, and P. minor. Among them, P. major, P. falcatus and P. tectus are important pest mites on gramineous crops, which often occur in low-temperature and high-humidity environments. These three mites are difficult to distinguish in the field due to their small size and similar shape. Furthermore, they exhibit different resistance to chemical pesticides, making it challenging to control their outbreak. Worryingly, we only saw reports of frequent occurrence of P. major in winter wheat regions in China, and no other species of blue oat mites were found. Understanding the distribution pattern, genetic diversity, and genetic differentiation among populations of blue oat mite species in winter wheat regions of China is essential for developing effective control programs. In this study, we evaluated the distribution of blue oat mites in the major wheat regions in China and assessed the level of genetic diversity and genetic structure of blue oat mites from winter wheat regions of China using mitochondrial cytochrome c oxidase I (COI) sequences. Blue oat mite species, including Penthaleus major and P. tectus, are pests widely distributed across China that cause damage to winter wheat. This study evaluated the genetic diversity of P. major and P. tectus on Triticum hosts collected from 23 geographic locations based on mitochondrial cytochrome c oxidase subunit I (COI) sequences. We identified nine haplotypes in 438 P. major individuals from 21 geographic locations and five haplotypes in 139 P. tectus individuals from 11 geographic locations. Meanwhile, P. major exhibits high values of haplotype diversity (Hd) and nucleotide diversity (Pi) (Hd = 0.534 > 0.5 and Pi = 0.012 > 0.005), representing a large stable population with a long evolutionary history. P. tectus shows low values of Hd and Pi (Hd = 0.112 < 0.5 and Pi = 0 < 0.005), which suggest recent founder events. Moreover, demographic analysis suggested that P. major and P. tectus have not undergone a recent population expansion. The lowest genetic variation was observed in Xiangzhou (XZ-HB), Zaoyang (ZY-HB), Siyang (SY-JS), and Rongxian (RX-SC), with only one species and one haplotype identified in over 30 individuals. Robust genetic differentiation was found in P. major compared to P. tectus, which provides a theoretical basis for the widespread distribution of P. major in China. [ABSTRACT FROM AUTHOR]

The brown planthopper, Nilaparvata lugens (Stål) is a major sucking insect pest of rice. This insect has long been considered as migratory; however, its route in India is still unknown. Hence, to find out its migration route genetic diversity, genetic structure and gene flow of 16 N. lugens populations from major rice growing regions of India was studied based on mitochondrial cytochrome oxidase I (COI). The results revealed a high genetic homogeneity among the populations on the basis of genetic diversity statistics and neutrality tests. There was a prevalence of a single major haplotype across the country. No spatial relevance was found with the genetic structure of the populations indicating presence of excessive gene flow among them. Extensive gene flow among populations was also confirmed with the presence of higher number of immigrants in North, Central, and East India. To further clarify the migration sources, 48 h air-mass reverse trajectory was performed for Varanasi just aftermath of cyclones Amphan and Yaas, which disclosed Eastern/Northeastern states along with Bangladesh and Myanmar as the possible source areas. Overall, the results revealed a single panmictic homogeneous population of N. lugens in India with extensive gene flow as a consequence of their migration. These findings will help in better forecasting enabling efficient regional management of this important rice pest. [ABSTRACT FROM AUTHOR]

Stored grain insect pests are of economic importance, and they spread rapidly through the grain trade. Hence, it is important to identify these pests accurately. Although several morphological methods exist, it is tedious and timeconsuming. DNA barcoding using mitochondrial COI is an alternative approach that aids in precise species identification. In this study, 13 stored grain insect pests belonging to the order Coleoptera and Lepidoptera were collected from different storage structures in Coimbatore. A fragment of mt-COI was amplified and sequenced. Sequence analyses were carried out with BOLD (Barcode of Life Data System) and ABGD (Automatic Barcode Gap Discovery) tools. The barcoding gap analysis revealed that the inter-species genetic distance is greater than the intraspecies genetic distance. The ABGD analysis for species delimitation partitioned the Coleopteran and Lepidopteran datasets into 9 and 4 putative species, respectively. The barcode gap was absent in more closely related species. However, analysis of their sequences revealed significant variations. Our results showed the ability of the mt-COI to discriminate between the species, thus provide a complementary technique for the diagnosis of stored grain insect pests. [ABSTRACT FROM AUTHOR]

Grouper aquaculture is rapidly expanding in both tropical and subtropical regions. The presence of marine leeches (Pterobdella arugamensis; previously named Zeylanicobdella arugamensis) infesting cultured groupers, however, can have a fatal effect on grouper aquaculture production and cause significant economic loss. Understanding the marine leech's population structure is therefore important to determine its possible distributional origin and distributional mechanisms, which will help monitor and mitigate the infestation. In this study, a total of 84 marine leeches collected from cultured hybrid groupers Epinephelus spp. in Brunei Darussalam, Malaysia and Indonesia were identified as P. arugamensis, based on morphological and mitochondrial cytochrome c oxidase subunit I gene sequence analyses. These leech samples, together with additional sequences from the GenBank database, were grouped into four genetically distinct haplogroups: (1) Asia Pacific, (2) Borneo, (3) Surabaya and (4) Iran. The four populations were found to be highly diverged from each other. The results also suggested that the samples from the Asia Pacific population could be dispersed and transported from Indonesia. [ABSTRACT FROM AUTHOR]

Revealing cryptic diversity constitutes the backbone of the future identification and description of a new lineage. For the genus Ophisops (Lacertidae), previous studies indicated that this genus is characterized by cryptic diversity as three clades were obtained for O. elegans and O. occidentalis that do not fit the classical systematics. Notably, we were interested to delineate the distribution range of the two clades of O. elegans described in Iran. We sequenced 65 individuals of Ophisops mainly from northwestern Iran for one mitochondrial (cytochrome c oxidase subunit 1 [COI]; 686 base pairs [bp]) and three nuclear genes (R35, MC1R, and PKM2; 1,857 bp). Phylogeographic analysis from mitochondrial and nuclear genes confirmed that Iranian samples belong to two major haplogroups (divergence of 13% for COI) that are also split into several subclades (divergence of 6%–10% for COI), revealing an unsuspected diversity within Iranian Ophisops. Divergence dating and biogeographical analysis indicated that most clades arose through vicariance and dispersal processes during the Mio‐Pliocene (between 7.2 and 1 Myr). However, a scenario of expansion/regression is also advocated for explaining the distribution and contact between three subclades in North Iran. We propose to recognize the three major clades as three potential candidate species. Moreover, we found some correspondence between several phylogenetic clades or subclades identified and six of the nine subspecies described for O. elegans. Both species and subspecies delimitation and identification would deserve additional investigations (including morphology, ecology, biogeography, and behavior) to fulfill the conditions of integrative taxonomy. [ABSTRACT FROM AUTHOR]

Leafhoppers (Cicadellidae) occur in all biogeographical regions and feed on a wide range of host plants. Classic identification of leafhoppers is based only on the morphology of male genitalia, while DNA barcoding is an effective tool to identify different leafhopper species in any stage of their life cycle. Phylogenetic relationships among leafhoppers remain unexplored, but recent phylogenies have begun to interpret the evolutionary relationships of previously recognized tribes and subfamilies. Here we used mitochondrial Cytochrome Oxidase I sequences to barcode and determine the phylogenetic relationship among eight leafhopper genera collected from Egypt. Eight leafhopper samples from eight different genera were collected from various plants using a sweeping net and aspirator. Males of seven species were available while females only were available from the one genus (Austroagallia sp.). Novel leafhopper-specific mtDNA COI degenerate primers were designed. Our results showed that the mean of intraspecific COI sequence divergence in leafhoppers was 0.45%, with a range from 0.15% to 1.23%, and the divergences of most species were over 2%. For the first time, we were exploring the evolutionary relationships among surveyed genera (Austroagallia, Empoasca, Eupteryx, Exitianus, Macrosteles, Nephotettix, and Orosius) using a robust phylogenetic supertree. Our results demonstrate that DNA barcoding is likely to be a powerful tool for identifying and understanding leafhoppers' evolution. [ABSTRACT FROM AUTHOR]

Srinu, G.; Padmavathi, P., and Chatla, D., 2019. Identification and validation of Anabas spp. (Osteichthyes: Anabantidae) through morphology and DNA barcoding from lake Kolleru, Andhra Pradesh, India. In: Jithendran, K.P.; Saraswathy, R.; Balasubramanian, C.P.; Kumaraguru Vasagam, K.P.; Jayasankar, V.; Raghavan, R.; Alavandi, S.V., and Vijayan, K.K. (eds.), BRAQCON 2019: World Brackishwater Aquaculture Conference. Journal of Coastal Research, Special Issue No. 86, pp. 142–148. Coconut Creek (Florida), ISSN 0749-0208. Anabas Cuvier, 1816 (Pisces: Osteichthyes: Anabantidae) constitutes a major proportion of fisheries with great ecological and commercial importance in lake Kolleru, Andhra Pradesh, India. Morphological analysis based on 54 adult specimens from different parts of the lake revealed two putative species, Anabas testudineus (Bloch, 1795) and A. oligolepis (Bleeker, 1855) which are very closely related to and barely distinguished by few characteristics (e.g. body depth) exclusively in fully matured specimens. In juvenile and sub-adult specimens, considerable overlap in the range of colour patterns has been observed, inturn it may lead to taxonomic confusion. DNA barcoding is a promising technique for accurate species level identification based on partial mitochondrial cytochrome oxidase I (COI) gene. A total of five DNA barcode sequences were generated using universal primers. The average Kimura 2 Parameter (K2P) distances within species and genera were 0.5% and 1.3% respectively. These barcodes discriminated the congeneric species with lower genetic distance when compared to other taxa. This may aid in deciding the threshold value to delineate the species complex of Anabas from lake Kolleru. Neighbour joining (NJ) tree formed two major clades each shared with accessions to the same species. The maximum likelihood (ML) phylogenetic tree was constructed using COI sequences of Anabantidae which were complemented with the sequences of six other families of order Anabantiformes and the phylogenetic position of Anabantidae was discussed. Future studies are needed for deeper insights into genetic diversity and evolutionary relationships of Anabantiformes using multiple molecular markers. [ABSTRACT FROM AUTHOR]

The Philippines is the center of radiation of the land snail subfamily Helicostylinae, with around 253 recognized species. Despite their morphological diversity, research on their biology and taxonomy is lacking. We present here the first mitochondrial COI profiles of 32 species of Philippine helicostyline land snails. With the addition of sequences downloaded from GenBank, we tested the utility of the COI for species identification. Relative distributions of intraspecific and interspecific distances overlapped; hence, no barcoding gap was observed. However, 90% of uncorrected interspecific comparisons can distinguish species at 14% genetic distance or lower. Furthermore, the COI barcodes could not discriminate several co-distributed species that have similar conchological features, which should be flagged for taxonomic re-evaluation. [ABSTRACT FROM AUTHOR]

Biofouling by the coronuloid barnacle Chelonibia patula (synonimized as C. testudinaria) was observed during the collection and maintenance of live broodstock of a few species of commercially important crustaceans at Mandapam Regional Centre of ICAR-CMFRI located along the Coromandel coast of Tamil Nadu, India. We assessed the genetic identity of C. patula found on the various crustacean hosts (Panulirus polyphagus, Portunus pelagicus and Charybdis natator) and turtle, olive ridley (Lepidochelys olivacea) from the Gulf of Mannar Biosphere Reserve. The phylogeographic studies of turtle barnacles from all potential hosts, carried out across world oceans about their species paradox, do not represent collection record from India and hosts like lobsters and olive ridleys. The mitochondrial Cytochrome c oxidase subunit 1 (COI) analysis of this epibiont from various hosts indicated that species from the south-east coast of India belong to the Indian Ocean/Western Pacific clade of C. testudinaria. The very low K2P genetic distance (0.008) between C. patula and C. testudinaria, on comparison with existing GenBank records, supports this finding. The phylogeographic structuring and evolutionary divergence between the clades of C. testudinaria with COI sequence data indicated that the Indian Ocean/Western Pacific and Eastern Pacific clades are the most divergent. [ABSTRACT FROM AUTHOR]

In the present study, two pod borer larvae were found to damage pods of the host plant Senna alata. As pods (seeds) are commercially important, it prompted the investigators to find out the adult of pod borer larvae by molecular sequencing and reported in this article for the first time from S. alata. Successful amplification was achieved for chloroplast DNA isolated from leaves of host plant S. alata and tissue DNA from unknown larvae and sequenced. The sequences were submitted to NCBI domain, and the taxonomic position of host plant of pod borer larvae was confirmed as S. alata (L.) Roxb. Of the two sequences belonging to pod borer larvae, specimen 1 (S1) matched with already available three sequences of Thylacoptila sp. AA. But, specimen 2 (S2) showed significant variation in its genetic distance in Neighborhood Joining tree and maximum likelihood tree; these factors imply that specimen 2 (S2) is distinct from Thylacoptila sp. AA. Therefore, it is reported that specimen 2 may be represented as Thylacoptila sp. BB where in BB indicates variant from other available sequences. Nonetheless, reporting species of Thylacoptila as insect pest in pods of S. alata is an important contribution to the annals of insect–pest–plant interaction more importantly in medicinally important plant species S. alata. [ABSTRACT FROM AUTHOR]

The identities of what have been referred to as Epigrapsus notatus (Heller, 1865) and E. villosus Ng, 2003, are evaluated using morphological and molecular methods. Morphological comparisons showed that the main characters used to diagnose E. villosus (viz. the setation of the carapace, the condition of the epigastric cristae, the granulation on the branchial region, and proportions of the ambulatory legs) can be attributed to the small size of the type specimen; and the extreme condition of these structures can be connected to adult forms of E. notatus through transitional phases. One morphological feature (the laterally expanded first epibranchial tooth) is seen only in the holotype of E. villosus, but it can also be regarded as a character state specific to small individuals of E. notatus, and signifying the extreme end of possible variation in that species. The analysis of mitochondrial cytochrome c oxidase subunit I (COI) reveals three haplotypes in the E. notatus/E. villosus complex, but with only as much as 0.61% of nucleotide divergence between them. In conclusion, it is clear that E. notatus undergoes dramatic morphological change during its growth, and that E. villosus should be considered a junior synonym of E. notatus. [ABSTRACT FROM AUTHOR]

Pink bollworm (PBW),Pectinophora gossypiellais one of the most destructive pest’s globally inflicting huge economic losses in cotton even during later stages of crop growth. In the present investigation, the population genetic structure, distribution, and genetic diversity ofP. gossypiellain cotton growing zones of India using partial mitochondrial DNA cytochrome oxidase-I (COI) gene was addressed. The overall haplotype (Hd),number of nucleotide differences (K), and nucleotide diversity (π) were 0.3028, 0.327, and 0.00047, respectively which suggest that entire population exhibited low level of genetic diversity. Zone-wise clustering of population revealed that central zone recorded low level ofHd(0.2730) as compared to north (0.3619) and south (0.3028) zones. The most common haplotype (H1) reported in all 19 locations could be proposed as ancestral/original haplotype. This haplotype with one mutational step formed star-like phylogeny connected with 11 other haplotypes. The phylogenetic relationship studies revealed that most haplotypes of populations are closely related to each other. Haplotype 5 was exclusively present in Dharwad (South zone) shared with populations of Hanumangarh and Bathinda (North zone). The result indicated that there is no isolation by distance effect among the Indian populations of PBW. The present study reports a low genetic diversity among PBW populations of India and H1, as ancestral haplotype from which other haplotypes have evolved suggests that the migration and dispersal over long distance and invasiveness are major factors. [ABSTRACT FROM PUBLISHER]

Sequencing of environmental DNA (eDNA) for community analysis (i.e., eDNA metabarcoding) is already well established and applied in molecular microbial ecology. However, molecular methods for use in soil animal studies require further development before high-throughput sequencing can be considered a reliable technique in community ecology. To aid in this effort, we compare primers that target two frequently used genetic markers, mitochondrial cytochrome oxidase (COI) and ribosomal 18S genes, to determine their utility in broad soil animal eDNA studies. DNA was analyzed from individually identified invertebrates to test the efficiency of the primer sets in successfully targeting animal DNA and identification through sequencing. Primers were also tested for amplification of faunal genes from forest soil and leaf litter eDNA. Targeting the 18S gene resulted in the most successful amplification and correct identification of a wide range of individual invertebrate taxa, and was the most reliable primer set for use in eDNA analysis of invertebrate communities spanning several phyla. In contrast, the selected COI primers were inefficient in identifying a wide range of invertebrates, and amplified mostly bacterial sequences from eDNA. [ABSTRACT FROM AUTHOR]

Partial mitochondrial cytochrome c oxidase subunit I (mt COI) sequences were generated from: Toxoplasma gondii (strains CTG, GTI, MAS, ME49, PTG, TgCatBr5, TgCat, Br64, TgCgCal, TgToucan); Neospora caninum (Strain NC1); Hammondia hammondi (Strain H.H-20); H. heydorni; H. cf. triffittae; Cystoisospora felis; C. suis; C. canis; C. rivolta; C. cf. ohioensis; Caryospora bigenetica; Sarcocystis rileyi; and S. neurona. Nuclear 18S rDNA sequences were generated for H. heydorni, H. hammondi, C. suis, C. canis, C. felis, C. rivolta, C. cf. ohioensis, S. neurona, and S. rileyi. Aligned, concatenated 18S rDNA and COI sequences were Bayesian analysed using partitioned nucleotide substitution models [HKY + I +G for 18S; GTR+ I +G codon (code = metmt) for COI]. Phylogenetic hypotheses supported a monophyletic Sarcocystidae and its subfamilie with two major clades within the Toxoplasmatinae: (1) a monophyletic clade of Cystoisospora spp. with Nephroisospora eptesici; and (2) a clade of Toxoplasma, Neospora and Hammondia. Within the latter, Hammondia was shown to be paraphyletic; H. heydorni and H. triffittae were monophyletic with N. caninum [canine definitive hosts (DHs)], whereas H. hammondi was monophyletic with T. gondii (feline DHs). A new genus is erected to resolve the paraphyly of the genus Hammondia confirmed using mt COI and combined 18S/COI sequence datasets. [ABSTRACT FROM AUTHOR]

The whitefly Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae), with its global distribution and extensive genetic diversity, is now known to be a complex of over 35 cryptic species. However, a satisfactory resolution of the systematics of this species complex is yet to be achieved. Here, we designed experiments to examine reproductive compatibility among species with different levels of mitochondrial cytochrome oxidase I (mtCOI) divergence. The data show that putative species with mtCOI divergence of >8% between them consistently exhibited complete reproductive isolation. However, two of the putative species, Asia II 9 and Asia II 3, with mtCOI divergence of 4.47% between them, exhibited near complete reproductive compatibility in one direction of their cross, and partial reproductive compatibility in the other direction. Together with some recent reports on this topic from the literature, our data indicates that, while divergence in the mtCOI sequences provides a valid molecular marker for species delimitation in most clades, more genetic markers and more sophisticated molecular phylogeny will be required to achieve adequate delimitation of all species in this whitefly complex. While many attempts have been made to examine the reproductive compatibility among genetic groups of the B. tabaci complex, our study represents the first effort to conduct crossing experiments with putative species that were chosen with considerations of their genetic divergence. In light of the new data, we discuss the best strategy and protocols to conduct further molecular phylogenetic analysis and crossing trials, in order to reveal the overall pattern of reproductive incompatibility among species of this whitefly complex. [ABSTRACT FROM AUTHOR]

Amphistomiasis, a neglected trematode infectious disease of ruminants, is caused by numerous species of amphistomes belonging to six families under the Superfamily Paramphistomoidea. In the present study, four frequently used DNA markers, viz. nuclear ribosomal 28S (D1-D3 regions), 18S and ITS2 and mitochondrial COI genes, as well as sequence motifs from these genes were evaluated for their utility in species characterization of members of the amphistomes' Family Gastrothylacidae commonly prevailing in Northeast India. In sequence and phylogenetic analyses the COI gene turned out to be the most useful marker in identifying the gastrothylacid species, with the exception of Gastrothylax crumenifer, which showed a high degree of intraspecific variations among its isolates. The sequence analysis data also showed the ITS2 region to be effective for interspecies characterization, though the 28S and 18S genes were found unsuitable for the purpose. On the other hand, sequence motif analysis data revealed the motifs from the COI gene to be highly conserved and specific for their target species which allowed accurate in silico identification of the gastrothylacid species irrespective of their intraspecific differences. We propose the use of COI motifs generated in the study as a potential tool for identification of these species. [ABSTRACT FROM AUTHOR]

We used an integrative-taxonomy approach to help resolve taxonomic issues within the genus Micropsectra (Diptera:Chironomidae). We used partial cytochrome c oxidase subunit I (COI) and nuclear carbamylphosphate synthetase (CAD) sequences and morphological data to provide a framework for better understanding of North American species in this group of nonbiting midges. As part of our results, we describe 3 new species: Micropsectra neoappendica, n. sp., Micropsectra penicillata, n. sp., and Micropsectra subletteorum, n. sp., and 1 species new to the north-central USA, Micropsectra xantha (Roback). Two of the species, M. neoappendica n. sp. and M. subletteorum n. sp., initially appeared to be morphologically identical to species known from the Palearctic. However, molecular data indicated that they were genetically distinct, and reexamination of adult and pupal morphology revealed slight but consistent diagnostic differences. The implications of using species-level identifications for cryptic-species complexes in biological monitoring and conservation management are briefly discussed with reference to our findings. Our results emphasize the importance of using molecular tools in conjunction with traditional morphological techniques when studying Chironomidae diversity, especially when relying on diagnoses from other geographic regions. [ABSTRACT FROM AUTHOR]

Dysaphis ( Pomaphis) pyri (Boyer de Fonscolombe, 1841) and Dysaphis ( Pomaphis) reaumuri (Mordvilko, 1928) are two holocyclic aphid species alternating between Pyrus (Rosaceae) and Galium (Rubiaceae). Comparative phylogenetic analysis was performed using partial mitochondrial cytochrome oxidase subunit I (COI) and nuclear elongation factor 1 alpha (EF-1α) sequences. Partial COI data indicate the possibility of the early divergence in the D. pyri- D. reaumuri stem, which might have occurred even before the splitting of the common ancestral species of the D. reaumuri-D. plantaginea complex. Such a conclusion seems to be compatible with the available data on host specificity, life cycles and distribution of both species. This introductory phylogenetic analysis based on partial COI and EF-1α sequences indicates the need for reconsideration of the subgeneric structure in the genus Dysaphis. [ABSTRACT FROM AUTHOR]

We use DNA molecular marker technology to correct the deficiency of traditional morphological taxonomy. Totality 770 Pelteobagrus fish from Poyang Lake were collected. After preliminary morphological classification, random selected eight samples in each species for DNA extraction. MitochondrialCOIgene sequence was cloned with universal primers and sequenced. The results showed that there are four species of Pelteobagrus living in Poyang Lake. The average of intraspecific genetic distance value was 0.003, while the average interspecific genetic distance was 0.128. The interspecific genetic distance is far more than intraspecific genetic distance. Besides, phylogenetic tree analysis revealed that molecular systematics was in accord with morphological classification. It indicated thatCOIgene is an effective DNA molecular marker in Pelteobagrus classification. Surprisingly, the intraspecific difference of some individuals (P. e6, P. n6, P. e5, andP. v4) from their original named exceeded species threshold (2%), which should be renewedly classified intoPelteobagrus fulvidraco. However, another individualP. v3 was very different, because its genetic distance was over 8.4% difference from original namedPelteobagrus vachelli. Its taxonomic status remained to be further studied. [ABSTRACT FROM AUTHOR]

A diagnostic polymerase chain reaction (PCR) method is presented for differentiating among the North American internal apple-feeding pests codling moth, Cydia pomonella (L.); oriental fruit moth, Grapholita molesta (Busck); lesser appleworm, Grapholita prunivora (Walsh); and cherry fruitworm, Grapholita packardi Zeller. An ≈470-bp fragment of mitochondrial cytochrome oxidase subunit I (COI) was sequenced in three to six specimens of each species. Consistent and diagnostic differences were observed among the species in two regions of COI from which forward and reverse primers were designed to amplify a 112-116-bp segment of the gene. The primer sets were used to selectively amplify DNA from specimens of diverse geographic origin for each corresponding target species. Protocols were adapted for conventional and quantitative PCR, the latter being substantially faster. The method was validated as a decision-making tool for quarantine identifications for Mexico by representatives of their phytosanitary agency (Sanidad Vegetal). The method can facilitate identification of intercepted internal feeding Lepidoptera in apple and pear for many other importing nations. [ABSTRACT FROM AUTHOR]

Metabarcoding and high-throughput sequencing methods have greatly improved our understanding of protist diversity. Although the V4 region of small subunit ribosomal DNA (SSU-V4 rDNA) is the most widely used marker in DNA metabarcoding of eukaryotic microorganisms, doubts have recently been raised about its suitability. Here, using the widely distributed ciliate genus Pseudokeronopsis as an example, we assessed the potential of SSU-V4 rDNA and four other nuclear and mitochondrial markers for species delimitation and phylogenetic reconstruction. Our studies revealed that SSU-V4 rDNA is too conservative to distinguish species, and a threshold of 97% and 99% sequence similarity detected only one and three OTUs, respectively, from seven species. On the basis of the comparative analysis of the present and previously published data, we proposed the multilocus marker including the nuclear 5.8S rDNA combining the internal transcribed spacer regions (ITS1-5.8S-ITS2) and the hypervariable D2 region of large subunit rDNA (LSU-D2) as an ideal barcode rather than the mitochondrial cytochrome c oxidase subunit 1 gene, and the ITS1-5.8S-ITS2 as a candidate metabarcoding marker for ciliates. Furthermore, the compensating base change and tree-based criteria of ITS2 and LSU-D2 were useful in complementing the DNA barcoding and metabarcoding methods by giving second structure and phylogenetic evidence. [ABSTRACT FROM AUTHOR]