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ABSTRACT 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 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.

Propylea japonica (Coleoptera: Coccinellidae) is a natural enemy insect with a wide range of predation in Chinese mainland and is commonly used in pest management. However, its genetic pattern (i.e., genetic variation, genetic structure, and historical population dynamics) is still unclear, impeding the development of biological control of insect pests. Population genetic research has the potential to optimize strategies at different stages of the biological control processes. This study used 23 nuclear microsatellite sites and mitochondrial COI genes to investigate the population genetics of Propylea japonica based on 462 specimens collected from 30 sampling sites in China. The microsatellite dataset showed a moderate level of genetic diversity, but the mitochondrial genes showed a high level of genetic diversity. Populations from the Yellow River basin were more genetically diverse than those in the Yangtze River basin. Propylea japonica has not yet formed a significant genealogical structure in China, but there was a population structure signal to some extent, which may be caused by frequent gene flow between populations. The species has experienced population expansion after a bottleneck, potentially thanks to the tri-trophic plant–insect–natural enemy relationship. Knowledge of population genetics is of importance in using predators to control pests. Our study complements existing knowledge of an important natural predator in agroecosystems through estimating its genetic diversity and population differentiation and speculating about historical dynamics.

Abstract The cryptic invasion of golden apple snails (Pomacea canaliculata and P. maculata) in Taiwan has caused significant ecological and economical damage over the last few decades, however, their management remains difficult due to inadequate taxonomic identification, complex phylogeny, and limited population genetic information. We aim to understand the current distribution, putative population of origin, genetic diversity, and potential path of cryptic invasion of Pomacea canaliculata and P. maculata across Taiwan to aid in improved mitigation approaches. The present investigation conducted a nationwide survey with 254 samples collected from 41 locations in 14 counties or cities across Taiwan. We identified P. canaliculata and P. maculata based on mitochondrial COI and compared their genetic diversity across Taiwan, as well as other introduced and native countries (based on publicly available COI data) to understand the possible paths of invasion to Taiwan. Based on mitochondrial COI barcoding, sympatric and heterogeneous distributions of invasive P. canaliculata and P. maculata were noted. Our haplotype analysis and mismatch distribution results suggested multiple introductions of P. canaliculata in Taiwan was likely originated directly from Argentina, whereas P. maculata was probably introduced from a single, or a few, introduction event(s) from Argentina and Brazil. Our population genetic data further demonstrated a higher haplotype and genetic diversity for P. canaliculata and P. maculata in Taiwan compared to other introduced regions. Based on our current understanding, the establishment of P. canaliculata and P. maculata is alarming and widespread beyond geopolitical borders, requiring a concerted and expedited national and international invasive species mitigation program.

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.

Nerita yoldii is a euryhaline species commonly found in the intertidal zone. To investigate the genetic diversity of 233 N. yoldii individuals from eight locations along the coast of China, we utilized the mitochondrial COI gene as a molecular marker. A total of 34 haplotypes were detected, exhibiting a mean haplotype diversity (Hd) of 0.5915 and a mean nucleotide diversity (Pi) of 0.0025, indicating high levels of genetic diversity among all populations. An analysis of molecular variance (AMOVA) indicated that the primary source of genetic variation occurs within populations. In addition, neutral tests and mismatch analyses suggested that N. yoldii populations may have experienced bottleneck events. Moderate genetic differentiation was observed between Xiapu and other populations, excluding the Taizhou population, and may be attributed to the ocean currents. Intensively studying the genetic variation and population structure of N. yoldii populations contributes to understanding the current population genetics of N. yoldii in the coastal regions of China. This not only provides a reference for the study of other organisms in the same region but also lays the foundation for the systematic evolution of the Neritidae family.

Background Trichoptera are one of the most diverse groups of freshwater insects worldwide and one of the main bioindicators for freshwater quality. However, in many areas, caddisflies remain understudied due to lack of taxonomic expertise. Meanwhile, globally increasing anthropogenic stress on freshwater streams also threatens Trichoptera diversity. Methods To assess the Trichoptera diversity of the area within and around the Mount Halimun Salak National Park (MHSNP or Taman Nasional Gunung Halimun Salak) in West Java (Indonesia), we conducted a molecular-morphological study on Trichoptera diversity using larvae from a benthic survey and adults from hand-netting. In addition to morphological identification, we applied four different molecular taxon delimitation approaches (Generalized Mixed Yule Coalescent, Bayesian Poisson Tree Processes, Automatic Barcode Gap Discovery and Assemble Species by Automatic Partitioning) based on DNA barcoding of Cytochrome-C-Oxidase I (COI). Results The molecular delimitation detected 72 to 81 Operational Taxonomic Units (OTU). Only five OTUs could be identified to species level by comparing sequences against the BOLD database using BLAST, and four more to the genus level. Adults and larvae could be successfully associated in 18 cases across six families. The high diversity of Trichoptera in this area highlights their potential as bioindicators for water quality assessment. Conclusions This study provides an example of how molecular approaches can benefit the exploration of hidden diversity in unexplored areas and can be a valuable tool to link life stages. However, our study also highlights the need to improve DNA barcode reference libraries of Trichoptera for the Oriental region.

Abstract Background The white-backed planthopper (WBPH), Sogatella furcifera (Horváth) (Hemiptera, Delphacidae), is a migratory pest of rice in Asia. Shandong Province, in northern China, is located on the migration pathway of WBPH between southern and northeast China. The potential sources of WBPH in northern China are poorly understood. We studied the sources of WBPH in Shandong Province by determining the population genetic structure of WBPH in 18 sites distributed in Shandong and in six regions of the Greater Mekong Subregion (GMS). We used mitochondrial gene and single-nucleotide polymorphism (SNP) markers for analysis. Results All of the WBPH populations studied in the seven regions had low genetic diversity. Pairwise FST values based on mtDNA ranged from − 0.061 to 0.285, while FST based on SNP data ranged from − 0.007 to 0.009. These two molecular markers revealed that 4.40% (mtDNA) and 0.19% (SNP) genetic variation could be explained by the interpopulation variation, while the rest came from intrapopulation variation. The populations in the seven geographic regions comprised four hypothetical genetic clusters (K = 4) not associated with geographic location. Eighty-four of 129 individuals distributed across the given area were designated as recent migrants or of admixed ancestry. Although the substantial migration presented, a weak but significant correlation between genetic and geographic distances was found (r = 0.083, P = 0.004). Conclusion The Greater Mekong Subregion was the main genetic source of WBPH in Shandong, while other source populations may also exist. The genetic structure of WBPH is shaped by both migration and geographic barriers. These results help clarify the migration route and the source of WBPH in northern China.

Based on research carried out from 2016 to 2018 there are twenty-six species of aphids of the tribe Eulachnini in Lithuania. Seventeen are members of the subgenus Cinara of the genus Cinara, three of the subgenus Cupressobium and two of the subgenus Schizolachnus. In addition, there are four species of the genus Eulachnus. Summarized information on the distribution and host specificity of Eulachnini in Lithuania is presented. Nine species were in all climatic regions of Lithuania: C. (Cinara) brauni, C. (Cinara) hyperophila, C. (Cinara) neubergi, C. (Cinara) piceicola, C. (Cinara) pinea, C. (Cinara) pini, C. (Cinara) pruinosa, C. (Cupressobium) cupressi and C. (Cupressobium) juniperi. Five species of Lithuanian Eulachnini inhabit host plants of the genus Picea, three live on Larix, thirteen on Pinus, one on Abies, one on Thuja and three on Juniperus. Cinara (Cinara) piniphila was recorded on Pinus mugo and C. (Cinara) nuda on Pinus heldreichii for the first time. Partial COI and EF-1α sequences of C. (Cinara) hyperophila, C. (Cinara) pilosa and C. (Cinara) piceicola were obtained for the first time. Partial sequences of mitochondrial (COI) and nuclear (EF-1α) DNA of Lithuanian samples were used to explore molecular diversity using NJ trees, Automatic Barcode Gap Discovery (ABGD), General Mixed Yule Coalescent (GMYC) and Poisson Tree Processes (PTP). Species delimitation using GMYC (both on COI and EF-1α sequences), PTP (BI on COI) and ABGD (EF-1α) were the most consistent with traditional classifications. Pairwise between-species sample divergences (COI and EF-1α fragments) of the samples of the species complex C. (Cinara) pinea - C. (Cinara) piniphila indicate it is composed of a single species. Of the species of aphids that live on conifers, there are seven species of the tribe Eulachnini that are likely to shortly colonize Lithuania.

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.

Neotropical species of the Elachista praelineata species group are reviewed. Five species are recognized in the Neotropics. A new species, Elachista stonisi sp. nov., and female of E. albisquamella Zeller, 1877 are described and illustrated with photographs of the adults, some of the immature stages, female genitalia, and leaf mines. The female of E. lata Sruoga, 2010 is associated with the male based on morphology and the comparison of partial mitochondrial COI sequences. Identification keys to the Neotropical species of E. praelineata species group, based on male and female genitalia, are provided.

Several European freshwater crayfish species are currently included in one of the IUCN Red list categories. In the Iberian Peninsula, the native Austropotamobius pallipes species complex (the white clawed crayfish, WCC) has experienced a drastic decline since 1973. Implementing conservation management strategies for this species requires a better understanding of the patterns and structure of its genetic diversity. In this study, we assessed the levels and patterns of genetic variation in 71 populations along the whole distributional range of the WCC in the Iberian Peninsula. The two mitochondrial markers analyzed (Cytochrome Oxidase subunit I and 16S rRNA genes) indicated high levels of genetic diversity, which are significantly geographically structured in three main genetic groups, two corresponding to Northern and one to Central-Eastern and the westernmost Iberian Peninsula. The diversity found included new private haplotypes, and revealed the potential effect of paleogeographic barriers and last glaciations in the population structure observed. Current conservation and management programs for the WCC in the Iberian Peninsula should consider these three phylogeographic groups as essential management units in order to preserve the remaining genetic diversity in the species.

Aphid species Aphis pomi (de Geer, 1773) is oligophagous on pomoideous host plants, whilst Aphis spiraecola Patch, 1914 is a polyphagous species alternating between Spiraea spp., its primary host, and a wide variety of secondary hosts, also including pomoideous species. Despite the biological distinction, these species are difficult to separate using their morphological characters. Partial sequences of mitochondrial COI and nuclear EF-1α genes were analyzed for samples from Central and Eastern Europe, Germany, Bulgaria, Italy, Turkey, China together with available data from GenBank. Interspecific pairwise sample divergences of the COI fragment ranged from 3.1 to 4.3%. One COI haplotype of A. pomi was predominant (n = 24), with a pan European distribution. The most abundant COI haplotype of A. spiraecola (n = 16) occurred in Lithuania, Latvia, Poland, Italy, Turkey and China. Interspecific pairwise sample divergences of the EF-1α fragment ranged from 0.6 to 1.2%. Analyzed partial sequences of EF-1α were identical in A. pomi. The most abundant EF-1α haplotype of A. spiraecola (n = 14) occurred in Lithuania, Poland, Italy, Turkey and China. The length of ultimate rostral segment appeared to be the most reliable morphological character for discrimination between apple and spirea aphid species. It allowed a 100% correct identification of A. pomi (n = 143) and 91.5% of A. spiraecola (n = 94) specimens in the European samples used for the molecular analysis. The existence of A. spiraecola in the Eastern Baltic region of Europe is documented for the first time.

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.

Ancient Egyptians used pigeons not only as food in the form of squab but also as a messenger by virtue of their strong homing ability. Pigeons are bred for many purposes like meat in the form of squabs, exhibition as fancy and ornamental, flying and sports like racing competition, and finally for laboratory experiments of cognitive sciences. In this study, a total of 133 pigeon samples of six Egyptian breeds (n=110) and Japanese racing pigeons (n=23) were surveyed. One sample from each breed was sequenced for mitochondrial COI gene and all samples were genotyped across 11 microsatellites loci. From COI sequence, all the seven studied populations were found to belong to same the species (Columba livia). By the analysis of 11 microsatellite loci a total of 89 alleles were observed with an average of 8.1 alleles per locus. The expected heterozygosities of the six Egyptian breeds and Japanese racing pigeons were 0.580 and 0.630, respectively. FST showed a relatively high mean of 0.203 which indicated that there is a great differentiation among the seven pigeon populations. Zagel breed and Japanese racing pigeons showed the lowest values for both pairwise FST (0.108) and Nei's genetic distance (0.154). The information from this study would be useful for genetic characterization and provide a foundation for developing sustainable genetic improvement and conservation programs of this agriculturally and commercially important species.

The Entiminae are broad-nosed weevils constituting the most diverse subfamily of Curculionidae, with over 50 tribes. We performed Bayesian and Maximum Parsimony combined phylogenetic analyses with the main objective of testing higher-level relationships and the naturalness of the major Neotropical and Southern South American (Patagonia and Andes) tribes, including some members from other regions. We compiled a data matrix of 67 terminal units with 63 Entiminae species, as well as four outgroup taxa from Cyclominae, by 3522 molecular (from nuclear 18S rDNA and 28S rDNA, and mitochondrial 16S rDNA and COI gene sequences) and 70 morphological characters. The resulting trees recover a clade Entiminae with a monophyletic Cylydrorhinini and Premnotrypes branching off early. The tree resulting from parsimony analysis shows a clade of Leptopiini from the Australian region and another clade including taxa mainly distributed in the Palaearctic and Neotropical regions, but in the Bayesian tree the South American and Australian Leptopiini are grouped together. The mainly Palaearctic Entiminae (e.g., Brachyderini, Laparocerini, Otiorhynchini, Peritelini, Polydrusini, Phyllobiini and Sciaphylini) form a subclade separated from Southern Hemisphere taxa. Among the latter, the well-supported Naupactini are the sister group of the South American Tanymecini, excluding Platyaspistes, herein transferred to Leptopiini (new placement). Another well-justified clade is Eustylini–Geonemini, which also includes the enigmatic Galapagonotus, and the genus Artipus, thus corroborating its recent exclusion from Naupactini.

The phylogenetic relationships of the subgenera Polyommatus and Plebicula, within the Palaearctic butterfly genus Polyommatus, were inferred from a combined analysis of the nuclear marker ITS2 and the barcoding section of the mitochondrial gene COI. Eight major clades were recovered within Polyommatus s. l., which correspond closely to subgenera based on traditional systematics and are of late Pliocene to early Pleistocene origin. Extraordinary chromosomal evolution occurred independently in three of these clades. The disputed position of several species formerly placed in the subgenus Plebicula is clarified. A group of Central Asian species (Bryna) was recovered as a monophyletic clade within Polyommatus s. str. The Kurdistanian endemic P. buzulmavi appears as a sister species to P. icarus. P. celina replaces P. icarus in NW Africa and the Canary Islands, and split from the last common ancestor with P. icarus back in the early Pleistocene.