Phylogeography of Parnassius citrinarius Based on Mitochondrial DNA Reveals Large Differences in Genetic Structure between the Eastern and Western Japan.

The Japanese Archipelago hosts a rich butterfly fauna, and elucidating the genetic structures of multiple species is necessary to clarify their formation processes. This study aimed to reveal the genetic structure and distribution formation process of Parnassius citrinarius, which is widely distributed across the Japanese Archipelago from Hokkaido to Shikoku, through phylogeographic analysis based on the mitochondrial cytochrome c oxidase subunit I (COI) gene sequence. Thirty haplotypes were revealed from 311 individuals from 47 sites, indicating significant differences in the genetic structures between the eastern and western parts of the Japanese Archipelago. In Eastern Japan, multiple genetic clusters were found, with some sites harboring two clusters. The divergence times among populations in Eastern Japan were relatively recent, and no genetic differentiation was observed between regions, including between Hokkaido and Honshu, which are separated by a narrow strait. In contrast, in Western Japan, including Shikoku, unique genetic clusters were observed in each region. The phylogenetic relationships among populations were regionally clustered, and the divergence times were relatively ancient. The distribution and genetic structure of P. citrinarius in the Japanese Archipelago have been significantly influenced by temperature fluctuations and the presence of geographical barriers during the Pleistocene glacial-interglacial cycles, including the potential formation of refugia in Western Japan.

Integrating wing morphometrics and mitochondrial DNA analysis to assess the filaria vector Mansonia uniformis (Diptera: Culicidae) populations in Thailand.

Mansonia uniformis (Diptera: Culicidae) is recognized as a vector of Brugia malayi and has been reported to transmit Wuchereria bancrofti, both causing lymphatic filariasis in humans. This study employed geometric morphometrics (GM) to investigate wing shape variation and analyzed genetic diversity through cytochrome c oxidase subunit 1 (COI) gene analyses in Ma. uniformis populations across Thailand. Wing GM analyses indicated significant differences in wing shape based on Mahalanobis distances among nearly all population pairs (p < 0.05), with no significant correlation between wing shape and geographic distance (r = 0.210, p > 0.05). Genetic analyses identified 63 haplotypes and 49 polymorphic sites, with the overall population exhibiting a nucleotide diversity of 0.006 (± 0.001) and a haplotype diversity of 0.912 (± 0.017). Deviations from neutrality, as indicated by Tajima's D and Fu's FS tests for the overall Ma. uniformis populations in Thailand, were statistically significant and negative, suggesting population expansion (both p < 0.05). Analysis of molecular variance revealed no significant genetic structure when all populations were categorized based on collection sites and geographic regions. However, significant differences in FST values were observed between some populations. These findings enhance our understanding of the geographical and genetic factors influencing Ma. uniformis populations, which are crucial for developing effective control strategies in Thailand.

The origin and insecticide resistance of Aedes albopictus mosquitoes established in southern Mozambique.

BACKGROUND: The Aedes albopictus mosquito is of medical concern due to its ability to transmit viral diseases, such as dengue and chikungunya. Aedes albopictus originated in Asia and is now present on all continents, with the exception of Antarctica. In Mozambique, Ae. albopictus was first reported in 2015 within the capital city of Maputo, and by 2019, it had become established in the surrounding area. It was suspected that the mosquito population originated in Madagascar or islands of the Western Indian Ocean (IWIO). The aim of this study was to determine its origin. Given the risk of spreading insecticide resistance, we also examined relevant mutations in the voltage-sensitive sodium channel (VSSC). METHODS: Eggs of Ae. albopictus were collected in Matola-Rio, a municipality adjacent to Maputo, and reared to adults in the laboratory. Cytochrome c oxidase subunit I (COI) sequences and microsatellite loci were analyzed to estimate origins. The presence of knockdown resistance (kdr) mutations within domain II and III of the VSSC were examined using Sanger sequencing. RESULTS: The COI network analysis denied the hypothesis that the Ae. albopictus population originated in Madagascar or IWIO; rather both the COI network and microsatellites analyses showed that the population was genetically similar to those in continental Southeast Asia and Hangzhou, China. Sanger sequencing determined the presence of the F1534C knockdown mutation, which is widely distributed among Asian populations, with a high allele frequency (46%). CONCLUSIONS: These results do not support the hypothesis that the Mozambique Ae. albopictus population originated in Madagascar or IWIO. Instead, they suggest that the origin is continental Southeast Asia or a coastal town in China.

Integrative taxonomy of the genus Coridius Illiger, 1807 (Hemiptera: Heteroptera: Dinidoridae) reveals hidden diversity and three new species from North-East India.

The genus Coridius Illiger, 1807 (Heteroptera: Dinidoridae) comprises a group of phytophagous terrestrial bugs consisting of 36 species distributed in the Afrotropical and Indo-Malayan regions. In several communities in northeastern India, insects are recognised as a delicacy, medicine, and a nutritional supplement, with Coridius being a popular delicacy. However, Coridius has received little taxonomic attention to date due to large intraspecific variations, inadequate taxonomic treatments, and the rarity of many species. To address this gap, an integrative taxonomy of the genus was performed. Two mitochondrial genes, viz., cytochrome oxidase subunit 1 (COI) and 16S rRNA, were sequenced to reconstruct the phylogenetic relationships within Coridius. We performed both maximum likelihood (ML) and Bayesian inference (BI) to develop a species tree, followed by the Bayesian implementation of the Poisson tree process (bPTP) and Assemble Species by Automatic Partitioning (ASAP) as an additional test to assess species boundaries and delimit operational taxonomic units. A linear discriminant analysis (LDA) of four key morphological characters was then performed to identify species groups. Overall, our analysis supported the establishment of three new species: Coridius adii sp. nov., Coridius esculentus sp. nov., and Coridius insperatus sp. nov., and revealed six distinct lineages within Coridius chinensis (Dallas, 1851). Linear discriminant analysis of morphological characters indicated the clustering of eight species. The species status of Coridius nigriventris (Westwood, 1837) stat. rev, formerly synonymized under Coridius nepalensis (Westwood, 1837), is reinstated in this study. Further, we revised the genus Coridius from India and rediscovered Coridius assamensis (Distant, 1902) and Coridius fuscus (Westwood, 1837) after 100 years.

Population genetic diversity and structure of Tephritis angustipennis and Campiglossa loewiana (Diptera: Tephritidae) based on COI DNA barcodes in the three-river source region, China.

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 (mtCOI) 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.

An analysis of morphological and genetic diversity of mango fruit flies in Pakistan.

Fruit flies of genus Bactrocera are important insect pests of commercially cultivated mangos in Pakistan limiting its successful production in the country. Despite the economic risk, the genetic diversity and population dynamics of this pest have remained unexplored. This study aimed to morphologically identify Bactrocera species infesting Mango in major production areas of the country and to confirm the results with insect DNA barcode techniques. Infested mango fruits from the crop of 2022, were collected from 46 locations of 11major production districts of Punjab and Sindh provinces, and first-generation flies were obtained in the laboratory. All 10,653 first generation flies were morphologically identified as two species of Bactrocera; dorsalis and zonata showing geography-based relative abundance in the two provinces; Punjab and Sindh. Morphological identification was confirmed by mitochondrial cytochrome oxidase gene subunit I (mt-COI) based DNA barcoding. Genetic analysis of mtCOI gene region of 61 selected specimens by the presence of two definite clusters and reliable intraspecific distances validated the results of morphological identification. This study by morphological identification of a large number of fruit fly specimens from the fields across Pakistan validated by insect DNA barcode reports two species of Bactrocera infesting mango in the country.

An integrative approach to the study of Kempnyia Klapálek, 1914 (Plecoptera: Perlidae) from Brazil: Support for the description of four new species and a basis for future studies.

Kempnyia (Plecoptera: Perlidae) is an endemic genus of Brazilian stoneflies that has 36 valid species and is distributed primarily in the Atlantic Forest and the mountainous areas of Central Brazil, particularly in Goiás and Tocantins states. Despite being the Brazilian genus with the most DNA sequences available on GenBank, integrative studies on the genus began only recently, in 2014. In this context, herein we studied the morphology and molecular data of Kempnyia specimens deposited in the Aquatic Biology Laboratory (UNESP, Assis) and the Entomology Museum of the Federal University of Viçosa (UFVB, Viçosa) collections. For the integrative approach adopted, in addition to studying the specimens morphologically, we used sequences of the COI mitochondrial gene combined with the following species delimitation methods: Automatic Barcode Gap Discovery (ABGD), both primary (ABGDp) and recursive (ABGDr) partitions; Assemble Species by Automatic Partitioning (ASAP); Poisson Tree Processes (PTP) and the Bayesian implementation of the Poisson Tree Processes (bPTP). As a result, we provided 28 new COI sequences of 21 species and support the description of four new species, namely, K. guarani sp. nov., K. tupiniquim sp. nov., K. una sp. nov., and K. zwickii sp. nov., consequently increasing the known diversity of the genus to 40 species. We also discuss the morphological variations observed in other species of the genus and provide several new geographic records. Therefore, our study brings new insights into the values of intra- and interspecific molecular divergence within Kempnyia, serving as a basis for new studies.

DNA barcodes from over-a-century-old type specimens shed light on the taxonomy of a group of rare butterflies (Lepidoptera: Nymphalidae: Calinaginae).

We analyzed COI barcode sequences from 138 over-a-century old specimens of Calinaga including 36 name-bearing type specimens stored at the Natural History Museum London. These new data, combined with previously available RPS5 sequences, divide the Calinaga samples into four well-supported mitochondrial lineages that together with a novel wing-pattern analysis, support the recognition of six species (lhatso, buddha, brahma, aborica, formosana and davidis), with all other names subsumed either as subspecies or synonyms. One new taxon is described, Calinaga aborica naima Vane-Wright, ssp. n.

Phylogenetic and population genetic analyses of Thrips tabaci Lindeman (Thysanoptera: Thripidae) on Allium host in India.

Background: Onion thrips (Thrips tabaci) is a complex of cryptic species with subtle morphological differences and distinct genetic backgrounds; thus, species identification using traditional methods remains challenging. The existence of different haplotypes and genotypes within a species can significantly influence various aspects of its biology, including host preference, reproductive capacity, resistance to pesticides, and vector competence for plant viruses. Understanding the genetic diversity and population structure of cryptic species within T. tabaci will not only aid in the development of more effective control strategies tailored to specific genetic variants but also in monitoring population dynamics, tracking invasive species, and implementing quarantine measures to prevent the spread of economically damaging thrips biotypes. Methods: This study aims to explore intraspecies genetic diversity and molecular evolutionary relationships of the mitochondrial cytochrome oxidase gene subunit I (mtCOI) in T. tabaci populations from India. To capture diversity within the Indian T. tabaci populations, amplicon sequencing was performed for the thrips mtCOI gene from eight diverse localities in India. A total of 48 sequences retrieved for the mtCOI gene from the NCBI Nucleotide database were analysed. Results: Multiple insertions and deletions were detected at various genomic positions across the populations from different localities, with the highest variation observed in the 300-400 genome position range. Molecular diversity analyses identified 30 haplotypes within the population, with certain subpopulations exhibiting higher gene flow. Analysis of single nucleotide polymorphism patterns within the mtCOI gene across diverse Indian locales revealed significant intrapopulation genetic heterogeneity and its potential repercussions on gene functionality. Elevated F statistics (Fst) values in the northern-western subpopulations suggested high genetic variability, particularly evident in haplotype networks originating mainly from the northern region, notably Delhi. While most populations displayed stable and ancient evolutionary histories, thrips populations from northern, western, and north-eastern regions indicated rapid growth.

Cytochrome oxidase requirements in Bordetella reveal insights into evolution towards life in the mammalian respiratory tract.

Little is known about oxygen utilization during infection by bacterial respiratory pathogens. The classical Bordetella species, including B. pertussis, the causal agent of human whooping cough, and B. bronchiseptica, which infects nearly all mammals, are obligate aerobes that use only oxygen as the terminal electron acceptor for electron transport-coupled oxidative phosphorylation. B. bronchiseptica, which occupies many niches, has eight distinct cytochrome oxidase-encoding loci, while B. pertussis, which evolved from a B. bronchiseptica-like ancestor but now survives exclusively in and between human respiratory tracts, has only three functional cytochrome oxidase-encoding loci: cydAB1, ctaCDFGE1, and cyoABCD1. To test the hypothesis that the three cytochrome oxidases encoded within the B. pertussis genome represent the minimum number and class of cytochrome oxidase required for respiratory infection, we compared B. bronchiseptica strains lacking one or more of the eight possible cytochrome oxidases in vitro and in vivo. No individual cytochrome oxidase was required for growth in ambient air, and all three of the cytochrome oxidases conserved in B. pertussis were sufficient for growth in ambient air and low oxygen. Using a high-dose, large-volume persistence model and a low-dose, small-volume establishment of infection model, we found that B. bronchiseptica producing only the three B. pertussis-conserved cytochrome oxidases was indistinguishable from the wild-type strain for infection. We also determined that CyoABCD1 is sufficient to cause the same level of bacterial burden in mice as the wild-type strain and is thus the primary cytochrome oxidase required for murine infection, and that CydAB1 and CtaCDFGE1 fulfill auxiliary roles or are important for aspects of infection we have not assessed, such as transmission. Our results shed light on the environment at the surface of the ciliated epithelium, respiration requirements for bacteria that colonize the respiratory tract, and the evolution of virulence in bacterial pathogens.

Mitochondrial genome of the fluke pond snail, Austropeplea cf. brazieri (Gastropoda: Lymnaeidae).

BACKGROUND: Lymnaeid snails of the genus Austropeplea are an important vector of the liver fluke (Fasciola hepatica), contributing to livestock production losses in Australia and New Zealand. However, the species status within Austropeplea is ambiguous due to heavy reliance on morphological analysis and a relative lack of genetic data. This study aimed to characterise the mitochondrial genome of A. cf. brazieri, an intermediate host of liver fluke in eastern Victoria. METHODS: The mitochondrial genome was assembled and annotated from a combination of second- and third-generation sequencing data. For comparative purposes, we performed phylogenetic analyses of the concatenated nucleotide sequences of the mitochondrial protein-coding genes, cytochrome c oxidase subunit 1 and 16S genes. RESULTS: The assembled mt genome was 13,757 base pairs and comprised 37 genes, including 13 protein-coding genes, 22 transfer RNA genes and 2 ribosomal RNA genes. The mt genome length, gene order and nucleotide compositions were similar to related species of lymnaeids. Phylogenetic analyses of the mt nucleotide sequences placed A. cf. brazieri within the same clade as Orientogalba ollula with strong statistical supports. Phylogenies of the cox1 and 16S mt sequences were constructed due to the wide availability of these sequences representing the lymnaeid taxa. As expected in both these phylogenies, A. cf. brazieri clustered with other Austropeplea sequences, but the nodal supports were low. CONCLUSIONS: The representative mt genome of A. cf. brazieri should provide a useful resource for future molecular, epidemiology and parasitological studies of this socio-economically important lymnaeid species.

[Molecular tracing of Biomphalaria straminea in China].

OBJECTIVE: To investigate the origin of Biomphalaria straminea in China, so as to provide insights into assessment of schistosomiasis mansoni transmission risk and B. straminea control. METHODS: Guanlan River, Dasha River, Shenzhen Reservoir, upper and lower reaches of Kuiyong River, and Xinzhen River in Shenzhen, China, were selected as sampling sites. Ten Biomphalaria samples were collected from each site, and genomic DNA was extracted from Biomphalaria samples. DNA samples were obtained from 15 B. straminea sampled from 5 sampling sites in Minas Gerais State, Pará State, Federal District, Pernambuco State, and Sao Paulo State in Brazil, South America. Cytochrome c oxidase I (COI) and mitochondrial 16S ribosomal RNA (16S rRNA) genes were sampled using the above DNA templates, and the amplified products were sequenced. The COI and 16S rRNA gene sequences were downloaded from GenBank, and the sampling sites were acquired. All COI and 16S rRNA gene sequences were aligned and evolutionary trees of B. straminea were created based on COI and 16S rRNA gene sequences to identify the genetic similarity and evolutionary relationship between B. straminea samples from China and South America. RESULTS: A total of 60 COI gene sequences with a length of 529 bp and 3 haplotypes were obtained from B. straminea sampled from China. There were 165 COI gene sequences of B. straminea retrieved from GenBank, and following alignment with the above 60 gene sequences, a total of 33 haplotypes were obtained. Phylogenetic analysis showed that the three haplotypes of B. straminea from China were clustered into one clade, among which the haplotype China11 and three B. straminea samples from Brazil retrieved from GenBank belonged to the same haplotype. Geographical evolution analysis showed that the B. straminea samples from three sampling sites along eastern coasts of Brazil had the same haplotype with China11, and B. straminea samples from other two sampling sites were closely, genetically related to China11. A total of 60 16S rDNA gene sequences with approximately 322 bp in length were amplified from B. straminea in China, with 2 haplotypes identified. A total of 70 16S rDNA gene sequences of B. straminea were captured from GenBank. Phylogenetic analysis showed that Biomphalaria snails collected from China were clustered into a clade, and the haplotype China64 and the haplotype 229BS from Brazil shared the same haplotype. The 49 16S rDNA gene sequences of B. straminea from 25 sampling sites in southern Brazil, which were captured from GenBank, were included in the present analysis, and the B. straminea from 3 sampling sites shared the same haplotype with China64 in China. Geographical evolution analysis based on COI and 16S rRNA gene sequences showed that B. straminea sampled from eastern coastal areas of Brazil shared the same haplotypes in two gene fragment sequences with Biomphalaria snails collected from China. CONCLUSIONS: The Biomphalaria snails in China are characterized as B. straminea, which have a low genetic diversity. The Biomphalaria snails in China have a high genetic similarity with B. straminea sampled from eastern coastal areas of Brazil, which may have originated from the eastern coastal areas of Brazil.

Population genetic structure of Culex tritaeniorhynchus in different types of climatic zones in China.

BACKGROUND: Culex tritaeniorhynchus is widely distributed in China, from Hainan Island in the south to Heilongjiang in the north, covering tropical, subtropical, and temperate climate zones. Culex tritaeniorhynchus carries 19 types of arboviruses. It is the main vector of the Japanese encephalitis virus (JEV), posing a serious threat to human health. Understanding the effects of environmental factors on Culex tritaeniorhynchus can provide important insights into its population structure or isolation patterns, which is currently unclear. RESULTS: In total, 138 COI haplotypes were detected in the 552 amplified sequences, and the haplotype diversity (Hd) value increased from temperate (0.534) to tropical (0.979) regions. The haplotype phylogeny analysis revealed that the haplotypes were divided into two high-support evolutionary branches. Temperate populations were predominantly distributed in evolutionary branch II, showing some genetic isolation from tropical/subtropical populations and less gene flow between groups. The neutral test results of HNQH (Qionghai) and HNHK(Haikou) populations were negative (P < 0.05), indicating many low-frequency mutations in the populations and that the populations might be in the process of expansion. Moreover, Wolbachia infection was detected only in SDJN (Jining) (2.24%), and all Wolbachia genotypes belonged to supergroup B. To understand the influence of environmental factors on mosquito-borne viruses, we examined the prevalence of Culex tritaeniorhynchus infection in three ecological environments in Shandong Province. We discovered that the incidence of JEV infection was notably greater in Culex tritaeniorhynchus from lotus ponds compared to those from irrigation canal regions. In this study, the overall JEV infection rate was 15.27 per 1000, suggesting the current risk of Japanese encephalitis outbreaks in Shandong Province. CONCLUSIONS: Tropical and subtropical populations of Culex tritaeniorhynchus showed higher genetic diversity and those climatic conditions provide great advantages for the establishment and expansion of Culex tritaeniorhynchus. There are differences in JEV infection rates in wild populations of Culex tritaeniorhynchus under different ecological conditions. Our results suggest a complex interplay of genetic differentiation, population structure, and environmental factors in shaping the dynamics of Culex tritaeniorhynchus. The low prevalence of Wolbachia in wild populations may reflect the recent presence of Wolbachia invasion in Culex tritaeniorhynchus.

Taxonomic revision of two species in the genus Ptychorhynchus Simpson, 1900 (Bivalvia: Unionidae: Gonideinae), with description of a new species.

Accurate identification and precise classification of freshwater mussel species that are among the most threatened freshwater taxa in the world, play a crucial role in informing conservation and management efforts for these organisms. However, due to the variability in shell morphology, relying solely on shell characteristics for species taxonomy poses significant challenges, thereby impeding effective conservation planning and management. The freshwater mussel genus Ptychorhynchus Simpson, 1900 is one such group in need of study. We integrate molecular phylogeny, shell morphology and soft-body anatomy to examine the classification of Ptychorhynchus denserugata (Haas, 1910) and Ptychorhynchus resupinatus (von Martens, 1902). The COI barcoding data support the clustering of P. denserugata and Nodularia douglasiae within a single clade, and P. denserugata shares the diagnostic feature of the genus Nodularia , i.e. knobs or bumps on the inner mantle surface in the excurrent aperture. Therefore, by integrating molecular data and anatomical characteristics, we confirm that the nominal species P. denserugata syn. nov. is a new synonym for N. douglasiae . The multi-locus (COI + ND1 + 16S rRNA + 18S rRNA + 28S rRNA ) phylogeny and mitochondrial phylogenomics support the transfer of P. resupinatus from Ptychorhynchus to the newly elevated genus Cosmopseudodon stat. rev., as Cosmopseudodon resupinatus stat. rev. that is still considered the designated type species. We also describe a new species based on integrative taxonomy, i.e. Cosmopseudodon wenshanensis sp. nov. The comprehensive understanding of the taxonomy and diversity of the revised Cosmopseudodon species, and shell heteromorphism of N. douglasiae (=P. denserugata syn. nov.), will serve as a crucial foundation for further scientific assessment and conservation strategies pertaining to these taxa. ZooBank: urn:lsid:zoobank.org:pub:E48968B1-DF0F-42AD-8F31-B8C95F23CE57.

A morpho-phylogenetic update on ixodid ticks infesting cattle and buffalos in Vietnam, with three new species to the fauna and a checklist of all species indigenous to the country.

BACKGROUND: Southeast Asia is regarded as a hotspot for the diversity of ixodid ticks. In this geographical region, Vietnam extends through both temperate and tropical climate zones and therefore has a broad range of tick habitats. However, molecular-phylogenetic studies on ixodid tick species have not been reported from this country. METHODS: In this study, 1788 ixodid ticks were collected from cattle, buffalos and a dog at 10 locations in three provinces of northern Vietnam. Tick species were identified morphologically, and representative specimens were molecularly analyzed based on the cytochrome c oxidase subunit I (cox1) and 16S rRNA genes. Fifty-nine tick species that are indigenous in Vietnam were also reviewed in the context of their typical hosts in the region. RESULTS: Most ticks removed from cattle and buffalos were identified as Rhipicephalus microplus, including all developmental stages. Larvae and nymphs were found between January and July but adults until December. Further species identified from cattle were Rhipicephalus linnaei, Rhipicephalus haemaphysaloides, Amblyomma integrum and Haemaphysalis cornigera. Interestingly, the latter three species were represented only by adults, collected in one province: Son La. The dog was infested with nymphs and adults of R. linnaei in July. Phylogenetically, R. microplus from Vietnam belonged to clade A of this species, and R. haemaphysaloides clustered separately from ticks identified under this name in China, Taiwan and Pakistan. Amblyomma integrum from Vietnam belonged to the phylogenetic group of haplotypes of an Amblyomma sp. reported from Myanmar. The separate clustering of H. cornigera from Haemaphysalis shimoga received moderate support. CONCLUSIONS: Three tick species (R. linnaei, A. integrum and H. cornigera) are reported here for the first time in Vietnam, thus increasing the number of indigenous tick species to 62. Clade A of R. microplus and at least R. linnaei from the group of Rhipicephalus sanguineus sensu lato occur in the country. There is multiple phylogenetic evidence that different species might exist among the ticks that are reported under the name R. haemaphysaloides in South and East Asia. This is the first report of A. integrum in Southeastern Asia.

Morphological and phylogenetic analyses reveal four species of myxomycetes new to China.

Four species of myxomycetes (Arcyria pseudodenudata, Diderma europaeum, Lycogala irregulare, and Trichia armillata) new to China were observed via light microscope and scanning electron microscope, and detailed descriptions and illustrations are provided, along with comparisons with related species. Among them, A. pseudodenudata was discovered for the first time outside of the type locality, D. europaeum was discovered for the first time outside of Europe, and L. irregulare and T. armillata were reported again after being named. Phylogenetic analyses based on nuclear 18S rDNA and elongation factor-1 alpha sequences or nuclear 18S rDNA and cytochrome oxidase subunit I sequences was performed to provide a molecular basis for morphological identification. These specimens were deposited in the Herbarium of Fungi of Nanjing Normal University.

Molecular identification and phylogenetic relationship of fishes belonging to the Family Danionidae from Brahmaputra Basin, Assam, Northeast India.

BACKGROUD: The Northeast India, being part of two global biodiversity hotspot namely the Indo-Burma and Eastern Himalayan Hotspots supports a wide variety of rich aquatic biodiversity including fishes. The family Danionidae is a widely diverse group inhabiting the upper colder stretches of river although few are abundant in the lower stretches. The persisting similarity in the morphological appearance and body colouration within the members of this family seeks an integrated method to identify the species correctly. METHODS AND RESULTS: In the present study, the mt-DNA barcode was generated for correct identification and confirmation of the species. A total of nine mitochondrial cytochrome c oxidase subunit I gene sequences were generated for each species under the study. The pairwise distance values ranged from 0.09 to 9.11% within species and 9.06-32.71% between species. A neighbour-joining tree was constructed based on the Kimura 2 parameter model. Two major groups were observed where Danioninae formed a sister group to the Chedrinae and Rasborinae. CONCLUSION: The present study is a preliminary work to document and identify the species under the family Danionidae from Brahmaputra basin, Assam, using molecular tools and establish the phylogenetic relationship.

Identification of pre-flexion fish larvae from the western South Atlantic using DNA barcoding and morphological characters.

Knowledge on species composition is the first step necessary for the proper conservation and management of biological resources and ecologically relevant species. High species diversity and a lack of diagnostic characters for some groups can impose difficulties for taxonomic identification through traditional methodologies, and ichthyoplankton (fish larvae and eggs) are a good example of such a scenario. With more than 35.000 valid species of fishes worldwide and overall similar anatomies in early developmental stages in closely related groups, fish larvae are often hard to be identified at the species or even more encompassing taxonomic levels. To overcome this situation, molecular techniques have been applied, with different markers tested over the years. Cytochrome c oxidase I (COI) is the most commonly used marker and now has the broadest public reference libraries, providing consistent results for species identification in different metazoan studies. Here we sequenced the mitochondrial COI-5P fragment of 89 fish larvae collected in the Campos Basin, coastal southeastern Brazil, and compared these sequences with references deposited in public databases to obtain taxonomic identifications. Most specimens identified are species of the Blenniiformes, with Parablennius and Labrisomus the most frequently identified genera. Parablennius included two species (P. marmoreus and P. pilicornis), while Labrisomus included three species (L. cricota, L. conditus and L. nuchipinnis). Anatomy of these molecularly identified specimens were then analyzed with the intention of finding anatomical characters that might be diagnostically informative amongst the early development stage (pre-flexion) larvae. Ventral pigmentation patterns are proposed as useful markers to identify Labrisomus species. However, additional specimens are needed to confirm if the character holds stability through the geographic distribution of the species.

Population genetic structure and phylogenetic analysis of Anopheles hyrcanus (Diptera: Culicidae) inferred from DNA sequences of nuclear ITS2 and the mitochondrial COI gene in the northern part of Iran.

BACKGROUND: The Anopheles hyrcanus group is distributed throughout the Oriental and Palaearctic regions and can transmit diseases such as malaria, Japanese encephalitis virus, and filariasis. This investigation marks the inaugural comprehensive study to undertake a phylogenetic analysis of the constituents of this malaria vector group in the northeastern region of Iran, juxtaposed with documented occurrences from different areas within Iran and worldwide. METHODS: Mosquitoes were collected using various methods from nine different locations in Golestan province from April to December 2023. The collected mosquitoes were identified morphologically using valid taxonomic keys. DNA was isolated using the Sambio™ Kit. COI and ITS2 primers were designed using Oligo7 and GeneRunner. PCR and purification were performed with the Qiagen kit. Subsequently, sequencing was carried out at the Mehr Mam GENE Center using an Applied Biosystems 3730XL sequencer. The nucleotide sequences were then analyzed and aligned with GenBank data using BioEdit. Kimura 2-parameter was Utilized for base substitutions. DNA models were selected based on AIC and BIC criteria. Bayesian and Maximum Likelihood trees were constructed, along with a haplotype network. Molecular diversity statistics computed using DnaSP software. RESULTS: In this study, a total of 819 adult mosquitoes were collected. An. hyrcanus was the second most abundant species, predominantly found in Kalaleh and Turkman counties. The sequenced and edited COI and ITS2 sequences were deposited in GenBank under specific accession numbers. Phylogenetic analyses using ML, BI, and NJ methods confirmed a monophyletic lineage for An. hyrcanus with strong support. Molecular analysis of Iranian An. hyrcanus found 11 diverse haplotypes, with the COI gene displaying low diversity. The ITS2 gene revealed two clades - one associating with Iran, Europe, and Asia; the other originating from southwestern Iran. The haplotype network showed two main groups - one from southwest Iran and the other from north Iran. Iran exhibited six distinct haplotypes, while Turkey showcased the highest diversity. CONCLUSIONS: An. hyrcanus in southwestern Iran exhibits a distinct haplogroup, suggesting possible subspecies differentiation. Additional studies are required to validate this phenomenon.

Molecular based identification and phylogenetic relationship of the leech Hirudinaria manillensis from India by using mitochondrial cytochrome c oxidase subunit I gene.

BACKGROUND: A molecular approach for the identification of unknown species by the using mitochondrial cox1 gene is an effective and reliable as compared with morphological-based identification. Hirudinaria manillensis referred to as Asian Buffalo Leech, is found in South Asia and traditionally used as medicine owing to its medicinal properties. METHODS AND RESULTS: The study aimed to isolate and identify the leech species using cox1 gene sequencing and their phylogenetic relationships. The nucleotide sequences of cytochrome c oxidase subunit I (cox1) mitochondrial genes were analyzed for species identification and the phylogenetic relationship of crucial therapeutic leech Hirudinaria manillensis. The isolated DNA from the leech sample was amplified with cox1 gene-specific primers. BLAST results with the H. manillensis sequence showed 89.24% homology with H. manillensis and phylogenetic tree analysis revealed the genetic relationship with other GenBank submitted sequences. CONCLUSION: The present study concluded that the cox1 gene could be an effective way to identify the leech H. manillensis and provided sufficient phylogenetic information to distinguish H. manillensis indicating a significant mtDNA-based approach to species identification.