Cytochrome C Oxidase I Research Articles

Digging in the sand: new species and records of mesopsammic sea slugs in the Western Mediterranean (Mollusca: Gastropoda)

ABSTRACT Marine interstitial habitats (mesopsammon) have been understudied due to complications of exploring its millimetric and highly adapted organisms. The restrictive ecological conditions of this neglected habitat include faint light and a limited amount of space. These have forced its fauna, with gastropod molluscs as one of the most common groups, to adapt by reducing body size to minute (often shell-less) vermiform organisms. These usually simplified heterobranch species are hard to collect and problematic to determine, often including cases of cryptic and pseudo-cryptic speciation. Here, we explore the interstitial heterobranch diversity alongside the Spanish and Catalan coast (Western Mediterranean) to shed light on the taxonomy of this group. Among the 15 localities, we have collected 315 specimens directly from coarse samples and associated habitats, belonging to ca. 39 different species, providing live photographs, species identification based on the current taxonomic knowledge, and species delimitations based on newly generated sequences of the cytochrome c oxidase I (COI). Results show the first records for Spain and the Catalan Coast for some of these neglected taxa, thereby reinforcing or expanding its distribution with molecular data. Moreover, two species are described, the lower heterobranch Rhodope salviniplaweni sp. n. and the nudibranch Embletonia mediterranea sp. n. and the cephalaspidean Laona vestita comb. nov. is reassigned to a different family. Overall, this study represents a significant advancement in the taxonomic and biogeographic knowledge of interstitial fauna, highlighting the value of continued research in these underexplored habitats. http://www.zoobank.org/urn:lsid:zoobank.org:pub:10B5A669-99B1-46E7-BBE3-42C36D4B3663

Morphological and molecular phylogeny of Clinostomum sp. (Digenea: Clinostomidae) metacercariae, using DNA barcode from a South American freshwater fish.

Here, we present a comprehensive morphological and molecular phylogenetic analysis of Clinostomum sp. (Digenea: Clinostomidae) metacercariae parasitizing two freshwater fish species from Southeast Brazil: Serrasalmus spilopleura (piranha) and Callichthys callichthys (tambuatá). The morphological examination revealed distinct characteristics of metacercariae in each host. Using the cytochrome c oxidase I (COI) gene barcode region, we obtained DNA sequences that allowed for accurate phylogenetic placement. Phylogenetic analyses revealed that Clinostomum sp. HM41 (metacercariae), isolated from S. spilopleura, exhibited 86% similarity to Ithyoclinostomum yamagutii, while Clinostomum sp. HM125 (metacercariae), from C. callichthys, showed 98.7% similarity to Clinostomum sp. Cr_Ha1. The phylogenetic trees constructed through Bayesian Inference and Maximum Likelihood methods indicated high biodiversity within the Clinostomum genus and strong support for distinct lineages. These findings enhance our understanding of the diversity and ecological distribution of Clinostomum species in South American freshwater environments.

Morphogenetic Identification of a New Record Condica capensis (Lepidoptera: Noctuidae) in Yunnan, China

Condica capensis (Lepidoptera: Noctuidae), a newly identified pest in Yunnan Province, China, poses a threat to safflower crops. Discovered in Nanhua County in November 2023, the pest damages safflower at multiple life stages, especially during its larval stage, when it feeds on leaves, tender stems, and flower filaments, sometimes causing the entire plant to die. Morphological and molecular analyses, including mitochondrial cytochrome C oxidase I (COI) gene sequencing, confirmed its identity as C. capensis, a new species record for Yunnan. The study also documented the pest’s life cycle, reproductive behavior, and natural enemies, highlighting the potential for biological control using parasitic wasps such as Cotesia sp. This research emphasizes the need for accurate pest identification and monitoring to develop effective, sustainable pest management strategies. As safflower cultivation grows in Yunnan, managing C. capensis is critical to safeguarding local agriculture and preventing broader agricultural threats.

M6a demethylase FTO regulates the oxidative stress, mitochondrial biogenesis of cardiomyocytes and PGC-1a stability in myocardial ischemia-reperfusion injury

ABSTRACT Objective Myocardial ischemia-reperfusion injury (MIRI) is a highly complex disease with high morbidity and mortality. Studying the molecular mechanism of MIRI and discovering new targets are crucial for the future treatment of MIRI. Methods We constructed the MIRI rat model and hypoxia/reoxygenation (H/R) injury cardiomyocytes model. RT–PCR and Western blot were used to investigate the expression of the fat mass and obesity-associated (FTO) gene. Electrocardiogram, echocardiography, triphenyltetrazolium chloride (TTC) staining and hematoxylin-eosin (HE) staining were used to assess the model and the effect of FTO overexpression. The generation of reactive oxygen species (ROS) and the levels of superoxide dismutase (SOD2), mitochondrial transcription factor (TFAM) and cytochrome c oxidase I (COXI) were detected to assess the oxidative stress and mitochondrial biogenesis. RNA immunoprecipitation (RIP) and RNA pulldown assays were used to identify the interaction of FTO and PGC-1a. The m6A dot blot, methylated RNA immunoprecipitation PCR (MeRIP-PCR) and RNA stability analysis were used to analyze the regulation of methylation of PGC-1a by FTO. Results FTO was downregulated in MIRI rats and H/R induced cardiomyocytes. Overexpression of FTO inhibited ROS level and increased the expression of SOD2, TFAM and COXI in vitro and in vivo. In addition, PGC-1a was identified as a downstream target of FTO. FTO enhanced the stability of PGC-1a mRNA through removing the m6A modification. Conclusion Our study revealed the role of FTO regulates the oxidative stress and mitochondrial biogenesis via PGC-1a in MIRI, which may provide a new approach to mitigating MIRI.

COI Metabarcoding as a Novel Approach for Assessing the Honey Bee Source of European Honey

Honey is a widely consumed food product frequently subjected to adulteration, with the mislabeling of its botanical or geographical origin being one of the most common practices. Determining the entomological origin of honey is particularly challenging but of high relevance for ensuring its authenticity, especially for products with protected designation of origin (PDO) labels. This study presents a novel DNA metabarcoding approach targeting a highly informative 406 bp fragment of the cytochrome c oxidase I (COI) gene to differentiate among the three major mitochondrial lineages (A, M, and C) of honey bees (Apis mellifera L.) native to Europe. The target region was selected based on the calculated fixation index (FST), which is frequently used in Population Genetics as a measure of differentiation between populations. The approach was validated with 11 honey samples of known entomological origin and applied to 44 commercial honeys from 13 countries. The approach demonstrated high sensitivity, accurately identifying the entomological origin of honey, including samples produced by honey bees of varying ancestries, which could not be resolved by previous methods based on real-time PCR coupled with high-resolution melting (PCR-HRM) analysis. The results demonstrate the effectiveness of COI metabarcoding in verifying honey authenticity and highlight the predominance of C-lineage honey bees in the production of commercial honeys from northwestern Europe. This finding suggests a limited presence of the native M-lineage ancestry, underscoring the need for conservation efforts.

Genetic and Haplotype Diversity of Hoplolaimus (Nematoda: Hoplolaimidae) Through Analysis of COI of mtDNA

Lance nematodes (Hoplolaimus spp.) feed on the roots of various plants, including key horticultural products. An investigation of the genetic diversity and structure of six Hoplolaimus species, utilizing the cytochrome c oxidase I (COI) of the mtDNA gene, was based on 174 sequences available on the NCBI. Based on the COI of mtDNA, the haplotype analysis revealed 44 haplotypes. Nucleotide diversity was low among all species of Hoplolaimus, except for H. magnistylus (π = 0.04915) and H. stephanus (π = 0.06746). In contrast, haplotype diversity (Hd) was high, especially for H. stephanus (Hd = 0.89) and H. pararobustus (Hd = 0.90). Phylogenetic analysis grouped the various populations into eight clades, and the result showed that H. magnistylus was placed in three different clades, which showed high variability in haplotype supported by the haplotype network. Neutrality tests and mismatch distribution based on the mtDNA supported the hypothesis of a constant population with no expansion in Hoplolaimus, except for H. concaudajuvencus (Tajima (D) = −0.84971) and H. columbus (Tajima (D) = −0.87674). In conclusion, genetic analysis showed a neutral evolution amongst the Hoplolaimus species. The result of the present study provides a better insight into the Hoplolaimus species toward species delimitation and managing this plant-parasitic nematode in various crops.

A new species of eight-gilled hagfish (Myxinidae: Eptatretus) from the deep waters of the Lakshadweep Sea, India.

A new species of eight-gilled hagfish genus Eptatretus (Myxinidae) is described based on five specimens trawled on the upper continental slope off Kollam, Kerala, India, northern Indian Ocean. Eptatretus gopali sp. nov. can be diagnosed by the presence of eight-gill pouches and gill apertures, 10-11 pre-brachial, 7 branchial, 50-55 trunk, 9-10 tail slime pores, 77-82 total pores, 3/2 multicusp teeth pattern, 40-44 total cusps, absence of nasal-sinus papillae, and a light brown body color. Detailed morphological comparisons with all known eight-gilled species of Eptatretus showed that the new species was distinct from all others by having 40-44 total cusps, characteristic body proportions, and the shortest tail length. E. gopali formed a district clade in the phylogenetic tree and exhibited a genetic distance of 3.6%-11.6% in Cytochrome C oxidase I (COI) sequences and 2.0%-4.9% in 16S rRNA sequences between the congeners. An identification key is also provided for the species of Eptatretus from the Indian Ocean.

Exploring Dietary Composition in an Invasive Apple Snail From Different Habitats Combining With Intestinal Microbiota and Metabolomics.

Pomacea canaliculata is recognized as a globally invasive aquatic species. Analyses of intestinal microbiota, dietary composition, and metabolism of invasive species can enhance our understanding of their feeding strategies and physiological adaptation strategies to the environment. Intestinal content samples were collected from P. canaliculata inhabiting three distinct environments including a pond, a river, and a ditch. These samples were subjected to 16S rRNA gene sequencing analysis and multiple metabarcoding analyses, including eukaryotic 18S rRNA, mitochondrial cytochrome c oxidase I (COI), and chloroplast rbcL genes. In addition, metabolomics analysis was conducted on the intestinal content samples to investigate metabolic change. The highest dietary diversity in P. canaliculata was observed in the ditch, and females exhibited a higher dietary diversity than males in the pond. The 18S rRNA gene has a high potential for identifying the dietary components of omnivorous species. The intestinal microbiota of P. canaliculata from different habitats displayed significant variations, attributed to differences in food resources and other environmental factors. Bacteria in the aquatic environment had minimal impact on the intestinal microbiota of P. canaliculata. Overall, P. canaliculata exhibited adaptive changes in physiological characteristics across different habitats, including alterations in diet, which, in turn, influence microbiota and metabolic pathways such as amino acid biosynthesis in the intestine. The present study investigated the physiological mechanisms that enable P. canaliculata to adapt to diverse habitats, considering various factors including diet, which is important for comprehending its invasive potential and the subsequent threats it poses to aquatic ecosystems.

Morphological identification and first molecular characterization of Lobothorax typus (Isopoda: Cymothoidae) parasitizing Trichiurus lepturus (Scombriformes: Trichiuridae) in the Philippines

Parasitic diseases pose a threat to fisheries, negatively affecting the livelihood and food security of billions of people worldwide. Among these, parasitic isopods of fish are not well documented and studied. Thus, we examined the buccal cavity of 385 randomly selected saltwater fishes sold in a fish market in Naic, Cavite, Philippines. Here, 7/385 (1.82%) saltwater fishes, exclusively Trichiurus lepturus, harbored a parasitic isopod. Morphological observation and sequencing analysis of cytochrome c oxidase I (COI) and 16S rRNA genes confirmed the identity as Lobothorax typus. Phylogenetic tree based on these sequences showed that it is closely related to isolates from India and Japan. As such, further studies on the risk factors associated with L. typus infestation are urgently needed.

Integrated taxonomy to advance species delimitation of the Anopheles maculipennis complex

The Anopheles maculipennis complex consists of several mosquito species, including some primary malaria vectors. Therefore, the presence of a species in a particular area significantly affects public health. In this study, 1252 mosquitoes were collected in northern Italy, representing four identified species of the Anopheles maculipennis complex (Anopheles daciae sp. inq., Anopheles maculipennis s. s., Anopheles atroparvus and Anopheles melanoon). The sequences of two DNA markers, mitochondrial cytochrome c oxidase I (COI) and nuclear internal transcribed spacer 2 (ITS2), were generated. DNA-based species delimitation analyses were performed, incorporating public sequences, with distance-based and coalescent tree-based methods to confirm the actual species boundaries within the complex. While some morphospecies were unequivocally delimited by all methods and markers, COI analysis splitted An. maculipennis s. s. into two well-supported groups. However, molecular delimitation failed in recognizing An. daciae sp. inq. and An. messeae as two separate evolutionary entities. Species delimitation was further tested with a morphometric approach, which clearly differentiated species collected in the survey area. These findings underscore how challenging the characterization of the taxonomy of the complex is, providing evidence of potential introgression events in An. maculipennis s. s. and suggesting the need for robust evidence to support An. daciae sp. inq. and An. messeae as distinct species.

Morphometric and Genetic Variations of Freshwater Eels (Anguilla spp.) in Poso River, Central Sulawesi: Implications for Conservation Strategies

The freshwater eel, Anguillids, is a valuable nutrition and commodity fish found in various freshwater environments. However, the world's population of Anguillids are declining because of habitat degradation, pollution, and barriers to migration, all of which are prevalent threats to freshwater ecosystems such as the Poso River in Central Sulawesi. Establishing conservation areas is one of the efforts to protect Anguillids and their habitats, which requires information on the anguillids morphometrics and genetics, where high morphometric and genetic variations are indicators of adaptation or evolution of the species to survive environmental changes. Therefore, the study aims to assess the morphometric and genetic variations in the Poso River, Central Sulawesi. Samples were collected along the Poso River in the range of May 2021 and August 2023 . Different fishing gears were used depending on the location and Anguillids’ phase of life. A total of 150 eel samples were used for morphometric analysis, out of which 38 were selected randomly for the genetic one. Genetic diversity analysis was performed based on the Cytochrome c Oxidase I (COI). The study successfully identified three species: A. bicolor, A. celebesensis, and A. marmorata. The key characteristic distinguishing the three species was ADL/TL ratio. Most coefficients of variation of morphometric characters of each species were above 10%, indicating medium to high variation. A total of 11 haplotypes were identified, of which six belong to A. marmorata and five to A. celebesensis. Generally, haplotype diversity was low, ranging from 0.2923 to 0.9333, and nucleotide diversity ranged from 0.0005 to 0.0046. The low genetic diversity observed in this study is likely a result of the migratory nature of Anguillids. Morphometric and genetic variations can support restocking as a conservation strategy to bolster wild populations. However, comprehensive studies must be conducted to understand all aspects impacting Anguillid resources for establishing conservation areas to protecti their populations and habitats.

Exploration and phylogenetic characterisation of Wolbachia in the peach fruit fly Bactrocera zonata (Saunders) (Diptera: Tephritidae)

ABSTRACT Bactrocera zonata (Saunders) is a significant pest for tropical and subtropical fruits and vegetables and has a detrimental impact on the global economy. Subsequent samples were successfully identified using the cytochrome c oxidase I (COI) gene. Wolbachia are naturally occurring endosymbiotic bacteria found in the reproductive tissues of arthropods. The prevalence of endosymbiont Wolbachia and the diversity of its strains in populations of B. zonata were explored. The screening of 422 samples revealed (29–40) positive samples for Wolbachia infection through 16S rRNA, wsp, and MLST genes. Phylogenetic analysis using neighbour-joining (NJ) and Bayesian inference (BI) methods exhibited that the identified Wolbachia strains were clustered with those belonging to supergroup A. The newly identified strain of Wolbachia, named wBzon, differs from previously published sequences of B. zonata, indicating its potential for the incompatible insect technique (IIT). This discovery offers an eco-friendly method for managing B. zonata populations and highlights the importance of Wolbachia in these populations.

Morphological description and molecular characterization of Heterospinus mccordi n. gen. n. sp. (Acanthocephala: Polymorphidae) from cystacanths infecting a non-native crayfish host, Procambarus clarkii (Decapoda: Cambaridae), in South Carolina, USA

A new genus and species within the family Polymorphidae Meyer, 1931 were erected to accommodate cystacanths recovered from the mesentery of individuals from a non-native population of the red swamp crayfish Procambarus clarkii (Girard), collected from South Carolina (USA). Morphological characteristics of the specimens collected included in both sexes a spindle-shaped body with a slender hindtrunk, two fields of markedly different sized spines on the foretrunk, the presence of a middle row of smaller scythe-shaped hooks on a proboscis armed with 19–20 longitudinal rows of 14–16 hooks; and in males, six cement glands, absence of genital spines, and a digitiform and spinose bursa (observed inverted). Sequencing portions of both the mitochondrial cytochrome c oxidase I (COI) and large subunit ribosomal RNA genes was completed, followed by phylogenetic analysis of a concatenated alignment. Sequences from our specimens appeared in a clade with those of Hexaglandula corynosoma (Travassos, 1915) and Ibirhynchus dimorpha (Schmidt, 1973) but were 27% divergent from both using the COI marker. The genetic divergence of this parasite from other polymorphid genera, along with unique morphological features, justified erecting a new genus and new species. Herein we describe Heterospinus mccordin. gen.n. sp. bringing the total number of genera within the family Polymorphidae to 16, and we emend and update the latest key that was provided for the genera within this family. This is the first record of polymorphids infecting P. clarkii outside of its native range. The definitive host remains unknown.

Genetic Diversity, Genetic Structure, and Demographic History of Giant Honeybee Apis dorsata Fabricius, 1793 (Hymenoptera: Apidae) in Thailand

Giant honeybee (Apis dorsata Fabricius, 1793) is native to Asia. It is an efficient pollinator contributing to ecosystem stability. Populations of A. dorsata have been decreasing continuously due to various factors; however, information on genetic diversity is very limited. Therefore, purposes of this study were to assess the genetic diversity, structure, and demographic history of A. dorsata based on the mitochondrial cytochrome c oxidase I (COI) sequences. Adult worker bees from 41 colonies were collected throughout Thailand. In addition, 24 COI sequences of A. dorsata from other countries available in public databases were also incorporated into the data analysis. Overall, high haplotype (0.958) and low nucleotide diversities (0.00536) with a maximum intraspecific genetic divergence of 1.65% were found within Thai specimens. Population pairwise FST revealed genetically significant differences among Northern, Central and Southern populations while those from Northeastern are not. Median-joining network analysis revealed a star-like shape, a characteristic of the recent expanding population. This is supported by unimodal mismatch distribution and significantly negative of Tajima’s D and Fu’s Fs tests. Population expansion time is estimated to be 83,000 – 177,000 years ago, possibly in response to interglacial Pleistocene climatic fluctuation.

A Novel Polymerase Chain Reaction (PCR)-Based Method for the Rapid Identification of Chrysodeixis includens and Rachiplusia nu.

Chrysodeixis includens and Rachiplusia nu are two species belonging to the Plusiinae subfamily within the Noctuidae family. Due to their morphological similarity, the identification of their larvae is difficult and time-consuming. A rapid and accurate identification of these two species is essential for their management as these species exhibit differential susceptibilities to insecticides and crops engineered to express Bacillus thuringiensis (Bt) proteins, and a molecular tool can easily provide this differentiation. Currently, molecular analysis can identify these species through genetic sequencing, an expensive and time-consuming process. In our study, after sequencing part of the mtDNA cytochrome c oxidase I (COI) gene and based on the differences found in the gene of each species, a set of species-specific primers was developed: one reverse primer common to both species and two forward primers, specific to each species, amplifying fragments of 199 base pairs (bp) for C. includens and 299 bp for R. nu. Our results indicate that the primers were specific for these species, enabling the identification of individuals directly through agarose gel. The new methodology proved to be accurate, rapid, and reliable for the correct identification of these two species of loopers.

Strong Small-Scale Differentiation but No Cryptic Species Within the Two Isopod Species Asellus aquaticus and Proasellus coxalis in a Restored Urban River System (Emscher, Germany).

Worldwide, humans have strongly altered river networks. Key changes resulted in modified hydromorphology, poor habitat quality and availability, migration barriers, and pollution. Restoration measures aim at mitigating anthropogenic stressors and at restoring connectivity, but the biological success of these measures is not guaranteed. Analyzing genetic diversity and metapopulation structure of target species in the river network with genetic markers can help to understand recolonization processes and to identify persisting gene flow barriers. Here, we studied the population genetic structure of the two pollution-tolerant detritivorous isopod species, Asellus aquaticus and Proasellus coxalis, in the former heavily degraded and polluted, but now mostly restored Emscher catchment in Germany. For both species, we analyzed mitochondrial cytochrome c oxidase I (COI) gene sequences and nuclear genome-wide single nucleotide polymorphism (SNP) data. Surprisingly, we found a strong metapopulation structure for both species with several isolated populations on a small-scale of few kilometers, but a still high genetic diversity, especially in the COI gene. For both taxa, potentially cryptic species are known, but our SNP data showed that the mitochondrial lineages represent only one species, each, in the study area. This highlights the importance of integrating high-resolution nuclear markers into species identification because species diversity may otherwise be overestimated. While we could identify some migration barriers and find indications for passive dispersal by birds or humans, these factors could not fully explain the local metapopulation structure, suggesting that also other drivers, such as isolation by adaptation, priority effects, or biotic interactions, play a role in shaping the population genetic structure.

Genetic and Haplotype Diversity of Clarias gariepinus (Burchell 1822) Based on Cytochrome c Oxidase I (COI) Gene

The evaluation of mitochondrial DNA and genetic analysis is helpful for economically significant species. Clarias gariepinus is a critical species in aquaculture. This study investigates the genetic diversity and population differentiation of C. gariepinus from 19 countries using 164 sequences of the mitochondrial DNA’s Cytochrome c oxidase I (COI) gene. The haplotype analysis revealed a total of 17 haplotypes, with a nucleotide diversity (π) of 0.012 and a haplotype diversity (Hd) of 0.87. The results of an AMOVA and fixation index indicated significant genetic variation and structure among the populations. Additionally, neutrality tests and mismatch distribution analysis supported the hypothesis of under-purifying selection in C. gariepinus. The findings suggested that the population did not experience expansion. In conclusion, the genetic analysis highlighted substantial variation among C. gariepinus populations from different locations, providing valuable insights for the global management of this species.

Faecal DNA Metabarcoding for Diet Analysis of Endangered Fish Species, Odontobutis obscurus.

Understanding the ecological characteristics of endangered species is essential for their management and conservation. The dark sleeper (Odontobutis obscurus) is a carnivorous freshwater fish that has been designated an endangered species in South Korea because of its highly restricted habitat range and small population size. However, ecological research on prey items for this species remains severely limited globally, posing challenges for establishing conservation measures such as translocation management. In this study, the food sources and feeding strategies of O. obscurus were identified through non-invasive faecal DNA metabarcoding. A total of 24 O. obscurus specimens were collected from Sanyang Stream on Geoje Island, and faecal DNA was amplified using mitochondrial cytochrome c oxidase I (COI) primer. As a result, 13 prey items were identified, comprising fish and benthic invertebrates. Dietary metrics revealed the overall food composition and major food source of O. obscurus, and the Costello method enabled graphical diet analysis, suggesting that O. obscurus is a generalist, feeding on a wide variety of food sources. The food selectivity index suggested that O. obscurus prefers specific prey items, such as genus Misgurnus, Zacco (fish), Radix (gastropod), and Ephemera (mayfly). This molecular method facilitated the diet analysis of an endangered fish species that cannot undergo dissection, providing crucial information for their management, particularly regarding translocation efforts.

Population genetic diversity of Culex tritaeniorhynchus in Jining City of Shandong Province based on the mitochondrial cytochrome C oxidase I gene

To understand the genetic basis of the adaptation of Culex tritaeniorhynchus to different environmental ecology in Jining City, Shandong Province, so as to provide insights into understanding of the population structure or isolation pattern of Cx. tritaeniorhynchus in the city. Seven sampling sites were selected from urban, suburban and rural areas of Jining City, Shandong Province from June to August 2023, and mosquitoes were collected using mosquito-trapping lamps. All collected adult mosquitoes were identified morphologically. Genomic DNA was extracted from a single female Cx. tritaeniorhynchus mosquito, and the mitochondrial cytochrome C oxidase I (COI) gene was amplified using a PCR assay, sequenced and subjected to molecular identification. The number of haplotypes, haplotype diversity (Hd), nucleotide diversity (Pi), and average number of nucleotide differences (K) of Cx. tritaeniorhynchus DNA sequences were estimated among different sampling sites using the software DnaSP 6, and a neutrality test was performed. The fixation index (FST), and gene flow (number of migrants, Nm) of Cx. tritaeniorhynchus populations were calculated using the software Arlequin 3.5.2, and subjected to analysis of molecular variance (AMOVA). In addition, a haplotype network diagrams and a phylogenetic tree of Cx. tritaeniorhynchus populations were created using the software PopART and MEGA 11, respectively. A total of 420 sequences were successfully amplified from the COI gene of Cx. tritaeniorhynchus samples collected from 7 sampling sites in Jining City, and a gene fragment sequence with a length of 603 bp was obtained, with 55 variable sites and 46 haplotypes and without insertion or deletion mutations. Of the 46 haplotypes, H01 was the dominant shared haplotype, and the haplotype frequency increased gradually from urban areas (34.00%) to rural areas (47.00%). The mean Hd, Pi and K values of Cx. tritaeniorhynchus COI genes were 0.814, 0.024 and 14.129, 0.489, 0.016 and 7.941 and 0.641, 0.016 and 10.393 in suburban, urban, and rural areas, respectively, with the highest population diversity of Cx. tritaeniorhynchus in suburban areas and the lowest in urban areas. Paired FST analysis among different types of sampling sites showed that the mean FST value was 0.029 between urban and suburban areas, indicating more frequent inter-population communication. AMOVA revealed that the percentage of intra-population variation (95.74%) was higher than that of inter-population variation (4.26%). Neutrality tests showed deviation from neutrality in Cx. tritaeniorhynchus populations collected from Nanyang Township (Tajima's D = 2.793, Fu's Fs = 6.429, both P values < 0.05). In addition, the mismatch distribution curves of Cx. tritaeniorhynchus COI gene appeared bimodal or multimodal patterns in Jining City, indicating a relatively stable overall population size. The mitochondrial COI gene may be used as a molecular marker to investigate the population genetic diversity of Cx. tritaeniorhynchus. The population genetic diversity of Cx. tritaeniorhynchus is higher in the suburban areas of Jining City than in rural and urban areas, and there are frequent genetic exchanges between Cx. tritaeniorhynchus populations from urban and suburban areas.

A DNA barcode reference library for the Tipulidae (Insecta, Diptera) of Germany.

Tipulidae, commonly known as true crane flies, represent one of the most species-rich dipteran families, boasting approximately 4,500 known species globally. Their larvae serve as vital decomposers across diverse ecosystems, prompting their frequent and close observation in biomonitoring programs. However, traditional morphological identification methods are laborious and time-consuming, underscoring the need for a comprehensive DNA barcode reference library to speed up species determination. In this study, we present the outcomes of the German Barcode of Life initiative focused on Tipulidae. Our DNA barcode library comprises 824 high-quality cytochrome c oxidase I (COI) barcodes encompassing 76 crane fly species, counting for ca. 54% of the German tipulid fauna. Our results significantly increased the number of European tipulid species available in the Barcode of Life Data System (BOLD) by 14%. Additionally, the number of barcodes from European tipulid specimens more than doubled, with an increase of 118%, bolstering the DNA resource for future identification inquiries. Employing diverse species delimitation algorithms - including the multi-rate Poisson tree processes model (mPTP), Barcode Index Number assignments (BIN), Assemble Species by Automatic Partitioning (ASAP), and the TaxCI R-script - we successfully match 76-86% of the morphologically identified species. Further validation through neighbor-joining tree topology analysis and comparison with 712 additional European tipulid barcodes yield a remarkable 89% success rate for the species identification of German tipulids based on COI barcodes. This comprehensive DNA barcode dataset not only enhances species identification accuracy but also serves as a pivotal resource for ecological and biomonitoring studies, fostering a deeper understanding of crane fly diversity and distribution across terrestrial landscapes.