Intraspecific Genetic Distances Research Articles

DNA barcoding and cryptic diversity in fishes from the Ili River Valley in China, Xinjiang.

The Ili River Valley, located in the northwest of China, serves as a vital repository for fish genetic resources. Its extensive water network and diverse climate have given rise to a unique fish composition and endemic species. In this study, we collected the cytochrome c oxidase subunit I (COI) sequences from 660 fish specimens in the Ili River Valley. The effectiveness of DNA barcoding in identifying fish species in the area was assessed by examining genetic distances, constructing phylogenetic trees, and performing ABGD (Automatic Barcode Gap Discovery) analyses, among other methods. In total, 20 species were identified, including one unidentified species (Silurus sp.). Except for Silurus asotus and Hypophthalmichthys molitrix (only one sample), the maximum intraspecific genetic distance among the remaining species was smaller than the minimum interspecific distance, which proves that the species exhibit obvious barcode gaps. In the Neighbor-Joining trees, 20 species formed separate monophyletic branches. According to ABGD analysis, 660 sequences were categorized into 19 Operational Taxonomic Units, with Silurus sp. and S. asotus grouped into a single OTU. The Silurus in this study exhibits shared haplotypes and significant genetic divergence, suggesting the potential presence of cryptic species. Furthermore, the nucleotide diversity across all species fell below the threshold level, indicating that the local fish population is gradually declining. In conclusion, this study has demonstrated the effectiveness of DNA barcoding in identifying fish species in the Ili River Valley, providing valuable data to support the conservation of local fish resources.

Genetic polymorphisms of common sandflies in selected areas of Henan Province based on DNA barcoding

To characterize the species of common sandflies in Henan Province using DNA barcoding with cytochrome c oxidase subunit I (COI) gene as the molecular marker, and to analyze the genetic polymorphisms of sandflies, so as to provide insights into visceral leishmaniasis prevention and control in Henan Province. Sandfly specimens were sampled from 13 sandflies surveillance sites from 2021 to 2023 in Anyang City, Zhengzhou, Luoyang and Xuchang cities (Zhengzhou-Luoyang-Xuchang areas) where visceral leishmaniasis cases were reported and in Jiaozuo and Xinxiang cities (Jiaozuo-Xinxiang areas) without visceral leishmaniasis cases reported. Genomic DNA was extracted from a single sandfly, and COI gene was amplified. The amplification product was subjected to bidirectional sequencing. Following sequence assembly, the species of sandflies was characterized through sequence alignment using the BLAST tool. The intra-specific and inter-specific genetic distances of sandflies were estimated among different areas using the software Mega 11, and phylogenetic trees were created. The polymorphisms of nucleotide sequences in the sandflies COI gene were estimated using the software DnaSP. The fixation index (FST) of different geographical isolates of sandflies was calculated using the Arlequin software, and the gene flow value (Nm) was used to measure the gene flow in the sandflies populations. In addition, the population genetic structure of different geographical populations of Phlebotomus chinensis was analyzed using the STRUCTURE software. A total of 978 sandflies were collected from 13 sandflies surveillance sites in Zhengzhou-Luoyang-Xuchang areas, Jiaozuo-Xinxiang areas and Anyang City of Henan Province from 2021 to 2023, and 475 sandflies were randomly sampled for subsequent detections. A total of 304 Ph. chinensis, 162 Se. squamirostris and 9 Se. bailyi were identified based on molecular biological detection of the COI gene, and Se. bailyi was reported for the first time in Henan Province. The intraspecific genetic distances of sandflies were 0.000 to 0.040, and the inter-specific genetic distances ranged from 0.133 to 0.161. Phylogenetic analysis revealed that each of the three sandfly species was clustered into a clade. The genetic polymorphisms of Ph. chinensis populations varied among different areas, with the highest haplotype diversity (0.966 ± 0.007) and the greatest nucleotide diversity (0.011) in Zhengzhou-Luoyang-Xuchang areas, and the lowest haplotype diversity (0.720 ± 0.091) and nucleotide diversity (0.004) in Anyang City. The dominant haplotype of Ph. chinensis populations was Pch_Hap_2 in Anyang City and Jiaozuo-Xinxiang areas, with moderate genetic differentiation (0.05 < FST < 0.15) and frequent gene exchange (Nm value > 1) between Ph. chinensis populations sampled from Anyang City, and Jiaozuo-Xinxiang areas. Population genetic structure analysis showed that the dominant component of Ph. chinensis populations was K5 in Anyang City and Jiaozuo-Xinxiang areas. No obvious dominant haplotype was observed in Ph. chinensis populations sampled from Zhengzhou-Luoyang-Xuchang areas, which had very high genetic differentiation (FST > 0.25) and little gene exchange (Nm value < 1) with Ph. chinensis populations from Anyang City, and Jiaozuo-Xinxiang areas, with K3 as the dominant component. In addition, there was no significant difference in the genetic polymorphism level among Se. squamirostris populations from the three areas. There are Ph. chinensis, Se. squamirostris and Se. bailyi in Henan Province, and S. bailyi is recorded for the first time in Henan Province by molecular biological assays. There are different levels of genetic differentiation and gene exchange among P. chinensis populations in different areas of Henan Province.

Delimiting species, revealing cryptic diversity in Molytinae (Coleoptera: Curculionidae) weevil through DNA barcoding.

The subfamily Molytinae (Coleoptera: Curculionidae), being the second largest group within the family Curculionidae, exhibits a diverse range of hosts and poses a serious threat to agricultural and forestry industries. We used 1,290 cytochrome c oxidase subunit I (COI) barcodes to assess the efficiency of COI barcodes in species differentiation and uncover cryptic species diversity within weevils of Molytinae. The average Kimura 2-parameter distances within species, genus, and subfamily were 2.90%, 11.0%, and 22.26%, respectively, indicating significant genetic differentiation at both levels. Moreover, there exists a considerable degree of overlap between intraspecific (0%-27.50%) and interspecific genetic distances (GDs; 0%-39.30%). The application of Automatic barcode gap discovery, Assemble Species by Automatic Partitioning, Barcode Index Number, Poisson Tree Processes (PTP), Bayesian Poisson Tree Processes (bPTP), and jMOTU resulted in the identification of 279, 275, 494, 322, 320, and 279 molecular operational taxonomic units, respectively. The integration of 6 methods successfully delimited species of Molytinae in 86.6% of all examined morphospecies, surpassing a threshold value of 3% GD (73.0%). A total of 28 morphospecies exhibiting significant intraspecific divergences were assigned to multiple MOTUs, respectively, suggesting the presence of cryptic diversity or population divergence. The identification of cryptic species within certain morphological species in this study necessitates further investigation through comprehensive taxonomic practices in the future.

Integrative studies of Microtus hartingi and Microtus guentheri (Cricetidae, Arvicolinae) and taxonomic problem of Microtus elbeyli

In this study, Taxonomic differences between Microtus guentheri and Microtus hartingi, which are similar in terms of external morphological features, were determined using linear morphometric, geometric morphometric, karyological and molecular analyses methods. Through classical morphometric (mean values) and geometric morphometric analyses of craniodental and mandible characters, influenced by various genetic, developmental, and functional processes, it was found that M. hartingi and M. guentheri exhibited notable differences in both mean linear measurements and shape. The Jackknife in classical morphometric analyses of samples, which underwent molecular and karyological species diagnoses, was higher than 88 % for each character. In geometric morphometric analyses, the Procrustes distance for shape follows the order Ventral < Dorsal < Mandible < M3<M2<M1. Based on cross-validation, the accurate classification rate is between 73 and 100 %. In both mean linear measurements and centroid size, M. hartingi specimens exhibited significantly larger mandibles and craniodentals compared to individuals of M. guentheri. According to PLS results, there was a significant relationship between the shape of M1, M2 and M3 and environmental variables, while the relationship between their size and environmental variables was not significant. However, neither the shape nor the size of the other parts had an sufficient relationship with environmental variables. Both species exhibited karyological similarity with a chromosome number of 2n = 54, but display polymorphism in chromosome arm number. There were differences in the C-banding of autosomal chromosomes and X chromosome. The classification of species in classical and geometric morphometric analyses, diverges from the outcomes of karyological and molecular assessments. In both species, the intraspecific genetic distance remained below 1.35 %. The cyt-b exhibits significant differentiation between the two species (K2P = 6.55 %). The divergence time for M. guentheri from the clade containing M. hartingi + M. qazvinensis + Microtus dogramacii was estimated to be 0.73 million years ago. Since no sample of Microtus elbeyli with karyological and cyt-b characteristics was found in the samples taken from the type locality, the taxonomic status of M. elbeyli remains unclear.

Morphological and Phylogenetic Analyses Reveal Three New Species of Entomopathogenic Fungi Belonging to Clavicipitaceae (Hypocreales, Ascomycota).

This study aims to report three new species of Conoideocrella and Moelleriella from Yunnan Province, Southwestern China. Species of Conoideocrella and Moelleriella parasitize scale insects (Coccidae and Lecaniidae, Hemiptera) and whiteflies (Aleyrodidae, Hemiptera). Based on the phylogenetic analyses of the three-gene nrLSU, tef-1α, and rpb1, it showed one new record species (Conoideocrella tenuis) and one new species (Conoideocrella fenshuilingensis sp. nov.) in the genus Conoideocrella, and two new species, i.e., Moelleriella longzhuensis sp. nov. and Moelleriella jinuoana sp. nov. in the genus Moelleriella. The three new species were each clustered into separate clades that distinguished themselves from one another. All of them were distinguishable from their allied species based on their morphology. Morphological descriptions, illustrations, and comparisons of the allied taxa of the four species are provided in the present paper. In addition, calculations of intraspecific and interspecific genetic distances were performed for Moelleriella and Conoideocrella.

Assessment of fish diversity in the East China Sea hairtail national aquatic germplasm resources conservation zone using DNA barcoding

Based on the fishery resource survey data from 2019 to 2022, the species composition and taxonomic diversity of fish in the East China Sea hairtail national aquatic germplasm resources conservation zone (ECSHZ) were studied through the analysis of 185 COI sequences. The results showed that the 185 fish samples collected belonged to 105 species, 21 orders, 46 families, and 89 genera in terms of their taxonomic status. There are discrepancies between morphological identification and genetic identification results for four fish species. The intraspecific genetic distance ranged from 0 % to 1.0 %, with an average value of 0.3 %. Meanwhile, the interspecies within genus distance ranged from 5.3 % to 22.7 %, with an average of 14.5 %. The outcomes of the division using Automatic Barcoding Gap Discovery (ABGD) and Bayesian phylogenetics and phylogeography (BPP) were concordant, identifying a total of 105 species groups. ABGD and BPP have shown that there was no overlap between intra-species variation and inter-species variation, supporting the existence of a barcoding gap. The results demonstrate the superiority of DNA barcoding in species identification. A phylogenetic tree was constructed using the maximum likelihood method based on the COI gene, which revealed certain limitations of COI in representing phylogenetic relationships at the genus and higher taxonomic levels. Another phylogenetic tree was constructed using 13 mitochondrial protein-coding genes with more genetic information, allowing for a better study of evolutionary relationships of ECSHZ fish species. The tree supported that Carangidae, Polynemidae, Paralichthyidae, Pleuronectidae, Sphyraenidae, and Cynoglossidae were clustered in an independent clade, with Priacanthidae and Lobotidae separating from Perciformes. This study offers DNA barcodes for ECSHZ fish species, providing crucial data support for conserving fish diversity.

Two new species of Platynothrus and Heminothrus (Oribatida: Crotoniidae) from Mexico

The oribatid mite genera Platynothrus and Heminothrus currently comprise 20 and 10 species, respectively, and collectively have a cosmopolitan distribution. They have been classified into three to five subgenera, depending on the classification. For Platynothrus, a couple of new species have been described in the last two years, while for Heminothrus, the last formal description of a new species was 26 years ago. In this study, we describe two new species of these crotoniid mites, discovered in the soil and litter of a tropical montane cloud forest in Mexico. One of these new species belongs to the genus Platynothrus, which can be distinguished from other species of this genus by the short clavate sensillus, curved smooth interlamellar seta, and the length of the notogastral setae. The second new species belongs to the genus Heminothrus, being characterized by a short rhomboidal sensillus and a very long interlamellar seta. Intraspecific genetic distances of the COX-1 mitochondrial marker were 2.15%, and 0.81% for the new Platynothrus and Heminothrus species, respectively.

Decoding the molecular phylogenetics of ornamental catfishes (siluriformes) of North East India using DNA barcoding approach.

Catfishes (order Siluriformes) are among the most diverse and widely distributed fish groups in the world. They are not only used for human consumption but are also a major part of the ornamental fish trade. Being a Biodiversity Hotspot, the North Eastern Region of India is home to a diverse population of ornamental fishes. Catfishes contain a humongous number of species; in this study, the authors have tried to elucidate the phylogenetic relationship of some important ornamental catfishes found in North East India using DNA barcodes. In this study, we have tried to explore the phylogenetic history of 13 species (41 specimens) of ornamental catfishes spanning 12 genera and 9 families of Siluriformes using DNA barcoding. Pairwise genetic distances using Kimura 2-Parameter (K2P) were calculated at intra-specific and inter-specific levels. A Neighbor-Joining tree was constructed to understand the phylogenetic relationship among the nine different catfish families. All the specimens under this study clustered with their respective species under the same family and formed three sub-clades. However, Olyra longicaudata, belonging to the Bagridae family, did not cluster with other species from the same family. In this study, the authors have suggested a revision of the classification of O. longicaudata back to its original family, Olyridae. In this study, the maximum intraspecific genetic distance of 0.03 and the minimum interspecific genetic distance of 0.14 were observed among the species. Therefore, it is evident that there is a barcoding gap among the species, which helped in the correct identification of the species. Thus, DNA barcoding helped complement the phenetic approach and also revealed a different phylogenetic relationship among the catfishes belonging to the Bagridae family.

When standard DNA barcodes do not work for species identification: intermixed mitochondrial haplotypes in the Jaera albifrons complex (Crustacea: Isopoda)

Here, we characterise the standard “Folmer region” of the mitochondrial cytochrome c oxidase subunit 1 (CO1) marker and a fragment of nuclear 28S marker in four species of the Jaera albifrons complex. Jaera albifrons (Leach, 1814), Jaera ischiosetosa Forsman, 1949, Jaera praehirsuta Forsman, 1949, and Jaera forsmani Bocquet, 1950 were collected from localities on the Norwegian coast and identified with morphological characters. We compared DNA sequences with sequences available in GenBank and BOLDsystems and calculated haplotype networks and interspecific versus intraspecific genetic distances. These analyses revealed low interspecific genetic distance (CO1 0.00–1.57%, 28S 0.00–0.39%) and extensive haplotype sharing between J. albifrons group species and specimens from both sides of the North Atlantic for both CO1 and 28S. Genetic distances between J. albifrons group species and other Jaera species, however, exceeded 29% for both CO1 and 28S, with no haplotype sharing. These assessments, together with taxonomically unconstrained analyses with software ABGD and ASAP, show that these markers are unable to distinguish between the J. albifrons group of morphospecies. The sequences do, however, clearly identify J. albifrons species complex from other Jaera species. Thus, a likely hypothesis is that taxa in this complex represent a single species. Our results corroborate previous finds where discordance between mitochondrial gene clusters, AFLP, and other data highlights the potential conflict between different “species criteria” and the well-established distinction between gene trees and species trees. In operational terms, common protocols for metabarcoding will potentially underestimate sympatric species diversity with cases like the J. albifrons complex, if the members of this complex indeed represent different species.

Phylogeography of a Widely Distributed Atlantic Species: The Case of the Ghost Crab Ocypode quadrata (Fabricius, 1787) (Decapoda: Brachyura: Ocypodidae)

Ocypode Weber, 1795 (Brachyura: Ocypodidae) is popularly known as ghost crab, and encompasses 21 valid species, including Ocypode quadrata (Fabricius, 1787). This species has wide distribution along the Atlantic coast of America, from the USA (Massachusetts) to Brazil (Rio Grande do Sul), Central America, and Antilles. Such distribution, along with some biological characteristics of its life cycle and the presence of geographic barriers, could lead to genetic structuring. Herein, we evaluate the hypothesis of the presence of geographic barriers using COI and 16S partial gene fragments. The Maximum Likelihood tree suggests the monophyly of O. quadrata, while the values of intraspecific genetic distance along with the star-shaped haplotype network suggested a lack of genetic structure in Brazilian, Panama, and French Guiana populations, probably caused by larval dispersion. USA and Mexico populations may be a new lineage, but we cannot say it with few sequences and with no morphological characters.

Identification of fish specimens of the Tocantins River, Brazil, using DNA barcoding.

The fish fauna of the Tocantins River possesses many endemic species; however, it is little studied in molecular terms and is quite threatened by the construction of several hydroelectric dams. Therefore, the objective of this study was to identify the ichthyofauna of the Tocantins River using DNA barcoding. For this, collections were carried out in five points of this river, which resulted in the capture of 725 individuals from which partial sequences of the cytochrome oxidase subunit I (COI) gene were obtained for genetic analysis. A total of 443 haplotypes were recovered with the mean intraspecific K2P genetic distance of 1.82%. Altogether, 138 species were identified based on morphological criteria, which was a quantity that was much lower than that indicated by the four molecular methods (assemble species by automatic partitioning [ASAP], barcode index number [BIN], generalized mixed Yule coalescent (GMYC), and Bayesian Poisson tree processes [bPTP]) through which 152-157 molecular entities were identified. In all, 41 unique BINs were obtained based on the data generated in the BOLDSystems platform. According to the result indicated by ASAP (species delimitation approach considered the most appropriate in the present study), there was an increase of 17 molecular entities (12.32%), when compared to the number of species identified through their morphological criteria, as it can show cryptic diversity, candidates for new species, and misidentifications. There were 21 incongruities indicated between the different identification approaches for species. Therefore, it is suggested that these taxonomic problems be cautiously evaluated by experts to solve such taxonomic issues.

Violins we see, species we don’t… Species delimitation of the spider genus Loxosceles Heineken &amp; Lowe (Araneae: Sicariidae) from North America using morphological and molecular evidence

In modern systematics, different sources of evidence are commonly used for the discovery, identification, and delimitation of species, especially when morphology fails to delineate between species or in underestimated species complexes or cryptic species. In this study, morphological data and two DNA barcoding markers—cytochrome c oxidase subunit I (COI) and internal transcribed spacer 2 (ITS2)—were used to delimit species in the spider genus Loxosceles from North America. The molecular species delimitation analyses were carried out using three different methods under the corrected p-distance Neighbor-Joining (NJ) criteria: 1) Assemble Species by Automatic Partitioning (ASAP), 2) General Mixed Yule Coalescent model (GMYC), and 3) Bayesian Poisson Tree Processes (bPTP). The analyses incorporated 192 terminals corresponding to 43 putative species of Loxosceles, of which 15 are newly recognized herein, as putative new species, based on morphology and congruence between molecular methods with COI. The average intraspecific genetic distance (p-distance) was &lt;2%, whereas the average interspecific genetic distance was 15.6%. The GMYC and bPTP molecular methods recovered 65-79 and 69 species respectively, overestimating the diversity in comparison with morphology, whereas the ASAP method delimited 60 species. The morphology of primary sexual structures (males palps and female seminal receptacles) was congruent with most of the molecular methods mainly with COI, showing that they are robust characters for identification at the species level. For species delimitation COI was more informative than ITS2. The diversity of Loxosceles species is still underestimated for North America, particularly in Mexico which holds the highest diversity of this genus worldwide.

Molecular characterisation of Amblyomma integrum circulating in southern India

Amblyomma integrum is a large gooseberry sized longirostrate tick (when fully repleted) found in India and Sri Lanka. In Kerala (India), this tick is commonly found in the forest and its fringe areas frequently infesting deer and hence it is locally known as “maan chellu / maanunny” (deer tick). In the present study, molecular characterisation and phylogenetic analysis of A. integrum collected from the area grazed by the sambar deer (Rusa unicolor) of Kerala, south India was performed using three molecular markers viz., the mitochondrial cytochrome c oxidase subunit 1 (COI), mitochondrial 16S ribosomal RNA, and nuclear 18S ribosomal RNA genes. Cytochrome c oxidase subunit 1 (COI) gene showed better resolving ability for elucidating the evolutionary relationship of A. integrum and identified two distinct clades, viz., A and B. The Tamil Nadu isolates of south India and Marayoor isolate 1 (from Idukki district of Kerala bordering with Tamil Nadu) belonged to clade A. Majority of Wayanad isolates from Kerala, occupied clade B. The intraspecific genetic distance among the A. integrum species ranged from 0.00 to 13.34%. Between clades A and B, the genetic distance observed was 11.49%. The clade B isolates were genetically close to A. geoemydae (GD: 1.22%). Morphological variations between the clades included darker exoskeletal coloration in clade A and distinct differences in the shape of basis capitulum. Further analysis using Assemble Species by Automatic Partitioning (ASAP) and Generalized Mixed Yule Coalescent (GMYC) provided additional insights. Assemble Species by Automatic Partitioning (ASAP) identified 26 Molecular Operational Taxonomic Units (MOTUs) at a threshold distance of 5.38%, supporting the species partition of A. integrum clade B. Generalized Mixed Yule Coalescent (GMYC) analysis retained the same species complex (A. integrum-geoemydae Complex) inferred from the ASAP analyses. It could be inferred from the present study that the A. integrum clades A and B could be two different putative pseudocryptic species.

Mitogenomes Provide Insights into the Species Boundaries and Phylogenetic Relationships among Three Dolycoris Sloe Bugs (Hemiptera: Pentatomidae) from China.

(1) Background: The three sloe bugs, Dolycoris baccarum, Dolycoris indicus, and Dolycoris penicillatus, are found in the Chinese mainland and are morphologically similar. The species boundaries and phylogenetic relationships of the three species remain uncertain; (2) Methods: In this study, we generated multiple mitochondrial genomes (mitogenomes) for each of the three species and conducted comparative mitogenomic analysis, species delimitation, and phylogenetic analysis based on these data; (3) Results: Mitogenomes of the three Dolycoris species are conserved in nucleotide composition, gene arrangement, and codon usage. All protein-coding genes (PCGs) were found to be under purifying selection, and the ND4 evolved at the fastest rate. Most species delimitation analyses based on the COI gene and the concatenated 13 PCGs retrieved three operational taxonomic units (OTUs), which corresponded well with the three Dolycoris species identified based on morphological characters. A clear-cut barcode gap was discovered between the interspecific and intraspecific genetic distances of the three Dolycoris species. Phylogenetic analyses strongly supported the monophyly of Dolycoris, with interspecific relationship inferred as (D. indicus + (D. baccarum + D. penicillatus)); (4) Conclusions: Our study provides the first insight into the species boundaries and phylogenetic relationships of the three Dolycoris species distributed across the Chinese mainland.

DNA barcoding, identification, and validation of the pufferfish (Order: Tetraodontiformes) in China coastal waters.

The order Tetraodontiformes are one of the most unique groups of teleostean fish, exhibiting highly derived and greatly diversified phenotypes. It is a difficult task for both professionals and nonprofessionals to accurately identify these species only according to morphological characteristics. DNA barcoding can identify species at the molecular level to overcome the limitations of morphological classification. In this study, we collected 616 specimens of pufferfish from the coastal waters of China. According to the morphological characteristics, they were preliminarily identified as 50 species. Further analysis using DNA barcodes identified these specimens as 46 species, belonging to 23 genera, 6 families. According to the species classification results of DNA barcoding, the three species identified by morphology as Takifugu pseudommus, Takifugu chinensis, and Takifugu rubripes should be the same species. Similarly, Lagocephalus wheeleri is the synonym of Lagocephalus spadiceus. Another important discovery of DNA barcoding analysis is that there are closer interspecific genetic distances within the genus Takifugu. If T. rubripes, T. pseudommus, and T. chinensis are taken as one species, the average interspecific to intraspecific genetic distance ratio of Takifugu is only 6.21 times, which does not reach the DNA barcoding threshold of more than 10 times proposed previously. Although the interspecific genetic distance in the genus Takifugu is relatively small, each species can be clustered into independent clades in the NJ tree. In conclusion, this study not only found that there are synonymous phenomena in the order Tetraodontiformes but also provided molecular evidence for the valid species names of Takifugu rubripes and Lagocephalus Spadiceus. The results can provide reliable DNA barcoding information for the identification of pufferfish species, help solve the problem of classification confusion in this order, and provide technical support for the identification of the original components of related commodities on the aquatic product market.

Screening of avian pathogens and genetic analysis of several mitochondrial genes in Dermanyssus gallinae populations in Türkiye&lt;/p &gt;

The poultry red mite, PRM, Dermanyssus gallinae (De Geer, 1778), is widely regarded as the primary ectoparasite affecting egg-laying hens around the world. Besides direct feeding damage, the transmission of avian pathogens by PRMs is also a significant concern. Therefore, we aimed to screen the presence of 14 common avian pathogens in 32 PRM populations from egg-laying hens in Türkiye. As a result, Salmonella enterica (n=3), Tsukamurella paurometabola (n=18), and Avian fowlpox virus (n=4) (3 of which were wild-type APV) were detected in tested PRM populations. The results showed that the prevalence of avian pathogens in Turkish PRM populations was low. Nevertheless, these findings suggested that PRM can harbor disease-causing agents and may act as a vector in the spread of these pathogens in farms. In addition, four mitochondrial genes (mtDNAs) of D. gallinae, namely ND4, ND5, cytb, and atp6, were amplified, and obtained sequences were submitted to the GenBank database for the first time, which will allow further analyses. The genetic variation within cytb sequences of Turkish PRMs was the highest (1.01%), whereas intra-specific genetic distance was 1.7% when compared to a Chinese population (only available cytb sequence in the GenBank), indicating increasing genetic differentiation by distance. Last, more sequences belonging to mitochondrial genes are required to reveal their potential as molecular markers.

DNA Barcoding of Catfishes (Order: Siluriformes) with New Records of Two Species from Eastern Nepal

The freshwater bodies of Nepal host a great deal of fish diversity; however, the molecular study of Nepalese fish is still in its early stages. Conservation management tactics require knowledge of the real species composition, often impeded by descriptions of intermittent species based on traditional taxonomy. Catfish, one of the most overfished taxa on the planet, are abundant and heavily exploited in the eastern region of Nepal. Hence, actual catfish diversity in this region needs to be examined using a combined morphological and molecular DNA barcoding approach. In this study, we sequenced the cytochrome oxidase subunit I (COI) gene in Nepalese fish species for the first time and used it for molecular identification. Altogether, 99 specimens representing 29 species belonging to 21 genera and 8 families were successfully barcoded. They were found to be 98.5–100% identical to the known sequences deposited in the GenBank and BOLD databases. Of these, two species, Amblyceps arunachalense and Erethistoides sicula, were recorded for the first time in Nepal. Out of 29 species, one species was found to be Endangered, one species under Vulnerable, two species under Near Threatened, one species under Data Deficient, and 24 species under Least Concern. The intraspecific genetic distances within species ranged from 0 to 5%. The highest intraspecific genetic distances (Kimura 2‐parameter) were found among the individuals of two species, namely, E. sicula (4%) and Glyptothorax telchitta (5%), indicating the presence of putative (sub)species. The interspecific genetic distances among the catfishes in eastern Nepal ranged between 8.47% (between Glyptothorax trilineatus and G. cavia) and 26.38% (between Amblyceps arunachalense and Chaca chaca). The GC content at the 1st, 2nd, and 3rd positions of the codons was found to be 42.4%, 49.3%, and 43.8%, respectively. These findings will support future initiatives for fish resource inventory, monitoring, and management. It is also an important input to the DNA barcode library of catfish in the eastern region of Nepal.

Delimiting Species with Single-Locus DNA Sequences.

DNA sequences are increasingly used for large-scale biodiversity inventories. Because these genetic data avoid the time-consuming initial sorting of specimens based on their phenotypic attributes, they have been recently incorporated into taxonomic workflows for overlooked and diverse taxa. Major statistical developments have accompanied this new practice, and several models have been proposed to delimit species with single-locus DNA sequences. However, proposed approaches to date make different assumptions regarding taxon lineage history, leading to strong discordance whenever comparisons are made among methods. Distance-based methods, such as Automatic Barcode Gap Discovery (ABGD) and Assemble Species by Automatic Partitioning (ASAP), rely on the detection of a barcode gap (i.e., the lack of overlap in the distributions of intraspecific and interspecific genetic distances) and the associated threshold in genetic distances. Network-based methods, as exemplified by the REfined Single Linkage (RESL) algorithm for the generation of Barcode Index Numbers (BINs), use connectivity statistics to hierarchically cluster-related haplotypes into molecular operational taxonomic units (MOTUs) which serve as species proxies. Tree-based methods, including Poisson Tree Processes (PTP) and the General Mixed Yule Coalescent (GMYC), fit statistical models to phylogenetic trees by maximum likelihood or Bayesian frameworks.Multiple webservers and stand-alone versions of these methods are now available, complicating decision-making regarding the most appropriate approach to use for a given taxon of interest. For instance, tree-based methods require an initial phylogenetic reconstruction, and multiple options are now available for this purpose such as RAxML and BEAST. Across all examined species delimitation methods, judicious parameter setting is paramount, as different model parameterizations can lead to differing conclusions. The objective of this chapter is to guide users step-by-step through all the procedures involved for each of these methods, while aggregating all necessary information required to conduct these analyses. The "Materials" section details how to prepare and format input files, including options to align sequences and conduct tree reconstruction with Maximum Likelihood and Bayesian inference. The Methods section presents the procedure and options available to conduct species delimitation analyses, including distance-, network-, and tree-based models. Finally, limits and future developments are discussed in the Notes section. Most importantly, species delimitation methods discussed herein are categorized based on five indicators: reliability, availability, scalability, understandability, and usability, all of which are fundamental properties needed for any approach to gain unanimous adoption within the DNA barcoding community moving forward.

Can Bergmann’s Rule and the Thermal Melanism Hypothesis explain the variation in colour and size observed in the wild bee Eoanthidium insulare (Apoidea: Megachilidae) across its Palaearctic range?

To analyse geographic variation patterns and assess the applicability of biogeographical rules in insects, the West Palaearctic anthidiine bee Eoanthidium insulare serves as an excellent model. This species showcases a highly variable black-and-yellow colour pattern, with a distribution spanning over 4400 km from the Adriatic Sea to the Pamir Mountains and extending over 1660 km in a south-north direction. We examined 214 specimens from all geographic regions of its distribution and identified a significant increase in both body size and the proportion of black in the integument from south to north. This phenomenon may be attributed to a more efficient exploitation of solar energy through a larger integument surface area (Bergmann’s Rule) and enhanced heat retention due to lower skin reflectance (Thermal Melanism Hypothesis). These two processes, therefore, complement each other. However, despite the cooler climates at higher altitudes, we found no correlations between body size and altitude. Furthermore, while we observed an increase in the proportion of black in the integument from east to west, there was no significant increase in body size. Notably, no correlation between body size and body colouration was identified, suggesting that size and colour adaptation are independent processes. Given that the various colour and size patterns were identified as clinal rather than discontinuous, and the intraspecific genetic distance across the distribution area was remarkably low (0.00138), featuring only four distinct haplotypes of the mitochondrial COI region DNA sequence, we suggest that the subspecies E. i. persicolum and E. i. lemesium be regarded as synonymous with the nominate subspecies (syn. nov.). These findings suggest that, although Bergmann’s Rule and the Thermal Melanism Hypothesis can account for certain aspects of geographic variation in the body size and colouration of E. insulare, their effects may be influenced or superimposed by factors such as genetic constitution.

First Record Sparus aurata Larvae in Teluk Penyu Beach Cilacap, Indonesia, revealed by DNA Barcoding

Morphological identification often fails to reveal the taxonomic status of fish larvae. DNA barcoding using particular DNA segment is frequently successful in solving the faulty and might reveal overlooked species, including species outside their recognized geographic ranges, such as Eastern Atlantic seabream Sparus aurata. This study aims to assess fish larvae diversity in Teluk Penyu Beach, Cilacap Central Java, Indonesia, through the cytochrome c oxidase 1 barcoding. Fish larvae were collected using a larva net with a diameter of 60 cm and height of 150 cm and horizontal towing during the field trips in March and April 2021. Larvae morphotypes were determined based on their general morphological performance observed under a magnification lens according the available references. Eighteen larvae morphotypes were successfully barcoded, and 5% genetic distance was used as a species border. Fourteen fish larvae species were revealed, with intraspecific genetic distances between 0.00% and 4.12%, while intrageneric genetic distances ranged from 5.50% to 19.29%. An interesting finding was that one larva morphotype was barcoded as S. aurata with high genetic identity (99.19% to 99.68%) and low genetic distance (0.32% to 0.81%). The discovery provides the first new data on S. aurata in Teluk Penyu Beach Cilacap, Central Java, outside its recognized geographic range. Overall, this study provides information about fish larvae in Teluk Penyu Beach, essential for estimating the number and species of fish recruited in the southern Cilacap, which is vital for fisheries management. Nevertheless, new research requires further clarification of S. aurata in Teluk Penyu Beach.