Monophyletic Research Articles

A new species of Hypancistrus Isbrücker & Nijssen 1991 (Loricariidae, Siluriformes) from the rapids of the middle Rio Tocantins.

The Hypancistrus genus is recognized in the Río Orinoco basin and Rio Xingu in the Guiana and Brazilian Shields, respectively. Some of its species are important in ornamental fishing. Despite this significance, many other undescribed species are still awaiting to be named. Here we describe a new species of Hypancistrus found on bedrock in the Rio Tocantins, representing an extension of the distribution of the genus. Also, a multigene phylogeny is presented to evaluate the taxonomic position of this species concerning congeners. The new species differs from all congeners by (1) hypertrophied odontodes on cheeks reaching beyond the cleithrum, (2) a supraoccipital crest conspicuously elevated, (3) a supraorbital crest slightly convex, (4) oblique bars on the anterior part of the body, (5) a dark E-shaped mark on the snout, (6) three oblique dark bars on the anterior part of the body and horizontal vermicular bars from the pectoral girdle to the posterior insertion of the dorsal fin, (7) a thin light gray bar on the posterior of the head extending across the branchial opening, (8) a tan background color, (9) a developed suspensorium with a diminished appendix in the metapterygoid, and (10) a dentary plate robust significantly fused with the angulo-articular bone. The molecular phylogenetic results show the new species forming a group with Hypancistrus zebra (Brazilian Shield-Rio Xingu) as a clade, a sister group of a monophyletic group consisting of all congeners from the Río Orinoco.

Identification and characterization of a novel Pavlovskyella (Acari: Argasidae) from Chile, parasite of the culpeo fox (Lycalopex culpaeus).

Soft ticks (Argasidae) of the subgenus Pavlovskyella Pospelova-Shtrom are worldwide distributed parasites of medical importance. However, the systematics of the subgenus are currently under debate because genetic data shows that the group is paraphyletic. Meanwhile, species of Pavlovskyella continue to be discovered. In this study a novel species of the subgenus is described from specimens collected on a fox in central Chile. The larva of this new species differentiates from other Pavlovskyella spp. by having the following combination of characters: subpyriform dorsal plate; 15 pairs of setae, 7 anterolateral, 3 central, and 5 posterolateral, and hypostome with denticles in the distal third. Nymphs and adults of the species lack cheeks, eyes or bulging structures on the flank, but exhibit dorsoventral grooves, and humps on tarsi I, II, and III. Moreover, a patch of glabrous integument appears on the distal portion of coxal folds. A phylogenetic analysis using the mitogenome indicates a monophyletic group composed by Ornithodoros (Pavlovskyella) brasiliensis Aragão, Ornithodoros (Pavlovskyella) furcosus Neumann, Ornithodoros (Pavlovskyella) improvisus Muñoz-Leal & Venzal, and Ornithodoros (Pavlovskyella) rostratus Aragão. Interestingly, a phylogeny using 18S-28S rDNA sequences shows that South American Pavlovskyella spp. are paraphyletic, as depicted in previous studies. Adding species of the subgenus from the Neotropical region to phylogenetic analyses could aid to solve this paraphyly. Furthermore, this is the fifth species of Pavlovskyella described in South America, and the second in Chile.

New mitochondrial genomes of three whip spider species from the Amazon (Arachnida, Amblypygi) with phylogenetic relationships and comparative analysis

The complete mitochondrial genomes of the whip spiders Charinus carajas, C. ferreus, and Heterophrynus longicornis were sequenced, annotated, and compared with other mitogenomes of whip spiders and arachnids. The three new mitogenomes have the 37 genes usually observed in Metazoa: 13 protein-coding genes (PCGs), 22 transfer RNAs (tRNAs), and two ribosomal RNAs (rRNAs), plus a non-coding control region (CR). Most PCGs presented an ATN start codon, except cox1 in both Charinus species, initiating with TTA. Most PCGs terminated with stop codons TAA or TAG, except nad5 of C. carajas and cox3 of H. longicornis, which presented an incomplete stop codon (T). The Ka/Ks ratios were less than one for all the PCGs, indicating these genes are under purifying selection. All the tRNAs, except for serine 1 (trnS1), had the typical cloverleaf-shaped secondary structure. All the phylogenetic analyses resolved Charinus carajas and C. ferreus as monophyletic groups. Nonetheless, we did not recover the monophyly of Heterophrynus longicornis. The phylogenies under partitioned models did not recover suprageneric taxonomic groups as clades, but the Bayesian inference under the CAT infinite mixture model recovered the family Phrynidae and the superfamily Phrynoidea as monophyletic groups.

Image-Based Quantitative Analysis of Epidermal Morphology in Wild Potato Leaves

The epidermal leaf patterns of plants exhibit remarkable diversity in cell shapes, sizes, and arrangements, driven by environmental interactions that lead to significant adaptive changes even among closely related species. The Solanaceae family, known for its high diversity of adaptive epidermal structures, has traditionally been studied using qualitative phenotypic descriptions. To advance this, we developed a workflow combining multi-scale computer vision, image processing, and data analysis to extract digital descriptors for leaf epidermal cell morphology. Applied to nine wild potato species, this workflow quantified key morphological parameters, identifying descriptors for trichomes, stomata, and pavement cells, and revealing interdependencies among these traits. Principal component analysis (PCA) highlighted two main axes, accounting for 45% and 21% of variance, corresponding to features such as guard cell shape, trichome length, stomatal density, and trichome density. These axes aligned well with the historical and geographical origins of the species, separating southern from Central American species, and forming distinct clusters for monophyletic groups. This workflow thus establishes a quantitative foundation for investigating leaf epidermal cell morphology within phylogenetic and geographic contexts.

A Novel Hyperactive Variant of the Sleeping Beauty Transposase Facilitates Non-Viral Genome Engineering

The Sleeping Beauty (SB) transposon system is a useful tool for genetic applications, including gene therapy. We discovered a hyperactive variant of the SB100X transposase, called SB200X. This mutant, resulting from a specific amino acid replacement (Q124C), showed a ∼2-fold increase in transposition activity in various human and murine cells. Other amino acid replacements in position 124 also led to a hyperactive phenotype. Position 124 is located at the very edge of the linker region that connects the DNA-binding and catalytic domains of the transposase. Consistent with a role of the linker in an autoregulatory mechanism called overproduction inhibition (OPI) in the monophyletic group of mariner transposases, we show that the hyperactivity of Q124C manifests at high concentrations of the transposase, suggesting a partial resistance of SB200X to OPI. We demonstrate that the hyperactive phenotype of Q124C can be combined with features of other useful mutations in the SB transposase. Namely, Q124C improves the transposition efficiency of the previously described K248R variant, while maintaining or even slightly improving its safer genome-wide integration profile. SB200X transposase could enhance the utility of SB transposon-mediated genome engineering in preclinical and clinical applications.

Taxonomic revision of the Vitex trifolia complex (Lamiaceae)

The Vitex trifolia complex is a well-circumscribed and monophyletic group. However, the species of the group are morphologically very similar and therefore, taxonomic boundaries within the group are uncertain. In addition, these medicinal plants have been cultivated and naturalised in many areas across the world and therefore the irnatural geographical distributions are not well known. A taxonomic account of the complexis presented in which nine species are recognized: Vitex agnus-castus, V. benthamiana, V. bicolor, V. negundo, V. pseudonegundo, V. rotundifolia and V. trifolia; a new species, Vitex collium Sengun, is described from frost prone regions of northern and Central China and an obscure name, V. hybrida Moldenke, is recognised for the Indian specimens previously recognized as V. negundo. In addition, Agnus-castus, V. cannabifolia, V. chinensis, V. latifolia, V. negundo f. alba, V. negundo f. intermedia, V. negundo f. laxipaniculata, V. rotundifolia and V. trifolia var. acutifolia are lectotypified, and V. leucoxylon Blanco has been neotypified. Also, many names have been placed in synonomy for the first time.

χ-Conotoxins are an evolutionary innovation of mollusk-hunting cone snails as a counter-adaptation to prey defense.

Mollusk-hunting (molluscivorous) cone snails belong to a monophyletic group in Conus, a genus of venomous marine snails. The molluscivorous lineage evolved from ancestral worm-hunting (vermivorous) snails ∼18 million years ago. To enable the shift to a molluscivorous lifestyle, molluscivorous cone snails must solve biological problems encountered when hunting other gastropods, namely: 1) preventing prey escape and 2) overcoming the formidable defense of the prey in the form of the molluscan shell, a problem unique to molluscivorous Conus. Here we show that χ-conotoxins, peptides exclusively expressed in the venoms of molluscivorous Conus, provide solutions to the above problems. Injecting χ-conotoxins into the gastropod mollusk Aplysia californica results in impaired locomotion and uncoordinated hyperactivity. Impaired locomotion impedes escape and a hyperactive snail will likely emerge from its shell, negating the protection the shell provides. Thus, χ-conotoxins are an evolutionary innovation that accompanied the emergence of molluscivory in Conus and provide solutions to problems posed by hunting other snails.

Hymenochaete ametzii sp. nov. (Hymenochaetales, Basidiomycota), an endangered bark-dwelling species inhabiting old Quercus pyrenaica trees from the Iberian Peninsula

Bark-dwelling fungi represent a group of ecologically highly specialized organisms. This study deals with an undescribed species of Hymenochaete characterized by specifically inhabiting the bark of Pyrenean oak (Quercus pyrenaica), and producing effuse-reflexed basidiomata and mainly globose to subglobose basidiospores. Maximum Likelihood and Bayesian analyses of the nuclear ITS-LSU regions revealed that the sequences of H. ametzii form a monophyletic group with a low intraspecific variation and substantially different from closest taxa, further supporting its recognition as a species. The seven localities H. ametzii is known from are old forests with a long ecological continuity, containing large old Q. pyrenaica trees, and are located in the supramediterranean belt of the Mediterranean biogeographical region. Based on habitat availability and field counts of colonized trees, the global population of H. ametzii is estimated at 8,670 mature individuals. Considering that the traditional use of old Q. pyrenaica stands ceased several decades ago in the Iberian Peninsula, with a consequent decline in habitat quality and availability, it is concluded that H. ametzii is Endangered (EN) according to the IUCN criteria.

Genomic epidemiology and evolutionary dynamics of the Omicron variant of SARS-CoV-2 during the fifth wave of COVID-19 in Pakistan.

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has posed extraordinary challenges to global health systems and economies. The virus's rapid evolution has resulted in several variants of concern (VOCs), including the highly transmissible Omicron variant, characterized by extensive mutations. In this study, we investigated the genetic diversity, population differentiation, and evolutionary dynamics of the Omicron VOC during the fifth wave of COVID-19 in Pakistan. A total of 954 Omicron genomes sequenced during the fifth wave of COVID-19 in Pakistan were analyzed. A Bayesian framework was employed for phylogenetic reconstructions, molecular dating, and population dynamics analysis. Using a population genomics approach, we analyzed Pakistani Omicron samples, revealing low within-population genetic diversity and significant structural variation in the spike (S) protein. Phylogenetic analysis showed that the Omicron variant in Pakistan originated from two distinct lineages, BA.1 and BA.2, which were introduced from South Africa, Thailand, Spain, and Belgium. Omicron-specific mutations, including those in the receptor-binding domain, were identified. The estimated molecular evolutionary rate was 2.562E-3 mutations per site per year (95% HPD interval: 8.8067E-4 to 4.1462E-3). Bayesian skyline plot analysis indicated a significant population expansion at the end of 2021, coinciding with the global Omicron outbreak. Comparative analysis with other VOCs showed Omicron as a highly divergent, monophyletic group, suggesting a unique evolutionary pathway. This study provides a comprehensive overview of Omicron's genetic diversity, genomic epidemiology, and evolutionary dynamics in Pakistan, emphasizing the need for global collaboration in monitoring variants and enhancing pandemic preparedness.

Comparative and phylogenetic analysis of the chloroplast genomes of four commonly used medicinal cultivars of Chrysanthemums morifolium

‘Boju’ and ‘Huaiju’ are cultivars of the Chrysanthemum (Chrysanthemum morifolium Ramat.) in the family Asteraceae, valued for their medicinal, tea, and ornamental properties, and valued by individuals. However, the yield and quality of medicinal chrysanthemums are limited by the characteristics of the germplasm resources, including the identification at the varieties and cultivation levels. Currently, research characterizing the chloroplast genomes of medicinal Chrysanthemum flowers is relatively limited. This study conducted chloroplast whole-genome sequencing on two cultivars of Chrysanthemum, ‘Boju’ and ‘Huaiju’, and compared them with the previously published chloroplast genomes of ‘Hangbaiju’ and ‘Gongju’. The study analyzed the chloroplast genome structures of these four medicinal Chrysanthemums, identifying mutation hotspots and clarifying their phylogenetic relationships. The chloroplast genome sizes of four medicinal Chrysanthemum cultivation products ranged from 151,057 to 151,109 bp, with GC content ranging from 37.45% to 37.76%. A total of 134 genes were identified, including 89 protein-coding genes, 37 ribosomal RNA genes, and 8 transfer RNA genes. Comparative genomic analysis revealed 159 large repeat sequences, 276 simple sequence repeats, 1 gene, and 8 intergenic regions identified as highly variable regions. Nucleotide diversity (Pi) values were high (≥ 0.004) for the petN-psbM, trnR-UCU-trnT-GGU, trnT-GGU-psbD, ndhC-trnV-UCA, ycf1, ndhI-ndhG, trnL-UGA-rpl32, rpl32-ndhF, and ndhF-ycf1 fragments, aiding in variety identification. Phylogenetic analysis revealed consistent results between maximum likelihood and Bayesian inference trees, showing that the four medicinal Chrysanthemum cultivars, along with their wild counterparts and related species, evolved as a monophyletic group, forming a sister clade to Artemisia and Ajania. Among the six Chrysanthemum species, the wild Chrysanthemum diverged first (Posterior probability = 1, bootstrap = 1,000), followed by Ajania, while C. indicum and C. morifolium clustered together (Bootstrap = 100), indicating their closest genetic relationship. The chloroplast whole-genome data and characteristic information provided in this study can be used for variety identification, genetic conservation, and phylogenetic analysis within the family Asteraceae.

Molecular phylogeny reveals a new genus and species of freshwater mussel (Bivalvia, Unionidae, Gonideinae) from Jiangxi, China

Freshwater mussels of the tribe Gonideini (Bivalvia: Unionidae: Gonideinae) constitute one of the most taxonomically diverse groups and serve as keystone species in riverine and lacustrine ecosystems across East Asia. A new genus and species of Gonideini (Bivalvia: Unionidae) is described from Jiangxi, China, as Pseudopostolata angulagen. et sp. nov. based on an integrative analysis of shell morphology and molecular data. The validity of the new genus and species is supported by distinct conchological features: a short, rounded anterior; a long, wide posterior; a slightly downward-curved dorsal margin; and a distinctly obtuse angle at the center of the posterior margin. The multi-locus (COI + 16S rRNA + 28S rRNA) phylogeny showed that the species formed a monophyletic group in the tribe Gonideini of the subfamily Gonideinae. Pseudopostolata angulagen. et sp. nov. is identified as the sister group to a clade comprising the genera Postolata, Cosmopseudodon, Obovalis, Ptychorhynchus, Parvasolenaia, and Koreosolenaia. We emphasize the significant morphological convergence in freshwater mussels, particularly within Gonideini, highlighting the necessity of an integrated taxonomic approach for accurate generic classification of this group.

Evidence for a Miocene pulse of diversification of the tropical American clade of the Brazil nut family (Lecythidaceae)

ABSTRACT The tropical American species of the Brazil nut family, Lecythidaceae, are abundant in the tree flora, with some economically important species. Yet, the phylogenetic relationships and pace of diversification within this family are understudied. Here, we used shotgun sequencing data for 86 of the 228 currently accepted species in the exclusively tropical American subfamily Lecythidoideae in a phylogenomic context. For each sampled species, we built the full plastid DNA sequence, and also extracted nuclear DNA for 571 regions using the new bioinformatics pipeline REFMAKER, which we used to produce a time-calibrated phylogenetic hypothesis. Our analysis shows that phylogenetic inference from plastid and nuclear DNA alignments resulted in different topologies. The nuclear DNA topology strongly suggests that genus Lecythis should be split into at least four genera. Samples of the genus Eschweilera formed a monophyletic group, with the exception of one sampled species (Eschweilera amazoniciformis S.A.Mori). The Bertholletia clade, which contains the majority of the Lecythidoideae species, and all Lecythis and Eschweilera species, started diversifying around 27.5 Ma, with an accelerated rate of diversification starting in the middle Miocene (c. 12 Ma). The clade sister to Bertholletia (including Corythophora, Eschweilera, and Corrugata, Chartacea and Poiteaui clades) includes at least 124 species and it has diversified less than 10 Ma. This time frame of diversification coincides with major changes in tropical American landscapes and climate associated with the Andean uplift.

The highly repetitive genome of Myxobolus rasmusseni, an emerging myxozoan parasite of fathead minnows.

Myxozoans are a monophyletic taxon of approximately 2,400 described species of parasites from the phylum Cnidaria. The recent focus on their negative impacts on fisheries, on their evolution from free-living ancestors, and on their emergence into new fish host populations has stressed the critical need for genomic resources for this parasitic group. Here, we describe the genome assembly and annotation of Myxobolus rasmusseni, an emerging parasite of fathead minnows in Alberta, Canada. The assembly is 174.6 Mb in size, 68% of which is made up of repetitive elements, making it one of the most repetitive animal genomes sequenced to date. Through comparisons to other myxozoans, we show that widespread gene loss, a known phenomenon of this group of parasites, is consistent with closely related species. Additionally, we assembled the M. rasmusseni mitochondrial genome which is nearly twice the size of the typical animal mitochondrial genome yet contains only five of the canonical mitochondrial protein-coding genes and open reading frames not found in other myxozoans. These results add to our understanding of the gene- and genome-level diversity observed in myxozoans.

Three new species of the genus ‘Carteria’ (Chlamydomonadales, Chlorophyceae) with the proposal of correct generic names

ABSTRACT ‘Carteria’ is a genus of quadriflagellate chlamydomonadalean green algae, however this genus is not thought to be a monophyletic group. In this study, we investigated nine strains of ‘Carteria’ newly isolated from China by morphological comparisons and molecular phylogeny of 18S rDNA and rbcL genes. Five strains could be identified as existing ‘Carteria’ species. Four strains with clearly different morphology and independent phylogenetic positions compared with the described ‘Carteria’ species were identified as three new species. Since the original type species, Carteria cordiformis, has been recognized as a prasinophyte alga, the generic name Carteria cannot be applied to other species previously assigned to ‘Carteria’. Six ‘Carteria’ species (including three new species) and one Pseudocarteria species of Carteria-Clade I were transferred to the genus Corbierea, and the description of this genus was emended. A new genus, Staurocarteria gen. nov. was proposed for Carteria-Clade II.

Three new species of Camaena Albers, 1850 (Gastropoda, Stylommatophora, Camaenidae) from Guizhou and Guangxi, China

AbstractThree new species of camaenid land snail, Camaena qiannanensis Chen, Dai, Wu & Ouyang sp. n., Camaena feichenyui Chen, Dai, Wu & Ouyang sp. n. and Camaena qinghuai Chen, Dai, Wu & Ouyang sp. n. are described from Guizhou and Guangxi of southern China based on shell and genital morphology, and molecular phylogeny. The phylogenetic result shows that the genus Camaena is not a monophyletic group, with Camaenella located within it. The discovery enhances the species diversity of Camaena in Southern China and fills the gap between distribution areas of known species.

The complete chloroplast genome of Erodium cicutarium (Linnaeus) l’ Héritier ex Aiton 1789 (Geraniaceae): genome characterization and phylogenetic consideration

Erodium cicutarium is an annual herbaceous plant valued for its applications in traditional medicine. However, the chloroplast genome of E. cicutarium has yet to be reported. In this study, we assembled chloroplast genomes of Erodium cicutarium using Illumina sequencing reads. The chloroplast genome was 114,652 bp long, harbored 111 complete genes, and its overall GC content was 39.1%. In Maximum Likelihood (ML) and Bayesian inference (BI) trees, the 13 Erodium species divided into three main clades, with E. cicutarium and E. carvifolium forming a monophyletic group, suggesting a close relationship between the two species. The E. cicutarium cp genome presented in this study lays a good foundation for the Erodium.

The complete chloroplast genome of Fuchsia standishii J. Harrison, 1840 (Onagraceae) from Yunnan, China

Fuchsia standishii J. Harrison, 1840, a perennial shrub, is renowned for its vividly colored and uniquely shaped blooms, which have an extended flowering season. Commonly cultivated as an ornamental potted plant, it is utilized in traditional Chinese medicine. In this study, we successfully sequenced and assembled the complete chloroplast genome of F. standishii using high-throughput Illumina sequencing technology. The assembled chloroplast genome displays a typical quadripartite structure, with a total length of 156,391 bp. It consists of a pair of inverted repeat regions (IRs), each measuring 25,069 bp, separated by a large single-copy region (LSC) of 87,754 bp and a small single-copy region (SSC) of 18,499 bp. The overall GC content of the genome is 37.60%. The genome includes a total of 129 genes, comprising 84 protein-coding genes, 37 tRNA genes, and eight rRNA genes. Phylogenetic analysis of 17 complete chloroplast genomes revealed that F. standishii forms a monophyletic group with the entire Circaea. This study provides a molecular foundation for future phylogenetic research on Fuchsia.

The complete mitochondrial genome of Chilean Jack Mackerel, Trachurus murphyi Nichols, 1920 (Teleostei, Carangidae)

The Chilean jack mackerel (Trachurus murphyi, Nichols, 1920) is a pelagic fish of high fishery interest, with a global capture of 828,358 mt in 2021. We report the complete mitochondrial genome of T. murphyi (Teleostei, Carangidae), collected in Bahia de Zapallar, Chile (32°33’02.59’’ S, 71°27’55.38’’ W). The size of the mitogenome is 16,561 bp (H-strand composition: 25.9% A, 16.7% C, 29.8% G, and 27.5% T). The mitogenome has the classic vertebrate gene content of 13 protein-coding, two rRNA, and 22 tRNA genes, as found in Carangidae and other Teleostei families. Phylogenetic analysis using mitochondrial genomes of 22 related species revealed that T. murphyi formed a well-supported monophyletic group with the other Trachurus species, being T. simmetricus its closest relative. Sequencing the mitochondrial genome from T. murphyi is the first step in developing traceability tools based on DNA analysis to enforce fishing quotas and trace the processed food and foodstuff containing Chilean jack mackerel following the objective of the South Pacific Regional Fisheries Management Organization (SPRFMO).

The complete mitochondrial genome of Taiwanaptera montana (Hemiptera: Aradidae)

Taiwanaptera montana is a apterous flat bug found in Yunnan Province, China. This study is the first to sequence, assemble, and annotate the complete mitochondrial genome of T. montana. The mitochondrial genome of T. montana has a total length of 15615 bp, which is a typical circular DNA, containing 13 protein-coding genes (PCGs), 22 tRNA genes, 2 rRNA genes and a control region, with A + T content of 69.4%. The phenomenon of gene rearrangement was found when the mitochondrial structure was compared with that of other Aradidae. The phylogenetic tree based on 37 mitochondrial genes showed that T. montana was most closely related to Libiocoris heissi. Aneurinae, Carventinae and Mezirinae are monophyletic groups. In addition, the results also confirmed that Aradinae and Calisiinae diverged earliest and were relatively primitive groups.

Characterization and phylogenetic analysis of the mitochondrial genome of Cylicostephanus longibursatus.

Cylicostephanus longibursatus is a common parasite in equine animals. Hosts infected by these nematodes might face disease or death. This study utilized next-generation sequencing technology to sequence the complete mitochondrial genome (mt genome) of C. longibursatus. Through bioinformatics techniques, the genomic base composition, codon usage, tRNA secondary structures, evolutionary relationships, and taxonomic status were analyzed. The results revealed that the mitochondrial genome of C. longibursatus is a double-stranded, 13,807-bp closed circular molecule with an AT content of 76.0%, indicating a clear preference for AT bases. The mitochondrial genome consisted of a total of 12 protein-coding genes, 22 tRNA genes, 2 rRNA genes, and 2 non-coding regions. Among the 12 protein-coding genes, TTG and ATT were the common start codons. TAA was the predominant termination codon, except for the ND3 and ND6 coding genes, and the COШ genes used TAG and "T" as termination codons, respectively. All tRNAs exhibited atypical clover-leaf secondary structures, except for tRNALys and tRNALeu2, where two tRNASer genes lacked DHU arms and DHU loops, tRNAmet lacked the TΨC-arm, tRNAIle lacked the TΨC-loop, and the remaining 16 tRNAs lacked the TΨC-arm and TΨC loop, which were substituted by the "TV-replacement loop". Phylogenetic analyses, based on the 12 protein-coding genes and utilizing maximum likelihood (ML) and Bayesian inference (BI) analyses, indicated that C. longibursatus did not form a monophyletic group with other Cylicostephanus but was instead more closely related to Cyathostomum. These research findings provide fundamental data for exploring the population classification and phylogeny of strongylid nematodes.