Protein-coding Gene Sequences Research Articles

Unraveling the phylogenetics of genetically closely related species, Haemaphysalis japonica and Haemaphysalis megaspinosa, using entire tick mitogenomes and microbiomes.

Ticks have a profound impact on public health. Haemaphysalis is one of the most widespread genera in Asia, including Japan. The taxonomy and genetic differentiation of Haemaphysalis spp. is challenging. For instance, previous studies struggled to distinguish Haemaphysalis japonica and Haemaphysalis megaspinosa due to the dearth of nucleotide sequence polymorphisms in widely used barcoding genes. The classification of H. japonica japonica and its related sub-species Haemaphysalis japonica douglasi or Haemaphysalis jezoensis is also confused due to their high morphological similarity and a lack of molecular data that support the current classification. We used mitogenomes and microbiomes of H. japonica and H. megaspinosa to gain deeper insights into the phylogenetic relationships and genetic divergence between two species. Phylogenetic analyses of concatenated nucleotide sequences of protein-coding genes and ribosomal DNA genes distinguished H. japonica and H. megaspinosa as monophyletic clades, with further subdivision within the H. japonica clade. The 16S rRNA and NAD5 genes were valuable markers for distinguishing H. japonica and H. megaspinosa. Population genetic structure analyses indicated that genetic variation within populations accounted for a large proportion of the total variation compared to variation between populations. Microbiome analyses revealed differences in alpha and beta diversity between H. japonica and H. megaspinosa: H. japonica had the higher diversity. Coxiella sp., a likely endosymbiont, was found in both Haemaphysalis species. The abundance profiles of likely endosymbionts, pathogens, and commensals differed between H. japonica and H. megaspinosa: H. megaspinosa was more diverse.

Sequencing and Analysis of the Complete Mitochondrial Genome of Lentipes ikeae.

We sequenced and analyzed the complete mitochondrial genome of Lentipes ikeae and explored the phylogenetic relationships among Sicydiinae based on mitochondrial genome sequences. The complete mitochondrial genome sequence of L. ikeae was determined using the Illumina HiSeq X Ten sequencing platform, and the gene structural characteristics and base composition were analyzed. Based on the mitochondrial genome sequences of 28 Sicydiinae species published in GenBank and mitochondrial protein-coding genes (PCGs), Acanthogobius flavimanus (Gobionellinae) was selected as an outgroup to construct phylogenetic trees of Sicydiinae using the maximum likelihood and Bayesian inference methods. The mitochondrial genome of L. ikeae (GenBank number: OP764680) has a total length of 16,498 bp and encodes 13 PCGs, 22 transfer RNA genes, two ribosomal RNA genes, and a D-loop (control) region. Gene rearrangement is not observed. The mitochondrial genome of L. ikeae exhibits an AT preference, with AT skew > 0 and GC skew < 0 across the entire genome. The phylogenetic relationships of Sicydiinae based on 13 mitochondrial PCG sequences are Sicydium + (Stiphodon + (Sicyopus + Lentipes)) + Sicyopterus, indicating that Sicydium, Sicyopterus, Lentipes, and Stiphodon are all monophyletic groups.

Compositional Features and Codon Usage Pattern of Genes Associated with Parkinson's Disease.

Codon usage bias (CUB) is the phenomenon of non-uniform usage of synonymous codons in which some codons are more used than others and it helps in understanding the molecular organization of genome. Bioinformatic approach was used to analyze the protein-coding sequences of genes associated with Parkinson's disease (PD) to explore compositional features and codon usage pattern as no details work was reported yet. The average improved effective number of codons (Nc) and Nc prime were 42.74 and 44.26 respectively, indicated that CUB was low in these genes. In most of the genes, the overall GC content was almost 50% and GC content at the 1st codon position was the highest while GC content at the 2nd codon position was lowest. Relative synonymous codon usage (RSCU) analysis elucidated over-represented (p > 1.6) and under-represented codons (p < 0.6). The GTG (Val) is the only codon over-represented in all genes. Over-represented codons except (GTG) were A or T ending while under-represented codons (except ACT) were G or C ending. The codons namely TTA (Leu), CTA (Leu), ATC (Ile), ATA (Ile), AGT (Ser), AAC (Asn), TGT (Cys), TGC (Cys), CGC (Arg), AGA (Arg), and AGG (Arg) were absent in SNCA1 to SNCA8 genes. The codon TCG (Ser) was absent in all genes except UCHL1 and PINK1. Correspondence analysis (COA) revealed that the pattern of codon usage differs among genes associated with PD. Neutrality plot analysis indicated some of the points are diagonal distribution suggested that mutation pressure influenced the CUB in genes associated with PD.

Comparative Mitogenome Analyses of Fifteen Ramshorn Snails and Insights into the Phylogeny of Planorbidae (Gastropoda: Hygrophila).

Ramshorn snails from the family Planorbidae are important freshwater snails due to their low trophic level, and some of them act as intermediate hosts for zoonotic trematodes. There are about 250 species from 40 genera of Planorbidae, but only 14 species from 5 genera (Anisus, Biomphalaria, Bulinus, Gyraulus, and Planorbella) have sequenced complete mitochondrial genomes (mitogenomes). In this study, we sequenced and assembled a high-quality mitogenome of a ramshorn snail, Polypylis sp. TS-2018, which represented the first mitogenome of the genus. The mitogenome of Polypylis sp. TS-2018 is 13,749 bp in length, which is shorter than that of most gastropods. It contains 13 protein-coding genes (PCGs), 22 transfer RNA (tRNA) genes, and 2 ribosomal RNA (rRNA). We compared mitogenome characteristics, selection pressure, and gene rearrangement among all of the available mitogenomes of ramshorn snails. We found that the nonsynonymous and synonymous substitution rates (Ka/Ks) of most PCGs indicated purifying and negative selection, except for atp8 of Anisus, Biomphalaria, and Gyraulus, which indicated positive selection. We observed that transpositions and reverse transpositions occurred on 10 tRNAs and rrnS, which resulted in six gene arrangement types. We reconstructed the phylogenetic trees using the sequences of PCGs and rRNAs and strongly supported the monophyly of each genus, as well as three tribes in Planorbidae. Both the gene rearrangement and phylogenetic results suggested that Polypylis had a close relationship with Anisus and Gyraulus, while Bulinus was the sister group to all of the other genera. Our results provide useful data for further investigation of species identification, population genetics, and phylogenetics among ramshorn snails.

Complete mitochondrial genome of larged-eye pygmy goby Trimma macrophthalmus (Teleostei, Gobiidae) and its phylogenetic implications

Trimma macrophthalmus is a colorful pygmy goby that is widely associated with coral and distributed in the Indo-Pacific Ocean. Here, the complete mitochondrial genome of T. macrophthalmus was first sequenced and annotated. The entire mitogenome is a typical circular molecule (16,943 bp in total length) containing 13 protein-coding genes, 2 ribosomal RNA genes, 22 transfer RNA genes, and 1 control region (CR). The overall base composition is 29.6% for A, 15.6% for G, 27.9% for C and 26.9% for T. Phylogenetic tree was reconstructed using concatenated 13 protein coding genes sequences of the 38 related taxa. The phylogenetic analysis indicated that T. macrophthalmus was most closely related to T. okinawae. These findings will contribute for further genetic studies such as evolution, DNA barcoding and molecular phylogeny of genus Trimma and related gobiid fishes.

The complete mitochondrial genome of Poraniopsis inflata (Asteroidea: Valvatida: Poraniidae) from Dokdo Island, Korea

This study presents the complete mitochondrial genome sequence of Poraniopsis inflata, providing valuable information on its genetic and taxonomic studies. Through next-generation sequencing, we successfully obtained the complete mitogenome of P. inflata, spanning a length of 16,322 bp. This genome structure encompasses 13 protein-coding genes (PCGs), 22 transfer RNA genes, and two ribosomal RNA genes. The phylogenetic analysis, based on a dataset of 13 PCG sequences, illuminated the phylogenetic relationships of P. inflata with other species of class Asteroidea and a species of echinoderm classes. The maximum likelihood phylogenetic tree showed that P. inflata closely clustered with Linckia laevigata. By revealing its mitochondrial genome and positioning it within the Asteroidea lineage, this study provides insights into the phylogenetic context of P. inflata.

Complete mitochondrial genome of Guigarracailaoensis Wang, Chen & Zheng, 2022 (Cypriniformes, Cyprinidae) and its phylogenetic implications.

Guigarracailaoensis is a member of family Cyprinidae, subfamily Labeoninae (Cypriniformes) which was recently discovered in southwestern China. Following its initial description, additional information on this species has remained notably scarce. In the current study, we assemble the complete mitochondrial genome (mitogenome) of G.cailaoensis using the Illumina sequencing platform. The mitogenome is identified as a circular, double-stranded DNA sequence of 16,593 base pairs, encompassing 13 protein-coding genes (PCGs), 22 transfer RNA genes, two ribosomal RNA genes, and a putative control region. Maximum-likelihood and Bayesian-inference approaches were used to construct phylogenetic trees for three datasets: (i) PCG sequences of the complete mitogenome (dataset 1); (ii) PCG sequences of the complete mitogenome combined with nuclear DNA (ncDNA) (Rag1) sequence (dataset 2); and (iii) ncDNA (Rag1) sequences (dataset 3). Phylogenetic analyses position G.cailaoensis as a sister taxon to the lineage consisting of Paraqianlabeolineatus Zhao, Sullivan, Zhang & Peng, 2014 and Pseudogyrinocheilusprochilus Fang, 1933 in dataset 1, and to Pseudogyrinocheilusprochilus in dataset 2, species lacking an oral disc on the lower lip. However, G.cailaoensis showed a close relationship to the lineage consisting of Discogobio and Discocheilus in dataset 3, species possessing an oral disc on the lower lip. Nonetheless, a variety of species with an oral disc on the lower lip are clustered into different lineages across the three datasets that may indicate that the development of the oral disc is homoplastic within the subfamily Labeoninae. The outcomes of this study have the potential to support conservation efforts for this species and to enrich our understanding of genetic resources in the area.

The complete chloroplast genome of an Arctic lycophyte, Huperzia arctica (Tolm.) Sipliv. 1973 (Lycopodiaceae)

Huperzia arctica (Tolm.) Sipliv. 1973 is a lycophyte species belonging to the Lycopodiaceae family, which is widely distributed in the Arctic region of Svalbard, Norway. To determine its taxonomic position, we sequenced the chloroplast genome of H. arctica. The complete chloroplast genome of H. arctica is 153,956 bp in length with 122 annotated genes, including 87 protein-coding genes, 31 tRNA genes, and 4 rRNA genes. To evaluate its evolutionary position, we performed phylogenetic analysis using 36 conserved orthologous protein-coding gene sequences from the chloroplast genomes of H. arctica and publicly available data from other Lycopodiaceae. H. arctica formed a monophyletic group with four other Huperzia species: H. lucidula, H. serrata, H. crispata, and H. javanica. However, it appeared as a separate species with a highly supported bootstrap value.

Overlaps Between CDS Regions of Protein-Coding Genes in the Human Genome: A Case Study on the NR1D1-THRA Gene Pair.

For several decades, it has been known that a substantial number of genes within human DNA exhibit overlap; however, the biological and evolutionary significance of these overlaps remain poorly understood. This study focused on investigating specific instances of overlap where the overlapping DNA region encompasses the coding DNA sequences (CDSs) of protein-coding genes. The results revealed that proteins encoded by overlapping CDSs exhibit greater disorder than those from nonoverlapping CDSs. Additionally, these DNA regions were identified as GC-rich. This could be partially attributed to the absence of stop codons from two distinct reading frames rather than one. Furthermore, these regions were found to harbour fewer single-nucleotide polymorphism (SNP) sites, possibly due to constraints arising from the overlapping state where mutations could affect two genes simultaneously.While elucidating these properties, the NR1D1-THRA gene pair emerged as an exceptional case with highly structured proteins and a distinctly conserved sequence across eutherian mammals. Both NR1D1 and THRA are nuclear receptors lacking a ligand-binding domain at their C-terminus, which is the region where these gene pairs overlap. The NR1D1 gene is involved in the regulation of circadian rhythm, while the THRA gene encodes a thyroid hormone receptor, and both play crucial roles in various physiological processes. This study suggests that, in addition to their well-established functions, the specifically overlapping CDS regions of these genes may encode protein segments with additional, yet undiscovered, biological roles.

Microbial symbionts are shared between ants and their associated beetles.

The transmission of microbial symbionts across animal species could strongly affect their biology and evolution, but our understanding of transmission patterns and dynamics is limited. Army ants (Formicidae: Dorylinae) and their hundreds of closely associated insect guest species (myrmecophiles) can provide unique insights into interspecific microbial symbiont sharing. Here, we compared the microbiota of workers and larvae of the army ant Eciton burchellii with those of 13 myrmecophile beetle species using 16S rRNA amplicon sequencing. We found that the previously characterized specialized bacterial symbionts of army ant workers were largely absent from ant larvae and myrmecophiles, whose microbial communities were usually dominated by Rickettsia, Wolbachia, Rickettsiella and/or Weissella. Strikingly, different species of myrmecophiles and ant larvae often shared identical 16S rRNA genotypes of these common bacteria. Protein-coding gene sequences confirmed the close relationship of Weissella strains colonizing army ant larvae, some workers and several myrmecophile species. Unexpectedly, these strains were also similar to strains infecting dissimilar animals inhabiting very different habitats: trout and whales. Together, our data show that closely interacting species can share much of their microbiota, and some versatile microbial species can inhabit and possibly transmit across a diverse range of hosts and environments.

Characterization and Molecular Phylogenetic Analysis of Subfamily Erebinae (Lepidoptera: Noctuoidea: Erebidae) Using Five Complete Mitochondrial Genomes.

In this study, the complete mitogenomes of Sympis rufibasis, Lacera noctilio, Oxyodes scrobiculata, Mocis undata, and Artena dotata were newly sequenced to bring up-to-date the database using the next-generation sequencing methods. The gene order of all sequenced mitogenomes was identical consisting of 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), two ribosomal RNA genes (rRNAs), and a non-coding A+T-rich region, which were common to other Lepidopteran insects. All protein-coding genes (PCGs) initiated with a canonical ATN codon and ended with TAN or an incomplete stop codon, single T. The A+T-rich region of S. rufibasis, L. noctilio, O. scrobiculata, M. undata, and A. dotata are 406bp, 462bp, 372bp, 410bp, and 406bp long, respectively, containing number of characteristics that are distinctive to Noctuoidea moths. We analyzed concatenated amino acid sequences of protein-coding genes not including rRNAs, using Maximum Likelihood and Bayesian Inference methods. The phylogenetic analyses indicated that the tribe relationships within Erebinae were reconstructed as (Sypnini+((Erebini 1+Poaphilini 1)+((Euclidiini+Catocalini+(Hypopyrini+Erebini 2))+((Hulodini+(Poaphilini 2+Ophiusini))))). Phylogenetic analyses supported and confirmed the monophyly of the subfamilies' relationships as follows: (Hypeninae+Lymantriinae)+((Scoliopterginae+((Calpinae+Erebinae)+((Herminiinae+Aganainae)+Arctiinae)))) within Erebidae.

Complete mitochondrial genome of Parasa sinica: New insights into the phylogeny of Limacodidae

In this study, the whole mitochondrial genome (mitogenome) of Parasa sinica was sequenced. It contains 15,306 base pairs (bp), 13 protein-coding genes (PCGs), two ribosomal RNAs (rRNAs), 22 transfer RNAs (tRNAs), and one non-coding regulatory area (CR), all of which are shared by other lepidopterans. It follows the same gene order as ordinary lepidopterans. Further, out of these 37 genes, 23 are present on the heavy strand whereas the remaining 14 are located on the light strand. The A + T composition of the mitogenome is relatively high. Although P. sinica has a negative AT-skew and GC-skew, the GC-skew value is significantly lower than the AT-skew value. All PCGs, with the exception of CO1, carry the same start codon (ATN). All tRNAs exhibit the usual cloverleaf secondary structure. We identified the conserved motif “ATAGA + poly-T″ found in other lepidopteran insects at the beginning of the CR. We collected the concatenated PCGs sequences in the mitochondrial genome of 15 species of Zygaenoidea, with the sequences of Geometridae as outgroups, including P. sinica, and constructed phylogenetic trees using Bayesian inference (BI) and maximum likelihood (ML) methods. The monolineage of each superfamily is usually well supported. Based on phylogenetic analysis, P. sinica is a member of family Limacodidae, strongly supporting the monophyly of the Zygaenoidea groups.

The complete mitochondrial genome and gene rearrangements in a gall wasp species, Dryocosmus liui (Hymenoptera: Cynipoidea: Cynipidae).

Mitochondrial genomes (mitogenomes) have been widely used in comparative and evolutionary genomics, molecular evolution, phylogenetics, and population genetics, but very limited information is available for the family Cynipidae. In this report, we describe the mitogenome of Dryocosmus liui Pang, Su et Zhu, providing the first complete mitogenomic data for a cynipid gall wasp species. The mitogenome of D. liui is 16,819 bp in length, and contains the typical set of 37 genes. Two control regions were detected, with the second being a perfect inverted repeat of the major portion of the first. Gene rearrangements were found in transfer RNA (tRNA) genes, protein-coding genes (PCGs) and ribosomal RNA (rRNA) genes, compared with the putative ancestral mitogenome. Similar to two other Cynipidae species with mitogenome data available, D. liui has a novel tRNA gene cluster trnL1-trnI-trnL2-trnW-trnM-trnQ between nad1 and nad2. Phylogenetic analysis based on sequences of PCGs and rRNA genes with D. liui included obtained topologies identical to previous studies supporting the a relationship of (Cynipoidea , (Platygastroidea, Proctotrupoidea)) within the monophyletic Proctotrupomorpha and (Cynipidae, Figitidae), Ibaliidae) within the Cynipoidea.

Genomic Characteristics of Okamejei kenojei and the Implications to Its Evolutionary Biology Study.

Okamejei kenojei is an important economic species widely distributed in shallow coastal waters of the western North Pacific. In this study, the whole-genome survey analysis of O. kenojei was conducted to reveal its genomic characteristics. The genome size was estimated to be 2027.44Mb, the repeat sequence content was 44.90%, and the heterozygous ratio was 1.04%. The mitochondrial genome excavated from the sequencing data was 16,974bp, and it can form the closed circular molecule. The phylogenetic tree based on 13 protein-coding gene sequences supported the validity of Okamejei and assisted the conclusion that Raja porosa was the junior synonym of O. kenojei. Plenty of potential microsatellite loci were identified, and the distribution frequency was estimated to be approximately 236.3 SSRs per Mb. Among all motif types of microsatellites, the dinucleotide repeats were dominant (82.59%), followed by the trinucleotide repeats (8.05%), tetranucleotide repeats (5.80%), pentanucleotide repeats (2.83%), and hexanucleotide repeats (0.72%). The results of the present study could not only provide useful information for understanding the genome structure and functional characteristics of O. kenojei, but also lay the foundation for the subsequent mapping of the whole genome.

Complete mitochondrial genome and the phylogenetic position of a new Annelida species belonging to the genus Syllis

The family Syllidae is the most taxonomically complex of the phylum Annelida. Although the gene order in the phylum Annelida's mitochondrial DNA (mtDNA) is well conserved, several exceptional cases have been reported. In this study, we describe the mitochondrial genome of a Syllis sp. that is 17,092 bp in length and contains 13 mitochondrial protein-coding genes (PCGs), 23 transfer RNA (tRNA) genes, including two tRNA-M, two ribosomal RNA (rRNA) genes, and a putative control region between tRNA-W and tRNA-G distinguished by a single short noncoding region. However, the gene order is not similar to those of other species in the family Syllidae. Phylogenetic analyses of 18S rRNA and 13 PCGs sequences demonstrated that this worm was clustered with other Syllis species in the family Syllidae. This is the first study to reveal the complete mitochondrial genome sequence of a previously unidentified Syllis sp. improving our understanding of the molecular biological characteristics of the poorly known genus Syllis.

Phylogenetic Analysis of Some Species of the Anopheles hyrcanus Group (Diptera: Culicidae) in China Based on Complete Mitochondrial Genomes.

Some species of the Hyrcanus group are vectors of malaria in China. However, the member species are difficult to identify accurately by morphology. The development of sequencing technologies offers the possibility of further studies based on the complete mitochondrial genome. In this study, samples of mosquitoes of the Hyrcanus group were collected in China between 1997 and 2015. The mitochondrial genomes of ten species of the Hyrcanus group were analyzed, including the structure and base composition, codon usage, secondary structure of tRNA, and base difference sites in protein coding regions. Phylogenetic analyses using maximum-likelihood and Bayesian inference were performed based on mitochondrial genes and complete mitochondrial genomes The mitochondrial genome of 10 Hyrcanus group members ranged from 15,403 bp to 15,475 bp, with an average 78.23% (A + T) content, comprising of 13 PCGs (protein coding genes), 22 tRNAs, and 2 rRNAs. Site differences between some closely related species in the PCGs were small. There were only 36 variable sites between Anopheles sinensis and Anopheles belenrae for a variation ratio of 0.32% in all PCGs. The pairwise interspecies distance based on 13 PCGs was low, with an average of 0.04. A phylogenetic tree constructed with the 13 PCGs was consistent with the known evolutionary relationships. Some phylogenetic trees constructed by single coding regions (such as COI or ND4) or combined coding regions (COI + ND2 + ND4 + ND5 or ND2 + ND4) were consistent with the phylogenetic tree constructed using the 13 PCGs. The phylogenetic trees constructed using some coding genes (COII, ND5, tRNAs, 12S rRNA, and 16S rRNA) differed from the phylogenetic tree constructed using PCGs. The difference in mitochondrial genome sequences between An. sinensis and An. belenrae was very small, corresponding to intraspecies difference, suggesting that the species was in the process of differentiation. The combination of all 13 PCG sequences was demonstrated to be optimal for phylogenetic analysis in closely related species.

Pharmaceuticals Promoting Premature Termination Codon Readthrough: Progress in Development.

Around 11% of all known gene lesions causing human genetic diseases are nonsense mutations that introduce a premature stop codon (PTC) into the protein-coding gene sequence. Drug-induced PTC readthrough is a promising therapeutic strategy for treating hereditary diseases caused by nonsense mutations. To date, it has been found that more than 50 small-molecular compounds can promote PTC readthrough, known as translational readthrough-inducing drugs (TRIDs), and can be divided into two major categories: aminoglycosides and non-aminoglycosides. This review summarizes the pharmacodynamics and clinical application potential of the main TRIDs discovered so far, especially some newly discovered TRIDs in the past decade. The discovery of these TRIDs brings hope for treating nonsense mutations in various genetic diseases. Further research is still needed to deeply understand the mechanism of eukaryotic cell termination and drug-induced PTC readthrough so that patients can achieve the greatest benefit from the various TRID treatments.

Characterization and comparative analysis of the complete plastid genomes of four Astragalus species.

Astragalus is the largest flowering plant genus. We assembled the plastid genomes of four Astragalus species (Astragalus iranicus, A. macropelmatus, A. mesoleios, A. odoratus) using next-generation sequencing and analyzed their plastomes including genome organization, codon usage, nucleotide diversity, prediction of RNA editing and etc. The total length of the newly sequenced Astragalus plastomes ranged from 121,050 bp to 123,622 bp, with 110 genes comprising 76 protein-coding genes, 30 transfer RNA (tRNA) genes and four ribosome RNA (rRNA) genes. Comparative analysis of the chloroplast genomes of Astragalus revealed several hypervariable regions comprising three non-coding sites (trnQ(UUG)-accD, rps7 -trnV(GAC) and trnR(ACG)-trnN(GUU)) and four protein-coding genes (ycf1, ycf2, accD and clpP), which have potential as molecular markers. Positive selection signatures were found in five genes in Astragalus species including rps11, rps15, accD, clpP and ycf1. The newly sequenced species, A. macropelmatus, has an approximately 13-kb inversion in IR region. Phylogenetic analysis based on 75 protein-coding gene sequences confirmed that Astragalus form a monophyletic clade within the tribe Galegeae and Oxytropis is sister group to the Coluteoid clade. The results of this study may helpful in elucidating the chloroplast genome structure, understanding the evolutionary dynamics at genus Astragalus and IRLC levels and investigating the phylogenetic relationships. Moreover, the newly plastid genomes sequenced have been increased the plastome data resources on Astragalus that can be useful in further phylogenomic studies.

Beginner's Guide on the Use of PAML to Detect Positive Selection.

The CODEML program in the PAML package has been widely used to analyze protein-coding gene sequences to estimate the synonymous and nonsynonymous rates (dS and dN) and to detect positive Darwinian selection driving protein evolution. For users not familiar with molecular evolutionary analysis, the program is known to have a steep learning curve. Here, we provide a step-by-step protocol to illustrate the commonly used tests available in the program, including the branch models, the site models, and the branch-site models, which can be used to detect positive selection driving adaptive protein evolution affecting particular lineages of the species phylogeny, affecting a subset of amino acid residues in the protein, and affecting a subset of sites along prespecified lineages, respectively. A data set of the myxovirus (Mx) genes from ten mammal and two bird species is used as an example. We discuss a new feature in CODEML that allows users to perform positive selection tests for multiple genes for the same set of taxa, as is common in modern genome-sequencing projects. The PAML package is distributed at https://github.com/abacus-gene/paml under the GNU license, with support provided at its discussion site (https://groups.google.com/g/pamlsoftware). Data files used in this protocol are available at https://github.com/abacus-gene/paml-tutorial.

Revalidation of Phalangomyia Dyar &amp;amp; Knab as a subgenus of Culex L. (Diptera: Culicidae) based on morphological and molecular evidence

Culex apicinus Philippi, 1865 and its synonyms, Phalangomyia debilis Dyar & Knab, 1914, Cx. escomeli Brèthes, 1920 and Cx. alticola Martini, 1931, were re-examined employing molecular and morphological phylogenetic methods. Archival and fresh specimens were used for morphological diagnosis and to generate fragments of two single-copy nuclear genes (CAD, HB) and one mitochondrial gene (COI). Additional sequences of Cx. apicinus and species of the subgenus Culex of Culex Linnaeus, 1758 and other subgenera of the genus were obtained from GenBank for molecular analysis. Diagnostic morphological characteristics of Cx. apicinus and other species of the subgenus Culex were contrasted. Phylogenetic relationships were generated using DNA sequences of protein coding genes under both Maximum Likelihood and Bayesian approaches. Culex apicinus showed unique morphological traits that allow its classification in a separate subgenus. The subspecific status is supported by its recovery as a monophyletic clade within the genus Culex based on morphological and molecular characters. The synonymous Phalangomyia debilis provides an available subgeneric name. Consequently, Phalangomyia Dyar & Knab, 1914 is resurrected from synonymy with Culex Linnaeus, 1758 as a monobasic subgenus of Culex.