Maximum Likelihood Phylogeny Research Articles

Resolving relationships despite past hybridization in Aliciella subsection Subnuda (Polemoniaceae)

AbstractPhylogenetics is crucial in the study of evolutionary processes and events transpiring in the course of species diversification. Phylogenetic studies within kingdom Plantae often reveal hybridization and introgression. Here, we study a subsection rife with historic hybridization and discuss the impacts of such processes on evolutionary trajectories. Aliciella subsection Subnuda comprises seven species of herbaceous plants occurring in Utah, the Navajo Nation, and the Four Corners region of North America. Previous molecular and morphological work left relationships in the subsection unresolved. Here, we use comparative DNA sequencing of nuclear ITS and chloroplast DNA regions and genome‐wide RAD‐seq data to clarify phylogenetic relationships and examine the role of hybridization in the subsection. We construct haplotype and nucleotype networks from chloroplast and nuclear ITS sequence matrices and compare nuclear and chloroplast phylogenies to identify multiple putative chloroplast capture events. The RAD‐seq maximum likelihood phylogeny and multispecies coalescent species tree robustly resolve relationships between six species‐level clades. We use STRUCTURE and HyDe on the RAD‐seq data to evaluate the influence of hybridization within the subsection. The HyDe results suggest that hybridization has occurred among all species in the subsection at some point in their history. Cytonuclear discordance reveals historic chloroplast capture, and we discuss potential causes of the observed discordance. Our study robustly resolves relationships in Aliciella subsection Subnuda and provides a framework for discussing its speciation despite a history of hybridization and introgression.

A tenuis relationship: traditional taxonomy obscures systematics and biogeography of the ‘Acropora tenuis’ (Scleractinia: Acroporidae) species complex

Abstract Molecular phylogenetics has fundamentally altered our understanding of the taxonomy, systematics and biogeography of corals. Recently developed phylogenomic techniques have started to resolve species-level relationships in the diverse and ecologically important genus Acropora, providing a path to resolve the taxonomy of this notoriously problematic group. We used a targeted capture dataset (2032 loci) to investigate systematic relationships within an Acropora clade containing the putatively widespread species Acropora tenuis and its relatives. Using maximum likelihood phylogenies and genetic clustering of single nucleotide polymorphisms from specimens, including topotypes, collected across the Indo-Pacific, we show ≥ 11 distinct lineages in the clade, only four of which correspond to currently accepted species. Based on molecular, morphological and geographical evidence, we describe two new species; Acropora rongoi n. sp. and Acropora tenuissima n. sp. and remove five additional nominal species from synonymy. Systematic relationships revealed by our molecular phylogeny are incongruent with traditional morphological taxonomy and demonstrate that characters traditionally used to delineate species boundaries and infer evolutionary history are homoplasies. Furthermore, we show that species within this clade have much smaller geographical ranges and, consequently, population sizes than currently thought, a finding with profound implications for conservation and management of reef corals.

First Report of Fusarium acuminatum Causing Dianthus chinensis root rot and foliage blight in China.

Dianthus chinensis is a popular ornamental plant that is widely cultivated in China. In May 2020, a disease was found at several landscape sites in Xuanwu District, Nanjing, China, causing symptoms of foliage blight and root discoloration onapproximately 52% of one-year old D. chinensis plants. To recover the causal pathogen, samples of infected roots and leaves were cut into 5×5 mm2 pieces, surface-disinfected in 75% ethanol for 30 sec, followed by 1% NaClO for 90 sec, rinsed with sterile water three times and placed on potato dextrose agar (PDA) with 0.1 mg/mL of ampicillin at 25 ⁰C. Hyphae growing on PDA were visible from both root and leaf tissues after three days. Individual hyphal tips were transferred to new PDA plates to obtain pure isolates. Three representative isolates were deposited in the China Forestry Culture Collection Center (CFCC 57545,57546, 57547). The hyphae grew radially, densely, and the aerial hyphae were velvety, white, yellow-white, or pink-white. Representative isolate Facu-DCY5 produced three types of conidia (microconidia, macroconidia, and chlamydospores). Macroconidia were sickle-shaped, measuring 25.7-55.4 µm × 3.2-4.6 µm (n=50). Microconidia were numerous, oval or kidney-shaped, measuring 6.8-11.9 µm × 3.5-4.8 µm (n=50). Conidia produced in the aerial mycelium were 16-34 × 2.2-5.3 µm (n=50). The ITS region, TEF1, calmodulin (CMDA), and RNA polymerase II second largest subunit (RPB2) were amplified with primers ITS1/ITS4, EF1/EF2, CL1/CL2A and 5F2/7CR , respectively and sequenced at Sangon Biotech (Nanjing, China). The ITS sequence of isolate Facu-DCY5 (GenBank No. ON307073.1) was identical to HQ165938.1, ON306850.1, OM964482.1. TEF1 (ON331997.1) was identical to LC546967.1, HQ165866.1, MZ158155.1. CMDA (ON331996.1) was identical to HQ412345.1, MZ921595.1 and MZ921597.1. RPB2 (ON331995.1) was identical to MZ997370.1. Maximum parsimony and maximum likelihood phylogenies of the Facu-DCY5 multilocus sequence data and those of several species within the F. tricinctum species complex identified the isolate from D. chinensis as F. acuminatum . Pathogenicity tests were performed using a conidial suspension (104conidia/mL). Each plant (approx. 0.3 m in -height) was inoculated with 1 mL of the conidial suspension by mixing it into the potting soil (500 g). Control plants were treated with sterile distilled water. All inoculated plants (n=9) in three repeats of the assay exhibited foliage blight and root rot after 15 days, whereas all control plants (n=9) remained asymptomatic. Fusarium isolates with identical morphological features and molecular marker sequences to those of Facu-DCY5 were recovered from foliage blight and root tissues of all the inoculated plants. In China, F. acuminatum has been reported as a pathogen of Cucurbita maxima, Actinidia arguta, Polygonatum odoratumand Schisandra chinensis. This is the first report of F. acuminatum on D. chinensis in China. Considering the importance of D. chinensis to both ornamental nurseries and landscaping industries, we recommend that diseased plants be removed to prevent the spread of F. acuminatum, and that identification of the infecting isolates from D. chinensis at other sites and landscape locations be performed.

DNA barcoding facilitates discovery and description of two new species of the Mystacides azureus Species Group (Trichoptera: Leptoceridae) in Japan.

We examined adult specimens of the Mystacides azureus Species Group (Trichoptera: Leptoceridae) collected in Japan and confirm three species including M. azureus Linnaeus 1761 and two new species, M. rivularis and M. moritai. Males and females of the new species are described. Mystacides azureus in Japan is shown to have a considerable variation in morphology of the male tergum X. We analyzed mitochondrial COI barcodes of the genus Mystacides including these three species to confirm their species status. A maximum likelihood phylogeny based on COI barcodes shows monophyly of the new species. It also supports the hypothesis that morphological variation of the male tergum X in Japanese populations is intraspecific in only M. azureus.

Hepatitis C virus transmission between eight high-income countries among men who have sex with men: a whole-genome analysis

Microelimination of the hepatitis C virus (HCV) among men who have sex with men (MSM) could be complicated by continuous external introductions and the emergence of phylogenetic clusters harbouring clinically significant resistance-associated substitutions (RAS). To investigate international clustering and the prevalence and transmission of RAS, we aimed to analyse whole-genome HCV sequences from MSM with a recently acquired infection who participated in a large, international HCV treatment trial. For this whole-genome analysis, we obtained HCV sequences from 128 MSM who had acquired HCV within the past 12 months and were participating in the REACT trial. The participants from whom sequences were obtained were recruited at 24 sites in eight countries. We inferred maximum-likelihood phylogenies and identified transmission clusters for HCV genotypes separately. We constructed time-scaled phylogenies to estimate cluster introduction dates and used a Bayesian Skygrid approach to estimate the effective population size over the past 50 years. We calculated the prevalence of RAS and the extent of RAS transmission in the study population. The majority of recent HCV infections were part of international networks that arose in the late 1990s and early 2000s. Sequences obtained in the same country clustered frequently, and in 36% of subclusters since 2015 we found evidence of international transmission. European MSM were more likely than non-European MSM to be in a cluster (odds ratio 11·9 [95% CI 3·6-43·4], p<0·0001). The effective population size decreased rapidly since around 2015 in Europe. RAS associated with substantially diminished cure rates were infrequently detected and transmission of highly resistant viruses was not observed. Despite antiviral treatment becoming widely available, international transmission of HCV among MSM has still occurred over the past 8 years, which could complicate microelimination of the virus in this population. RAS-enriched clusters and widespread RAS transmission are currently not a threat to elimination goals. These findings support an international approach for HCV microelimination among MSM. National Institutes of Health and Dr. C.J. Vaillant Fonds.

Emergence of a novel genetic lineage ‘A/ASIA/G-18/2019′ of foot and mouth disease virus serotype A in India: A challenge to reckon with

Foot and mouth disease (FMD) has engendered large scale socioeconomic crises on numerous occasions owing to its extreme contagiousness, transboundary nature, complicated epidemiology, negative impact on productivity, trade embargo, and need for intensive surveillance and expensive control measures. Emerging FMD virus variants have been predicted to have originated and spread from endemic Pool 2, native to South Asia, to other parts of the globe. In this study, 26 Indian serotype A isolates sampled between the year 2015 and 2022 were sequenced for the VP1 region. BLAST and maximum likelihood phylogeny suggest emergence of a novel genetic group within genotype 18, named here as ‘A/ASIA/G-18/2019′ lineage, that is restricted so far only to India and its eastern neighbour, Bangladesh. The lineage subsequent to its first appearance in 2019 seems to have displaced all other prevalent strains, in support of the phenomenon of ‘genotype/lineage turnover’. It has diversified into two distinct sub-clusters, reflecting a phase of active evolution. The rate of evolution of the VP1 region for the Indian serotype A dataset was estimated to be 6.747 × 10−3 substitutions/site/year. India is implementing a vaccination centric FMD control programme. The novel lineage showed good antigenic match with the proposed vaccine candidate A IND 27/2011 when tested in virus neutralization test, while the existing vaccine strain A IND 40/2000 showed homology with only 31% of the isolates. Therefore, in order to combat this challenge of antigenic divergence, A IND 27/2011 could be the preferred strain in the Indian vaccine formulations.

Escherichia coli encoding Shiga toxin subtype Stx2f causing human infections in England, 2015-2022.

Introduction. Shiga toxin-producing Escherichia coli (STEC) belong to a diverse group of gastrointestinal pathogens defined by the presence of Shiga toxin genes (stx) of which there are at least ten subtypes (Stx1a-Stx1d and Stx2a-Stx2g).Gap Statement. Initially thought to be associated with mild symptoms, more recently STEC encoding stx2f have been isolated from cases of haemolytic uraemic syndrome (HUS) and the clinical significance and public health burden require further investigation.Aim. We analysed clinical outcomes and genome-sequencing data linked to patients infected with STEC encoding-stx2f in England to assess the risk to public health.Methodology. One hundred and twelve E. coli (n=58 isolates encoded stx2f; n=54 isolates E. coli belonging to CC122 or CC722 that had eae but were negative for stx) isolated from patients' faecal specimens between 2015 and 2022 were genome sequenced and linked to epidemiological and clinical outcome data. All isolates were investigated for the presence of virulence genes and a maximum-likelihood phylogeny of isolates belonging to CC122 and CC722 was constructed.Results. There were 52 cases infected with STEC harbouring stx2f between 2015 and 2022, with the majority identified in 2022. Most cases resided in the North of England (n=39/52, 75 %), were female (n=31, 59.6 %) and/or aged five and under (n=29, 55.8 %). Clinical outcome data were available for 40/52 cases (76.9 %) and 7/40(17.5 %) were diagnosed with STEC-HUS. In the two most common clonal complexes, CC122 and CC722, the presence of the stx2f-encoding prophage correlated with the presence of additional virulence genes, astA, bfpA and cdt, located on an 85kbp IncFIB plasmid.Conclusions. Certain serotypes of E. coli harbouring stx2f cause severe clinical outcomes, including STEC-HUS. Public health advice and possible interventions are limited, as little is known about the animal and environmental reservoirs and transmission routes. We recommend more comprehensive and standardized collection of microbiological and epidemiological data, and routine sharing of sequencing data between public health agencies worldwide.

More than mcr: canonical plasmid- and transposon-encoded mobilized colistin resistance genes represent a subset of phosphoethanolamine transferases.

Mobilized colistin resistance genes (mcr) may confer resistance to the last-resort antimicrobial colistin and can often be transmitted horizontally. mcr encode phosphoethanolamine transferases (PET), which are closely related to chromosomally encoded, intrinsic lipid modification PET (i-PET; e.g., EptA, EptB, CptA). To gain insight into the evolution of mcr within the context of i-PET, we identified 69,814 MCR-like proteins present across 256 bacterial genera (obtained by querying known MCR family representatives against the National Center for Biotechnology Information [NCBI] non-redundant protein database via protein BLAST). We subsequently identified 125 putative novel mcr-like genes, which were located on the same contig as (i) ≥1 plasmid replicon and (ii) ≥1 additional antimicrobial resistance gene (obtained by querying the PlasmidFinder database and NCBI's National Database of Antibiotic Resistant Organisms, respectively, via nucleotide BLAST). At 80% amino acid identity, these putative novel MCR-like proteins formed 13 clusters, five of which represented putative novel MCR families. Sequence similarity and a maximum likelihood phylogeny of mcr, putative novel mcr-like, and ipet genes indicated that sequence similarity was insufficient to discriminate mcr from ipet genes. A mixed-effect model of evolution (MEME) indicated that site- and branch-specific positive selection played a role in the evolution of alleles within the mcr-2 and mcr-9 families. MEME suggested that positive selection played a role in the diversification of several residues in structurally important regions, including (i) a bridging region that connects the membrane-bound and catalytic periplasmic domains, and (ii) a periplasmic loop juxtaposing the substrate entry tunnel. Moreover, eptA and mcr were localized within different genomic contexts. Canonical eptA genes were typically chromosomally encoded in an operon with a two-component regulatory system or adjacent to a TetR-type regulator. Conversely, mcr were represented by single-gene operons or adjacent to pap2 and dgkA, which encode a PAP2 family lipid A phosphatase and diacylglycerol kinase, respectively. Our data suggest that eptA can give rise to "colistin resistance genes" through various mechanisms, including mobilization, selection, and diversification of genomic context and regulatory pathways. These mechanisms likely altered gene expression levels and enzyme activity, allowing bona fide eptA to evolve to function in colistin resistance.

Hidden species diversity and mito-nuclear discordance within the Mediterranean cone snail, Lautoconus ventricosus

The Mediterranean cone snail, Lautoconus ventricosus, is currently considered a single species inhabiting the whole Mediterranean basin and the adjacent Atlantic coasts. Yet, no population genetic study has assessed its taxonomic status. Here, we collected 245 individuals from 75 localities throughout the Mediterranean Sea and used cox1 barcodes, complete mitochondrial genomes, and genome skims to test whether L. ventricosus represents a complex of cryptic species. The maximum likelihood phylogeny based on complete mitochondrial genomes recovered six main clades (hereby named blue, brown, green, orange, red, and violet) with sufficient sequence divergence to be considered putative species. On the other hand, phylogenomic analyses based on 437 nuclear genes only recovered four out of the six clades: blue and orange clades were thoroughly mixed and the brown one was not recovered. This mito-nuclear discordance revealed instances of incomplete lineage sorting and introgression, and may have caused important differences in the dating of main cladogenetic events. Species delimitation tests proposed the existence of at least three species: green, violet, and red + blue + orange (i.e., cyan). Green plus cyan (with sympatric distributions) and violet, had West and East Mediterranean distributions, respectively, mostly separated by the Siculo-Tunisian biogeographical barrier. Morphometric analyses of the shell using species hypotheses as factor and shell length as covariate showed that the discrimination power of the studied parameters was only 70.2%, reinforcing the cryptic nature of the uncovered species, and the importance of integrative taxonomic approaches considering morphology, ecology, biogeography, and mitochondrial and nuclear population genetic variation.

Fusarium Keratitis From a Comprehensive Eye Health Care Facility in South India: Molecular Characterization by MALDI-TOF Versus Polymerase Chain Reaction Sequencing, Species Complex Distribution, and Clinical Correlation.

Fusarium keratitis possesses significant diagnostic and therapeutic challenges. Medically relevant Fusaria belong to various species complexes and show prominent differences in their antifungal susceptibility profile which may influence the clinical outcome. Rapid diagnostic methods are warranted for precise identification of species complexes for prompt initiation of correct antifungals. The aim of the study was to compare between matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) and polymerase chain reaction sequencing for correct species-level identification and to analyze the clinical outcome among different Fusarium species complexes. Twenty-nine culture-proven Fusarium keratitis cases were included in this study. A phylogenetic tree was constructed after TEF1α gene sequencing and isolates were subjected to MALDI-TOF MS, followed by database expansion and identification. Clinical outcome and risk association among species complexes were analyzed retrospectively. Maximum likelihood phylogeny categorized 68.9% isolates as Fusarium solani species complex (FSSC), 17.2% as Fusarium dimerum species complex (FDSC), followed by 13.7% as Fusarium fujikuroi species complex (FFSC). With extended database, MALDI-TOF MS could correctly speciate 96.5% (28/29) isolates. Previous antibiotic usage ( P = 0.034) and preoperative antifungal treatment with natamycin, voriconazole, or ketoconazole ( P = 0.025) were significantly higher in the FSSC group. The patients in the FFSC group had a significantly longer duration of symptoms at the time of clinical presentation to the clinic (15 days vs. 5 days, P = 0.030). Among 11 patients with a clinically poor outcome, 9 (31%) had FSSC infection. Patients infected with the FSSC had more aggressive infection with poor prognosis. MALDI-TOF MS can serve as the best alternative method to conventional molecular identification with reduced turnaround time, which may help the ophthalmologists to consider the appropriate antifungals or early surgical intervention for improved outcome.

The prevalence of Wolbachia in multiple cockroach species and its implication for urban insect management.

Cockroach management relies heavily on the use of conventional insecticides in urban settings, which no longer provide the anticipated level of control. Knowledge of cockroach endosymbionts, like Wolbachia, might provide novel avenues for control. Therefore, we screened 16 cockroach species belonging to 3 families (Ectobiidae, Blattidae, and Blaberidae) for the presence of Wolbachia. We mapped the evolution of Wolbachia-cockroach relationships based on maximum likelihood phylogeny and phylogenetic species clustering on a multi-loci sequence dataset (i.e., coxA, virD4, hcpA, and gatB) of Wolbachia genes. We confirmed the previous report of Wolbachia in 1 Ectobiid species; Supella longipalpa (Fab.), and detected the presence of Wolbachia in 2 Ectobiid species; Balta notulata (Stål) and Pseudomops septentrionalis Hebard, and 1 Blaberid species; Gromphadorhina portentosa (Schaum). All cockroach-associated Wolbachia herein detected were clustered with the ancestor of F clade Wolbachia of Cimex lectularius L. (bed bugs). Since Wolbachia provision C. lectularius with biotin vitamins that confer reproductive fitness, we screened the cockroach-associated Wolbachia for the presence of biotin genes. In toto, our results reveal 2 important findings: (i) Wolbachia is relatively uncommon among cockroach species infecting about 25% of species investigated, and (ii) cockroach-associated Wolbachia have biotin genes that likely provide nutritional benefits to their hosts. Thus, we discuss the potential of exploring Wolbachia as a tool for urban insect management.

DNA Barcodes of Mansonia (Mansonia) Blanchard, 1901 (Diptera, Culicidae).

Females of the genus Mansonia feed on the blood of humans, livestock, and other vertebrates to develop their eggs. The females' biting behavior may cause severe disturbance to blood hosts, with a negative impact on public health and economics. Certain species have been identified as potential or effective disease vectors. The accurate species identification of field-collected specimens is of paramount importance for the success of monitoring and control strategies. Mansonia (Mansonia) morphological species boundaries are blurred by patterns of intraspecific heteromorphism and interspecific isomorphism. DNA barcodes can help to solve taxonomic controversies, especially if combined with other molecular tools. We used cytochrome c oxidase subunit I (COI) gene 5' end (DNA barcode) sequences to identify 327 field-collected specimens of Mansonia (Mansonia) spp. The sampling encompassed males and females collected from three Brazilian regions and previously assigned to species based on their morphological characteristics. Eleven GenBank and BOLD sequences were added to the DNA barcode analyses. Initial morphospecies assignments were mostly corroborated by the results of five clustering methods based on Kimura two-parameter distance and maximum likelihood phylogeny. Five to eight molecular operational taxonomic units may represent taxonomically unknown species. The first DNA barcode records for Mansonia fonsecai, Mansonia iguassuensis, and Mansonia pseudotitillans are presented.

The First Complete Mitochondrial Genome of Genus Isocapnia (Plecoptera: Capniidae) and Phylogenetic Assignment of Superfamily Nemouroidea.

Capniidae are a family of stoneflies, also known as snow flies, who emerge in winter. The phylogeny of Capniidae is widely accepted to be based on morphological analysis. Until now, only five Capniidae mitochondrial genomes have been sequenced so far. In addition, sampling is required to determine an accurate phylogenetic association because the generic classification of this family is still controversial and needs to be investigated further. In this study, the first mitogenome of genus Isocapnia was sequenced with a length of 16,200 bp and contained 37 genes, including a control region, two rRNAs, 22 tRNAs, and 13 PCGs, respectively. Twelve PCGs originated with the common start codon ATN (ATG, ATA, or ATT), while nad5 used GTG. Eleven PCGs had TAN (TAA or TAG) as their last codon; however, cox1 and nad5 had T as their final codon due to a shortened termination codon. All tRNA genes demonstrated the cloverleaf structure, which is distinctive for metazoans excluding the tRNASer1 (AGN) that missed the dihydrouridine arm. A Phylogenetic analysis of the superfamily Nemouroidea was constructed using thirteen PCGs from 32 formerly sequenced Plecoptera species. The Bayesian inference and maximum likelihood phylogeny tree structures derived similar results across the thirteen PCGs. Our findings strongly supported Leuctridae + ((Capniidae + Taeniopterygidae) + (Nemouridae + Notonemouridae)). Ultimately, the best well-supported generic phylogenetic relationship within Capniidae is as follows; (Isocapnia + (Capnia + Zwicknia) + (Apteroperla + Mesocapnia)). These findings will enable us to better understand the evolutionary relationships within the superfamily Nemouroidea and the generic classification and mitogenome structure of the family Capniidae.

Genetic Characterization and Insular Habitat Enveloping of Endangered Leaf-Nosed Bat, Hipposideros nicobarulae (Mammalia: Chiroptera) in India: Phylogenetic Inference and Conservation Implication.

The Nicobar leaf-nosed Bat (Hipposideros nicobarulae) was described in the early 20th century; however, its systematic classification has been debated for over 100 years. This endangered and endemic species has achieved species status through morphological data in the last 10 years. However, the genetic information and phylogenetic relationships of H. nicobarulae remain neglected. The generated mitochondrial cytochrome b gene (mtCytb) sequences (438 bp) of H. nicobarulae contains 53.42-53.65% AT composition and 1.82% variable sites. The studied species, H. nicobarulae maintains an 8.1% to 22.6% genetic distance from other Hipposideros species. The genetic divergence estimated in this study is congruent with the concept of gene speciation in bats. The Bayesian and Maximum-Likelihood phylogenies clearly discriminated all Hipposideros species and showed a sister relationship between H. nicobarulae and H. cf. antricola. Current mtCytb-based investigations of H. nicobarulae have confirmed the species status at the molecular level. Further, the MaxEnt-based species distribution modelling illustrates the most suitable habitat of H. nicobarulae (294 km2), of which the majority (171 km2) is located on Great Nicobar Island. The present study suggests rigorous sampling across the range, taxonomic coverage, the generation of multiple molecular markers (mitochondrial and nuclear), as well as more ecological information, which will help in understanding population genetic structure, habitat suitability, and the implementation of appropriate conservation action plans for H. nicobarulae and other Hipposideros species.

The Evolution and Transmission Dynamics of Multidrug-Resistant Tuberculosis in an Isolated High-Plateau Population of Tibet, China.

On the Tibetan Plateau, most tuberculosis is caused by indigenous Mycobacterium tuberculosis strains with a monophyletic structure and high-level drug resistance. This study investigated the emergence, evolution, and transmission dynamics of multidrug-resistant tuberculosis (MDR-TB) in Tibet. The whole-genome sequences of 576 clinical strains from Tibet were analyzed with the TB-profiler tool to identify drug-resistance mutations. The evolution of the drug resistance was then inferred based on maximum-likelihood phylogeny and dated trees that traced the serial acquisition of mutations conferring resistance to different drugs. Among the 576 clinical M. tuberculosis strains, 346 (60.1%) carried at least 1 resistance-conferring mutation and 231 (40.1%) were MDR-TB. Using a pairwise distance of 50 single nucleotide polymorphisms (SNPs), most strains (89.9%, 518/576) were phylogenetically separated into 50 long-term transmission clusters. Eleven large drug-resistant clusters contained 76.1% (176/231) of the local multidrug-resistant strains. A total of 85.2% of the isoniazid-resistant strains were highly transmitted with an average of 6.6 cases per cluster, of which most shared the mutation KatG Ser315Thr. A lower proportion (71.6%) of multidrug-resistant strains were transmitted, with an average cluster size of 2.9 cases. The isoniazid-resistant clusters appear to have undergone substantial bacterial population growth in the 1970s to 1990s and then subsequently accumulated multiple rifampicin-resistance mutations and caused the current local MDR-TB burden. These findings highlight the importance of detecting and curing isoniazid-resistant strains to prevent the emergence of endemic MDR-TB. IMPORTANCE Emerging isoniazid resistance in the 1970s allowed M. tuberculosis strains to spread and form into large multidrug-resistant tuberculosis clusters in the isolated plateau of Tibet, China. The epidemic was driven by the high risk of transmission as well as the potential of acquiring further drug resistance from isoniazid-resistant strains. Eleven large drug-resistant clusters consisted of the majority of local multidrug-resistant cases. Among the clusters, isoniazid resistance overwhelmingly evolved before all the other resistance types. A large bacterial population growth of isoniazid-resistant clusters occurred between 1970s and 1990s, which subsequently accumulated rifampicin-resistance-conferring mutations in parallel and accounted for the local multidrug-resistant tuberculosis burden. The results of our study indicate that it may be possible to restrict MDR-TB evolution and dissemination by prioritizing screening for isoniazid (INH)-resistant TB strains before they become MDR-TB and by adopting measures that can limit their transmission.

DNA barcoding of some species of the genus Capoeta Valenciennes, 1842 from Kurdistan Region, Iraq.

The genus Capoeta belongs to the Cyprinidae family and can be found in many of the inland waters of Middle Eastern nations. They are systematically complex species, many of which cannot be distinguished based on morphology. Genetic techniques like DNA barcoding may be used to identify species due to the limits of such species’ morphological identification, although their genetic links have not yet been fully elucidated. The current study provides information on the genetic structure of three Capoeta species (C. damascina, C. trutta, and C. umbla) found in the Greater Zab River/ Bekhme Dam of the Kurdistan Region- Iraq. In order to confirm the identification of Capoeta species at the molecular level, the mitochondrial cytochrome oxidase I (COI) gene was sequenced. The interspecific genetic between species was (0.003) to (0.064). The maximum likelihood phylogeny indicates a consensus tree topology containing two branches, the first branch is divided into two subbranches including C. damascina and C. umbla and the second branch includes C. trutta. The results suggest that COI gene sequencing can be used to identify Capoeta species from streams in the Kurdistan Region, which is one of the first steps to improving the genetic understanding of Capoeta species.

Nanopore adaptive sampling for targeted mitochondrial genome sequencing and bloodmeal identification in hematophagous insects

BackgroundBlood-feeding insects are important vectors for an array of zoonotic pathogens. While previous efforts toward generating molecular resources have largely focused on major vectors of global medical and veterinary importance, molecular data across a large number of hematophagous insect taxa remain limited. Advancements in long-read sequencing technologies and associated bioinformatic pipelines provide new opportunities for targeted sequencing of insect mitochondrial (mt) genomes. For engorged hematophagous insects, such technologies can be leveraged for both insect mitogenome genome assembly and identification of vertebrate blood-meal sources.MethodsWe used nanopore adaptive sampling (NAS) to sequence genomic DNA from four species of field-collected, blood-engorged mosquitoes (Aedes and Culex spp.) and one deer fly (Chrysops sp.). NAS was used for bioinformatical enrichment of mtDNA reads of hematophagous insects and potential vertebrate blood-meal hosts using publically available mt genomes as references. We also performed an experimental control to compare results of traditional non-NAS nanopore sequencing to the mt genome enrichment by the NAS method.ResultsComplete mitogenomes were assembled and annotated for all five species sequenced with NAS: Aedes trivittatus, Aedes vexans, Culex restuans, Culex territans and the deer fly, Chrysops niger. In comparison to data generated during our non-NAS control experiment, NAS yielded a substantially higher proportion of reference-mapped mtDNA reads, greatly streamlining downstream mitogenome assembly and annotation. The NAS-assembled mitogenomes ranged in length from 15,582 to 16,045 bp, contained between 78.1% and 79.0% A + T content and shared the anticipated arrangement of 13 protein-coding genes, two ribosomal RNAs, and 22 transfer RNAs. Maximum likelihood phylogenies were generated to further characterize each insect species. Additionally, vertebrate blood-meal analysis was successful in three samples sequenced, with mtDNA-based phylogenetic analyses revealing that blood-meal sources for Chrysops niger, Culex restuans and Aedes trivittatus were human, house sparrow (Passer domesticus) and eastern cottontail rabbit (Sylvilagus floridanus), respectively.ConclusionsOur findings show that NAS has dual utility to simultaneously molecularly identify hematophagous insects and their blood-meal hosts. Moreover, our data indicate NAS can facilitate a wide array of mitogenomic systematic studies through novel ‘phylogenetic capture’ methods. We conclude that the NAS approach has great potential for broadly improving genomic resources used to identify blood-feeding insects, answer phylogenetic questions and elucidate complex pathways for the transmission of vector-borne pathogens.Graphical

Genotypic Characteristics and Antimicrobial Resistance of Escherichia coli ST141 Clonal Group

Escherichia coli ST141 is one of the ExPEC lineages whose incidence is rising in France, even if no epidemic situation involving multidrug resistant isolates has been reported so far. Nonetheless, in a 2015-2017 monocentric study conducted in our French University hospital, ST141 was the most frequent lineage after ST131 in our collection of phylogroup B2 ESBL-producing E. coli. The genomes of 187 isolates representing ST141 group, including 170 genomes from public databases and 17 from our local collection, of which 13 produced ESBL, were analyzed to infer the maximum likelihood phylogeny SNP-based (Single Nucleotide Polymorphism) free-recombinant tree defining the ST141 population structure. Genomes were screened for genes encoding virulence factors (VFs) and antimicrobial resistance (AMR). We also evaluated the distribution of isolates according to their origin (host, disease, country) and the distribution of VFs or AMR genes. Finally, the phylogenic tree revealed that ST141 isolates clustered into two main sublineages, with low genetic diversity. Contrasting with a highly virulent profile, as many isolates accumulated VFs, the prevalence of AMR was limited, with no evidence of multidrug resistant emerging lineage. However, our results suggest that surveillance of this clonal group, which has the potential to spread widely in the community, would be essential.

Mitochondriomics of Clarias Fishes (Siluriformes: Clariidae) with a New Assembly of Clarias camerunensis: Insights into the Genetic Characterization and Diversification

The mitogenome of an endemic catfish Clarias camerunensis was determined from the Cameroon water. This circular mitogenome was 16,511 bp in length and comprised 13 protein-coding genes, 2 ribosomal RNAs, 22 transfer RNAs, and a single AT-rich control region. The heavy strand accommodates 28 genes, whereas the light strand is constituted by ND6 and eight transfer RNA (tRNA) genes. The C. camerunensis mitochondrial genome is AT biased (56.89%), as showcased in other Clarias species. The comparative analyses revealed that most of the Clarias species have 6 overlapping and 11 intergenic spacer regions. Most of the PCGs were initiated and terminated with the ATG start codon and TAA stop codon, respectively. The tRNAs of C. camerunensis folded into the distinctive cloverleaf secondary structure, except trnS1. The placement of the conserved domains in the control region was similar in all the Clarias species with highly variable nucleotides in CSB-I. Both maximum likelihood and Bayesian-based matrilineal phylogenies distinctly separated all Clarias species into five clades on the basis of their known distributions (South China, Sundaland, Indochina, India, and Africa). The TimeTree analysis revealed that the two major clades (Indo-Africa and Asia) of Clarias species might have diverged during the Paleogene (≈28.66 MYA). Our findings revealed the separation of Indian species (C. dussumieri) and African species (C. camerunensis and Clarias gariepinus) took place during the Paleogene, as well as the South Chinese species (Clarias fuscus) and Sundaland species (Clarias batrachus) splits from the Indochinese species (Clarias macrocephalus) during the Neogene through independent colonization. This pattern of biotic relationships highlights the influence of topography and geological events in determining the evolutionary history of Clarias species. The enrichment of mitogenomic data and multiple nuclear loci from their native range or type locality will confirm the true diversification of Clarias species in African and Asian countries.

The Bryozoan Cauloramphus magnus (Cheilostomata: Calloporidae) in Northern Japan Includes Multiple, Co-occurring Cryptic Species.

The cheilostome bryozoan Cauloramphus magnus is common in the rocky intertidal habitat from southeastern Alaska to northern Japan. We examined its phylogeography by analyzing 576 bp of the mitochondrial COI (cox1) gene sequenced for 298 colonies from 16 localities in northern Japan. A maximum-likelihood phylogeny detected three main clades (A, B, C). Clades A and B occurred throughout the study area but differed in frequency, haplotype diversity, and haplotype distribution; each resolved into three divergent subclades (AI-III, BI-III). Clade A shared none among 15 haplotypes between the Pacific and Sea of Japan sides of Hokkaido. In contrast, Clade B (29 haplotypes) was thrice as common as Clade A among samples, with haplotype B28 common on both sides. Divergent Clade C (nine haplotypes) was detected only at Rumoi. K2P divergences of 12.3-28.3% among Clades A-C suggest these are distinct biological species, a conclusion supported by different inferred evolutionary histories. A bPTP species delimitation analysis indicated nine phylogenetic species among the sequences included in our phylogeny (AI-III, BI-III, C, and one specimen each from Alaska and the Commander Islands), with K2P divergences of 3.9-6.5% among subclades in A or B. Statistical and principal components analyses suggested weak morphological differentiation between Clades A + B and C, although overlapping ranges of measurements preclude identification to clade; these three clades are morphologically cryptic. For taxonomy, we suggest retaining the name C. magnus for lineages within this species complex across its range, followed by a clade designation, if known.