Phylogenetic Research Articles

Rare Pleurotus species with veiled basidiomata from the Neotropics: neotypification of Pleurotus magnificus and epityfication of Pleurotus rickii

Knowledge of Pleurotus in Brazil dates back to the 19th century, when European mycologists studied and described numerous Neotropical taxa. Some of these long-described species remain poorly known, mostly due to the lack of recent records, detailed morphological descriptions, and molecular data. During field surveys in Southeast and Southern Brazil, specimens of two rare Pleurotus species with veiled basidiomata, i.e. P. magnificus and P. rickii, were collected. These species are recorded for the first time in the state of Santa Catarina, Brazil, and P. magnificus for the first time in the states of Minas Gerais, Paraná and São Paulo. Updated and detailed morphological descriptions are provided. DNA sequences of the ITS and nLSU molecular markers were obtained for both species, and phylogenetic analyses were conducted to explore their relationships within Pleurotus. A neotype is designated for P. magnificus, and a lectotype and epitype are designated for P. rickii.

Major depressive disorder is associated with mitochondrial ND6 T14502C mutation in two Han Chinese families

BACKGROUND Globally, the World Health Organization ranks major depressive disorder (MDD) as the leading cause of disability. However, MDD molecular etiology is still poorly understood. AIM To explore the possible association between mitochondrial ND6 T14502C mutation and MDD. METHODS Clinical data were collected from two pedigrees, and detailed mitochondrial genomes were obtained for the two proband members. The assessment of the resulting variants included an evaluation of their evolutionary conservation, allelic frequencies, as well as their structural and functional consequences. Detailed mitochondrial whole genome analysis, phylogenetic, and haplotype analysis were performed on the probands. RESULTS Herein, we reported the clinical, genetic, and molecular profiling of two Chinese families afflicted with MDD. These Chinese families exhibited not only a range of onset and severity ages in their depression but also extremely low penetrances to MDD. Sequence analyses of mitochondrial genomes from these pedigrees have resulted in the identification of a homoplasmic T14502C (I58V) mutation. The polymorphism is located at a highly conserved isoleucine at position 58 of ND6 and distinct mitochondrial DNA (mtDNA) polymorphisms originating from haplogroups M10 and H2. CONCLUSION Identifying the T14502C mutation in two individuals with no genetic relation who exhibit symptoms of depression provides compelling evidence that this mutation may be implicated in MDD development. Nonetheless, the two Chinese pedigrees that carried the T14502C mutation did not exhibit any functionally significant mutations in their mtDNA. Therefore, the phenotypic expression of the T14502C mutation related to MDD may be influenced by the nuclear modifier gene(s) or environmental factors.

Infundibulicybe swatica (Basidiomycota, Agaricales), a new species from Pakistan

A new species, Infundibulicybe swatica is described and illustrated from District Swat, Khyber Pakhtunkhwa, Pakistan. The new taxon is characterized by pale yellow to pale yellow brown to brown pileus with a dark brown center; a bicolorous stipe, white at the apex and pale yellow brown hues at the base; hyaline, lacrymoid to sub-lacrymoid, small, inamyloid basidiospores (6.5 × 4.2 µm); and the absence of hymenial cystidia. Molecular phylogenetic analyses based on nuclear ribosomal Internal Transcribed Spacer (ITS) and nuclear ribosomal Large Subunit (LSU) regions confirm the novelty of the taxon.

Metabolic scaling, energy allocation tradeoffs, and the evolution of humans’ unique metabolism

All organisms use limited energy to grow, survive, and reproduce, necessitating energy allocation tradeoffs, but there is debate over how selection impacted metabolic budgets and tradeoffs in primates, including humans. Here, we develop a method to compare metabolic rates as quotients of observed relative to expected values for mammals corrected for size, body composition, environmental temperature, and phylogenetic relatedness. Contrary to previous analyses, these quotients reveal that nonhuman primates have total metabolic rates expected for similar-sized mammals in similar environments. In addition, data from several small-scale societies show that humans evolved exceptionally high resting, activity, and total metabolic rates apparently by overcoming tradeoffs between resting and active energy expenditures that constrain other primates. Enhanced metabolic rates help humans fuel expanded brains, faster reproductive rates, extended longevity, and high percentage of body fat.

Phenotypic and genomic characterization of ST11-K1 CR-hvKP with highly homologous bla KPC-2 -bearing plasmids in China

ABSTRACT Carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP) strains present a significant global public health threat due to their high mortality rates. This study investigated the genomic characteristics of seven ST11-K1 CR-hvKP isolates harboring highly homologous KPC-2-encoding multidrug-resistance plasmids. The strains were isolated from a Chinese tertiary hospital between 2017 and 2020. Whole-genome sequencing and bioinformatic analysis revealed various antibiotic resistance genes (ARGs) and virulence determinants. The bla KPC-2 -bearing plasmids that contain multiple antibiotic-resistance genes were also identified in these strains. ISfinder and Orifinder were applied to identify insertion sequences (IS) and conjugation-related factors among these bla KPC-2 -bearing plasmids. The bla KPC-2 was highly consistent in seven bla KPC-2 -bearing plasmids (IS Kpn6-bla KPC-2 -IS Kpn27 -IS Yps3 -IS 26 ). In addition, we found a region composed of IS IR , Tn 5393 , and IS 26 . It was located upstream of the bla CTX-M-15 gene and presented in six bla KPC-2 -bearing plasmids, with pCR-hvKP221-KPC-P3 as an exception. Conjugation experiments demonstrated the horizontal transfer of resistance plasmids pCR-hvKP128-KPC-P1 and pCR-hvKP132-KPC-P1 across species. Notably, pLVPK-like virulence plasmids carrying virulence gene clusters pCR-hvKP173-Vir-P1, and pCR-hvKP221-Vir-P1 were also detected. A fusional plasmid pCR-hvKP221-Vir-P2, which carries virulence gene clusters and ARGs, was also identified. Five CR-hvKP strains displayed enhanced biofilm formation and high virulence in vivo infection models. Phylogenetic and single nucleotide polymorphism (SNP) analyses indicated a close genetic relationship among the isolates, suggesting a subclade. These findings highlight the complex genetic profiles and potential transmission mechanisms of CR-hvKP strains. IMPORTANCE We reported seven CR-hvKP strains all carried a highly homologous bla KPC-2 integrated IncFⅡ-resistant plasmid, and two strains harbored virulence plasmids. Conjugation experiments confirmed the transferability of these plasmids, indicating a potential for resistance spread. Phylogenetic analysis clarified the relationship among the CR-hvKP isolates. This study provides insights into the phenotypic and genomic characteristics of seven ST11-K1 CR-hvKP strains. The high prevalence and potential for local outbreaks emphasize the need for effective control measures.

Ecological conditions of vineyards and the polymorphism of Saccharomyces cerevisiae strains as a prerequisite of terroir identification

Aim. The aim of the study was to assess whether the ecological conditions for vineyards and the phylogenetic analysis of Saccharomyces cerevisiae wine yeast strains isolated from several locations in the Odesa and Mykolayiv regions could be used for terroir identification. Methods. The study employed microbiological, analytical, expedition, cartographic, and comparative assessment methods. Phylogenetic analysis was conducted using the MEGA software program, and VIN-CAD-UKR software was used for the vineyard cadastre and the ampelecological characteristics data management. Results. The study highlighted differences in the ecological conditions of vineyards in the Odesa and Mykolayiv regions and within the Tairovo and Izmail territorial communities in the Odesa region, focusing on soil and microclimatic characteristics. Saccharomyces cerevisiae strains isolated in 2012 and 2013, years with varying weather conditions in temperature and precipitation, were primarily clustered by the year of isolation. Two notable exceptions included a strain isolated from the Isabela variety belonging to a hybrid group (direct producer, contrary to other varieties, which mainly represent Vitis vinifera or its hybrids with Vitis riparia, Vitis amurensis, Vitis berlandieri) and a strain isolated from the Odesa Black variety inoculated at the beginning of fermentation with a commercial strain of wine yeast. The 2013 isolates, collected under conditions of higher precipitation and lower temperature in the first half of the growing season, showed a more differentiated clustering pattern, with two strains isolated from the Sukholymansky White variety forming a distinct cluster, while the rest grouped together, irrespective of the region, formed of isolation. Conclusions. Phylogenetic analysis of 64 strains of Saccharomyces cerevisiae isolated from 36 technical grape varieties based on the ITS1_5.8S_ITS2 nucleotide sequence demonstrated that the year of isolation (reflecting agroclimatic factors) was the primary influence on sample clustering. These factors, as components of the ecological conditions of terroir, form the foundation for terroir identification.

Transposable element accumulation drives genome size increase in Hylesia metabus (Lepidoptera: Saturniidae), an urticating moth species from South America

Abstract We present the first nuclear genome assembly and a complete mitogenome for Hylesia metabus (Arthropoda; Insecta; Lepidoptera; Saturniidae). The assembled nuclear genome sequence is 1,271 Mb long, which is among the 10 largest lepidopteran genome assemblies published to date. It is scaffolded in 31 pseudo chromosomes, has a BUSCO score of 99.5%, and has a highly conserved synteny compared to phylogenetically close species. Repetitive elements make up 67% of the nuclear genome and are mainly located in intergenic regions, among which LINEs were predominant, with CR1-Zenon being the most abundant. Phylogenetic and comparative analyses of H. metabus assembly and 17 additional Saturniidae and Sphingidae assemblies suggested that an accumulation of repetitive elements likely led to the increased size of H. metabus’ genome. Gene annotation using Helixer identified 26,122 transcripts. The Z scaffold was identified using both a synteny analysis and variations of coverage for two resequenced male and female H. metabus. The H. metabus nuclear genome and mitogenome assemblies can be found and browsed on the BIPAA website and constitute useful resources for future population and comparative genomics studies.

Genetic species concept as applied to multilocus nuclear data

Using the example of multiple species groups and species complexes, it is empirically shown that the resolving power of a set of 10–20 nuclear loci is insufficient for the study of their genetic differentiation. For the adequate analysis of phylogenetic relationships (including reticulation events) and assessment of divergence levels within such groups of closely related species, it is necessary to multiply the number of nuclear loci and transit to high-throughput next-generation deep sequencing. However, the question of the genomic coverage required for the purposes of such study remains open. In other words, exactly how many genes in a set are needed to measure the genetic distance resolving the relationships between branches on the phylogenetic tree reconstruction of a complex of closely related species? We chose protein-coding sequences as a standard set of markers. Distances for 160–180 genes with a combined length of 270 000–300 000 bp were used to estimate the divergence levels of closely related mammalian species. In most cases, the interspecific distances are within the 0.15–0.75% range (median of 0.33%). The range of distances between semi-species is 0.12–0.28% (median of 0.14%). Intraspecific distances are always lower than 0.11%. Rodents show higher values of interspecific distances, 0.25–2.3% (median of 0.72%); distances between half-species range from 0.1 to 0.35%. To determine the number of nuclear loci and their combined length sufficient for the calculation of a genetic distance, we use simulations based on a model that included the following parameters: the average rate of gene evolution, its dispersion, and the level of polymorphism in the modern and ancestral populations. We performed a preliminary analysis of the distribution of loci evolution rates among mammalian taxa based on the data on ~50 thousand nuclear markers. It is shown that a relative error of 10–15%, comparable to the same value for mitochondrial distances between close species based on individual genes (approximately 1000 bp in length), is achieved using approximately 100 loci of 300 bp in length. Based on these data, we propose the following working hypothesis: the threshold of interspecific/intraspecific genetic distance calculated on the basis of the exons most frequently used in mammalian molecular phylogenetics is approximately 0.15%. This hypothesis assumes a species rank for the forms in statu nascendi. It should be noted that not all “good” species have significant genetic distances, since in some cases reproductive barriers form faster than substitutions accumulate in the genome.

Ichthyobodo necator infection as a first record in cultured Dicentrarchus labrax: morphomolecular characterization and infestation patterns in correlation to water quality parameters

Abstract Ichthyobodo necator is a damaging and economically important protozoan parasite; however, its clinical patterns and incidence, correlated to environmental factors, are still poorly understood. This study provides the first report of Ichthyobodo necator infestation among Dicentrarchus labrax. We recorded significant mortalities in the cultured D. labrax of average size (5.92 g weight, and 11.55 cm length) in El-Max Station, National Institute of Oceanography and Fisheries, NIOF, Egypt, during the period extending from early summer to early fall (June to October) 2023. Behavioral and gross lesions of the infested fish were recorded; besides, dead and moribund fish were collected. Wet smears of the skin and gill of the affected fish were examined by compound light microscopy. Moreover, water samples from the affected ponds were collected and examined for their physicochemical parameters. The bodonid biflagellated Ichthyobodo necator (Henneguy) C. Pinto, 1928 was isolated from the skin and gills and identified based on specific morphological characteristics. DNA sequencing of PCR products and phylogenetic analysis confirmed identification, with clustering of the current isolates with I. necator isolates from different fish species. The multivariate analysis revealed a positive correlation between the prevalence and infestation intensity of Ichthyobodo, increased temperature levels, decreased dissolved oxygen DO, and also correlated with higher ammonia, and pH values. Additionally, the elevated concentrations of iron, calcium, magnesium, zinc, and copper in pond water were positively correlated with infestation and mortalities. These water parameter levels promote respiratory distress, accelerated prevalence, and infestation intensities of I. necator, particularly during the infestation peak in August and September. This study bridges the knowledge gap regarding I. necator parasite infestations and their correlation with water quality parameters.

Genome-Wide Identification and Characterization of bHLH Gene Family in Hevea brasiliensis

The basic helix-loop-helix (bHLH) transcription factors play crucial roles in plant growth, development, and stress responses. However, their identification and insights into the understanding of their role in rubber trees remain largely uncovered. In this study, the bHLH gene family was explored and characterized in rubber trees using systematic bioinformatics approaches. In total, 180 bHLH genes were identified in the rubber tree genome, distributed unevenly across 18 chromosomes, and phylogenetic analysis classified these genes into 23 distinct subfamilies. Promoter regions revealed a high density of cis-elements responsive to light and hormones. Enrichment analysis indicated involvement in numerous biological processes, including growth, development, hormone responses, abiotic stress resistance, and secondary metabolite biosynthesis. Protein interaction network analysis identified extensive interactions between HbbHLH genes and other functional genes, forming key clusters related to iron homeostasis, plant growth, and stomatal development. Expression profiling of HbbHLH genes have demonstrated varied responses to endogenous and environmental changes. RT-qPCR of eleven HbbHLH genes in different tissues and under ethylene, jasmonic acid, and cold treatments revealed tissue-specific expression patterns and significant responses to these stimuli, highlighting the roles of these genes in hormone and cold stress responses. These findings establish a framework for exploring the molecular functions of bHLH transcription factors in rubber trees.

Microalgae-Assisted Treatment of Wastewater Originating from Varied Sources, Particularly in the Context of Heavy Metals and Antibiotic-Resistant Bacteria

The increasing prevalence of heavy metals and antibiotic-resistant bacteria in wastewater (WW) raises serious environmental and public health concerns. This study investigates the efficiency of the microalgal strain Chlorella vulgaris EV-465 in treating wastewater and evaluates the antibiotic resistance profile of bacterial strains obtained from WW samples. Chlorella vulgaris EV-465 was used to treat four types of wastewater—domestic, municipal, hospital, and industrial wastewater—through 21 days of incubation. The findings demonstrated pH stabilization and significant decreases in nutrients (total nitrogen—TN, total phosphorus—TP), biological oxygen demand (BOD), chemical oxygen demand (COD), heavy metal (HM) concentrations, and bacterial count. Copper (Cu) showed the highest reduction, decreasing by 80% in industrial wastewater within 14 days, while lead (Pb) proved more resistant to removal, with only a 50% decrease by day 21. Additionally, the algae decreased bacterial counts, lowering colony-forming units (Log CFU/mL) from 9.04 to 4.65 in municipal wastewater over the 21-day period. Antibiotic susceptibility tests for isolated bacterial strains revealed high levels of resistance, with seven out of nine bacterial strains exhibiting multidrug resistance. Notably, Enterococcus faecium (PBI08), Acinetobacter baumannii (YBH19), and Pseudomonas aeruginosa (NBH16) displayed resistance to all nine antibiotics tested. Among the tested antibiotics, Ciprofloxacin showed the highest efficacy, with 66% susceptibility of tested bacterial strains. Cluster and phylogenetic analyses showed that the majority of the isolated bacterial strains belonged to the genera Pseudomonas and Escherichia, highlighting their genetic diversity and varied resistance mechanisms.

Efficient Solar-Powered Bioremediation of Hexavalent Chromium in Contaminated Waters by Chlorella sp. MQ-1

Microalgae are known for their efficient removal of hexavalent chromium (Cr(VI)) through biosorption and bioaccumulation, yet the subsequent release of Cr(VI) upon cell death remains a challenge. The reduction of Cr(VI) to the less toxic trivalent chromium [Cr(III)] is another critical remediation strategy that mitigates the risk of Cr(VI) re-release, but research on microalgal reduction of Cr(VI) is scarce. In this study, a microalgal strain designated as MQ-1 was isolated from chromium-contaminated mine effluent, demonstrating the capability to tolerate and remove Cr(VI). Phylogenetic analysis revealed that MQ-1 is closely related to the genus Chlorella; hence, it is classified as Chlorella sp. MQ-1. This strain exhibited robust growth at Cr(VI) concentrations below 2 mg/L, achieving a removal rate higher than 82% for initial Cr(VI) concentrations between 0.5 and 1 mg/L after a 5-day incubation period. Mechanistic studies revealed that MQ-1 promoted the removal of Cr(VI) mainly through intracellular bioreduction and bioaccumulation processes, in which more than 60% of Cr(VI) was reduced to the less toxic Cr(III) and stocked in the cells. A two-stage cultivation strategy, involving initial biomass accumulation followed by Cr(VI) treatment, significantly enhanced the removal efficiency, which was further accelerated under illuminated conditions. Notably, MQ-1 cultures with initial OD680 values of 4 and 6 accomplished 84.28% and 91.31% Cr(VI) removal from 2 mg/L solutions, respectively, within 30 hours under light exposure. These findings highlight the potential of MQ-1 to utilize renewable solar energy to reduce Cr(VI) and to mitigate the risk of its re-release into the environment. This characteristic positions MQ-1 as a potentially sustainable and cost-effective solution for Cr(VI) remediation and suggests its significant potential for large-scale implementation in bioremediation strategies aimed at Cr(VI)-contaminated waters.

New mid‐Cretaceous macrothelids showing a similar living mode to extant Macrothelidae (Araneae: Mygalomorphae)

AbstractFive new spider fossils of the family Macrothelidae (Araneae: Mygalomorphae) are described from mid‐Cretaceous Kachin amber, Myanmar. A new genus Acanthomacrothele gen. nov. is established for three new species: Acanthomacrothele pauciverrucae sp. nov., Acanthomacrothele geminata sp. nov., and Acanthomacrothele longicaudata sp. nov. Both male and female specimens of A. pauciverrucae sp. nov. are described, representing a rare case of a fossil spider with both sexes known. We also describe an unidentified juvenile of Macrothelidae to document different developmental stages of Cretaceous species. To test the phylogenetic position of the new fossils, we undertook phylogenetic analyses using both topology‐unconstrained and topology‐constrained methods. Our analyses supported the placement of Acanthomacrothele gen. nov. in Macrothelidae, although its phylogenetic relationship to other genera of Macrothelidae differed among different analytical methods. The fossils documented here have elongated, widely spaced posterior lateral spinnerets, compact rectangular group of eyes on a tubercle, and more spines on male tibia of anterior legs than female, similar to extant macrothelids, suggesting that they probably have similar retreat construction behavior, habitat preference, and mating position to their living relatives.

Arachis mottle-associated virus, a new polerovirus infecting Pinto peanut.

A new polerovirus, named "arachis mottle-associated virus" (ArMoV), was identified by high-throughput sequencing in a Pinto peanut (Arachis pintoi) plant. The genome sequence was confirmed by Sanger sequencing and contains 5775 nucleotides and seven predicted open reading frames (ORFs), showing a typical polerovirus genome structure. All of the proteins encoded by ArMoV showed less than 90% amino acid sequence identity to those of other poleroviruses, the threshold to establish a new species in the genus. Phylogenetic analysis based on P1-P2 fusion protein and coat protein amino acid sequences showed that tobacco polerovirus 1 and chickpea chlorotic stunt virus, respectively, were the most closely related to ArMoV. These data suggest that ArMoV is a member of a new species of the genus Polerovirus, for which the binomial name "Polerovirus ARMOV " is proposed.

An efficient deep learning method for amino acid substitution model selection.

Amino acid substitution models play an important role in studying the evolutionary relationships among species from protein sequences. The amino acid substitution model consists of a large number of parameters; therefore, it is estimated from hundreds or thousands of alignments. Both general models and clade-specific models have been estimated and widely used in phylogenetic analyses. The maximum likelihood method is normally used to select the best fit model for a specific protein alignment under the study. A number of studies have discussed theoretical concerns as well as computational burden of the maximum likelihood methods in model selection. Recently, machine learning methods have been proposed for selecting nucleotide models. In this paper, we propose a method to measure substitution rates among amino acids (called summary statistics) from protein alignments to efficiently train a deep learning network of so-called ModelDetector for detecting amino acid substitution models. The ModelDetector network was trained from 2,246,400 alignments on a computer with 8 cores (without GPU) in about 3.3 hours. Experiments on simulation data showed that the accuracy of the ModelDetector was comparable with that of the maximum likelihood method ModelFinder. It was orders of magnitude faster than the maximum likelihood method in inferring amino acid substitution models and able to analyze genome alignments with millions of sites in minutes. The results indicate that the deep learning network can play as a promising tool for amino acid substitution model selection.

Structural Insights into Cold-Active Lipase from Glaciozyma antarctica PI12: Alphafold2 Prediction and Molecular Dynamics Simulation.

Cold-active enzymes have recently gained popularity because of their high activity at lower temperatures than their mesophilic and thermophilic counterparts, enabling them to withstand harsh reaction conditions and enhance industrial processes. Cold-active lipases are enzymes produced by psychrophiles that live and thrive in extremely cold conditions. Cold-active lipase applications are now growing in the detergency, synthesis of fine chemicals, food processing, bioremediation, and pharmaceutical industries. The cold adaptation mechanisms exhibited by these enzymes are yet to be fully understood. Using phylogenetic analysis, and advanced deep learning-based protein structure prediction tool Alphafold2, we identified an evolutionary processes in which a conserved cold-active-like motif is presence in a distinct subclade of the tree and further predicted and simulated the three-dimensional structure of a putative cold-active lipase with the cold active motif, Glalip03, from Glaciozyma antarctica PI12. Molecular dynamics at low temperatures have revealed global stability over a wide range of temperatures, flexibility, and the ability to cope with changes in water and solvent entropy. Therefore, the knowledge we uncover here will be crucial for future research into how these low-temperature-adapted enzymes maintain their overall flexibility and function at lower temperatures.

Widely acclaimed but poorly named: Phylogeny and systematics of the African daisy genus Dimorphotheca (Asteraceae, Calenduleae)

AbstractPhylogenetic relationships in the South African daisy genus Dimorphotheca have long been uncertain, with the taxonomy of the genus relying on a few morphological traits, most prominently capitulum sexual system (e.g., cypsela type) and ray colour, which may not be evolutionarily conserved. Here we present the first well‐sampled molecular phylogeny of Dimorphotheca, based on nuclear ribosomal (ITS, ETS) and plastid trnL‐trnF region DNA sequences from multiple accessions per species. Although the relationships suggested by these markers are broadly congruent, we do find some instances of incongruence which we resolve using a combined decomposition and deletion approach. Using our best estimate of phylogenetic relationships, we reconstruct the evolution of capitulum fertility and ray colour to assess the evolutionary conservatism of these traits and their taxonomic utility. We find support for the monophyly of Dimorphotheca, excluding the recently segregated Osteospermum polypterum, and our data thus support the modern, enlarged circumscription of the genus incorporating the former genus Castalis and Osteospermum sect. Acanthotheca and O. sect. Blaxium. Major subclades within Dimorphotheca are largely cohesive in terms of geographic distribution and morphological traits such as growth form and cypsela structure. While many species are resolved as monophyletic, the polyphyly of a few species suggests a need for taxonomic re‐evaluation. On the basis of both morphological and molecular data, we describe one new species, and elevate one variety to species level. A full taxonomic key to the enlarged genus is presented for the first time. Ancestral reconstructions show that capitulum sexual system and ray lamina colour are not evolutionarily conserved and that neither can therefore be used to delimit major lineages within Dimorphotheca. While our findings resolve some taxonomic problems, they also highlight the need for further species‐level taxonomic work on Dimorphotheca.

Repeat-rich regions cause false positive detection of NUMTs: a case study in amphibians using an improved cane toad reference genome.

Mitochondrial DNA (mtDNA) has been widely used in genetics research for decades. Contamination from nuclear DNA of mitochondrial origin (NUMT) can confound studies of phylogenetic relationships and mtDNA heteroplasmy. Homology searches with mtDNA are widely used to detect NUMTs in the nuclear genome. Nevertheless, false positive detection of NUMTs is common when handling repeat-rich sequences, whilst fragmented genomes might result in missing true NUMTs. In this study, we investigated different NUMT detection methods and how the quality of the genome assembly affects them. We presented an improved nuclear genome assembly (aRhiMar1.3) of the invasive cane toad (Rhinella marina) with additional long-read Nanopore and 10x linked-read sequencing. The final assembly was 3.47 Gb in length with 91.3% of tetrapod universal single-copy orthologs (n=5,310), indicating the gene-containing regions were well assembled. We used three complementary methods (NUMTFinder, dinumt and PALMER) to study the NUMT landscape of the cane toad genome. All three methods yielded consistent results, showing very few NUMTs in the cane toad genome. Furthermore, we expanded NUMT detection analyses to other amphibians and confirmed a weak relationship between genome size and the number of NUMTs present in the nuclear genome. Amphibians are repeat-rich, and we show that the number of NUMTs found in highly repetitive genomes is prone to inflation when using homology-based detection without filters. Together, this study provides an exemplar of how to robustly identify NUMTs in complex genomes when confounding effects on mtDNA analyses are a concern.

Taxonomy and phylogeny of rodents parasitic fleas in southeastern China with description of a new subspecies of Ctenophthalmus breviprojiciens.

Accurate differentiation and identification of flea species are essential for both basic and applied research on fleas, as well as for the diagnosis of flea-borne diseases. However, distinguishing between flea species can be challenging, especially among those with minimal morphological differences. Therefore, some scholars have suggested the necessity of comprehensive revisions to the classification of fleas, incorporating morphological, molecular, and phylogenetic data. In this study, we focused on classifying the rodents' parasitic fleas in southeastern China and provided molecular and phylogenetic data. We also described a new subspecies Ctenophthalmus breviprojiciens fujiansis n. ssp. A total of 392 fleas were collected from 8 species of rodents in 10 counties. Morphologically, they belonged to 10 species, 9 genera and 5 families. Barcode identification based on COI gene and phylogenetic analysis based on five genetic markers (18S rDNA, 28S rDNA, EF-1a, COI, COII) revealed that the molecular and morphological identification of Xenopsylla cheopis, Aviostivalius klossi bispiniformis, Leptopsylla segnis, Monopsyllus anisus and Ctenocephalides felis felis were consistent. The taxonomic status of Neopsylla specialis minpiensis and Peromyscopsylla himalaica sinica as subspecies is questionable due to significant intraspecific genetic distance, and further morphological and molecular data are required to determine if they should be elevated to species level. The molecular identification of C. breviprojiciens n. ssp., N. dispar fukienensis, and Nosopsyllus nicanus could not be completed at this time due to a lack of sequences for related species in existing GenBank databases. Additionally, phylogenetic relationships of 31 species from 9 genera and 5 families of Siphonaptera were inferred based on five molecular markers (18S rDNA, 28S rDNA, EF-1a, COI and COII) using Maximum Likelihood analyses. The analyses revealed that various taxa of Siphonaptera are monophyletic at the subspecies, species, and genus levels. However, at the family level, Leptopsyllidae, Ceratophyllidae, Pulicidae, and Pygiopsyllidae are all monophyletic, while Ctenophthalmidae is paraphyletic. we support the view of some authors that revising the catchall group Ctenophthalmidae and elevating each of its constituent subfamilies to family status.

Clonal and horizontal transmission of carbapenem-resistant Enterobacterales strains and genes via flies.

Antimicrobial resistance (AMR) is one of the most pressing global public health challenges; in particular, the rapid dissemination of carbapenem-resistant Enterobacterales (CRE) is emerging as a significant concern worldwide. Flies, serving as carriers of pathogens, pose a potential threat in the transmission of antibiotic-resistant bacteria (ARB) between animals and humans. The aim of this study was to evaluate and reveal the potential risk of AMR spread by flies. A total of 450 flies were collected from four farms, four rural areas, and four urban areas in Dengfeng, Henan, China. To select CRE strains on the surface of flies, three flies sampled from the same geographical location were arbitrarily selected and placed into one tube of brain heart infusion broth (BHI), and the supernatant was screened using CHROMagar™ mSuperCARBA culture medium. Different colors and shapes of colonies were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and 16S rRNA sequencing. Antimicrobial susceptibility testing for CRE strains was performed using broth microdilution. All CRE strains were whole-genome sequenced. Short-read sequencing was performed using MGISEQ-2000 and long-read sequencing was conducted using GridION. Totally, 150 BHI tubes were screened for CRE strains, and 33 strains were identified as CRE positive. In 24 mSuperCARBA plates, only one species of CRE strain was isolated from each plate. In three plates, two different species of CRE strains were identified in each plate. In one plate, three different species of CRE strains were simultaneously isolated. Carbapenem resistance genes were detected in 81.8% of CRE strains, and blaNDM-1 was predominant (66.7%). No significant correlations between carbapenem-resistant phenotypes and carbapenem resistance genes were observed. The complete genomes of all 33 strains were obtained. Genome analysis revealed that clonal transmission events may have occurred among different farms and rural areas. Phylogenetic analysis revealed that blaNDM-1 IncFII plasmids could break bacterial species barrier for cross-host transmission in diverse areas. To understand and control the transmission of AMR from the perspective of One Health, it is imperative to enhance surveillance of ARB, antibiotic resistance genes, and antibiotic-resistant plasmids in flies.