Search

Cytochrome P450 monooxygenases of the CYP79 family catalyze conversion of specific amino acids into oximes feeding into a variety of metabolic plant pathways. Here we present an extensive phylogenetic tree of the CYP79 family built on carefully curated sequences collected across the entire plant kingdom. Based on a monophyletic origin of the P450s, a set of evolutionarily distinct branches was identified. Founded on the functionally characterized CYP79 sequences, sequence features of the individual substrate recognition sites (SRSs) were analyzed. Co-evolving amino acid residues were identified using co-evolutionary sequence analysis. SRS4 possesses a specific sequence pattern when tyrosine is a substrate. Except for the CYP79Cs and CYP79Fs, substrate preferences toward specific amino acids could not be assigned to specific subfamilies. The highly diversified CYP79 tree, reflecting recurrent independent evolution of CYP79s, may relate to the different roles of oximes in different plant species. The sequence differences across individual CYP79 subfamilies may facilitate the in vivo orchestration of channeled metabolic pathways based on altered surface charge domains of the CYP79 protein. Alternatively, they may serve to optimize dynamic interactions with oxime metabolizing enzymes to enable optimal ecological interactions. The outlined detailed curation of the CYP79 sequences used for building the phylogenetic tree made it appropriate to make a conservative phylogenetic tree-based revision of the naming of the sequences within this highly complex cytochrome P450 family. The same approach may be used in other complex P450 subfamilies. The detailed phylogeny of the CYP79 family will enable further exploration of the evolution of function in these enzymes., (© 2024 The Author(s). The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

Contemporary glaciers are inhabited by streptophyte algae that balance photosynthesis and growth with tolerance of low temperature, desiccation and UV radiation. These same environmental challenges have been hypothesised as the driving force behind the evolution of land plants from streptophyte algal ancestors in the Cryogenian (720-635 million years ago). We sequenced, assembled and analysed the metagenome-assembled genome of the glacier alga Ancylonema nordenskiöldii to investigate its adaptations to life in ice, and whether this represents a vestige of Cryogenian exaptations. Phylogenetic analysis confirms the placement of glacier algae within the sister lineage to land plants, Zygnematophyceae. The metagenome-assembled genome is characterised by an expansion of genes involved in tolerance of high irradiance and UV light, while lineage-specific diversification is linked to the novel screening pigmentation of glacier algae. We found no support for the hypothesis of a common genomic basis for adaptations to ice and to land in streptophytes. Comparative genomics revealed that the reductive morphological evolution in the ancestor of Zygnematophyceae was accompanied by reductive genome evolution. This first genome-scale data for glacier algae suggests an Ancylonema-specific adaptation to the cryosphere, and sheds light on the genome evolution of land plants and Zygnematophyceae., (© 2024 The Author(s). New Phytologist © 2024 New Phytologist Foundation.)

Phylogenetic trees are believed to contain a wealth of information on diversification processes. However, comparing phylogenetic trees is not straightforward due to their high dimensionality. Researchers have therefore defined a wide range of low-dimensional summary statistics. Currently, it remains unexplored to what extent these summary statistics cover the same underlying information and what summary statistics best explain observed variation across phylogenies. Furthermore, a large subset of available summary statistics focusses on measuring the topological features of a phylogenetic tree, but are often only explored at the extreme edge cases of the fully balanced or imbalanced tree and not for trees of intermediate balance. Here, we introduce a new R package called 'treestats', that provides speed optimized code to compute 70 summary statistics. We study correlations between summary statistics on empirical trees and on trees simulated using several diversification models. Furthermore, we introduce an algorithm to create intermediately balanced trees in a well-defined manner, in order to explore variation in summary statistics across a balance gradient. We find that almost all summary statistics are correlated with tree size, and find that it is difficult, if not impossible, to correct for tree size, unless the tree generating model is known. Furthermore, we find that across empirical and simulated trees, at least three large clusters of correlated summary statistics can be found, where statistics group together based on information used (topology or branching times). However, the finer grained correlation structure appears to depend strongly on either the taxonomic group studied (in empirical studies) or the tree generating model (in simulation studies). Amongst statistics describing the (im)balance of a tree, we find that almost all statistics vary non-linearly, and sometimes even non-monotonically, with our generated balance gradient. This indicates that balance is perhaps a more complex property of a tree than previously thought. Furthermore, using our new imbalancing algorithm, we devise a numerical test to identify balance statistics, and identify several statistics as balance statistics that were not previously considered as such. Lastly, our results lead to several recommendations on which statistics to select when analyzing and comparing phylogenetic trees., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background and Aims: Progress in the systematic studies of the olive family (Oleaceae) during the last two decades provides the opportunity to update its backbone phylogeny and to investigate its historical biogeography. We also aimed to understand the factors underlying the disjunct distribution pattern between East Asia and both West Asia and Europe that is found more commonly in this family than in any other woody plant family., Methods: Using a sampling of 298 species out of ~750, the largest in a phylogenetic study of Oleaceae thus far, with a set of 36 plastid and nuclear markers, we reconstructed and dated a new phylogenetic tree based on maximum likelihood and Bayesian methods and checked for any reticulation events. We also assessed the relative support of four competing hypotheses [Qinghai-Tibet Plateau uplift (QTP-only hypothesis); climatic fluctuations (climate-only hypothesis); combined effects of QTP uplift and climate (QTP-climate hypothesis); and no effects (null hypothesis)] in explaining these disjunct distributions., Key Results: We recovered all tribes and subtribes within Oleaceae as monophyletic, but uncertainty in the position of tribe Forsythieae remains. Based on this dataset, no reticulation event was detected. Our biogeographical analyses support the QTP-climate hypothesis as the likely main explanation for the East-West Eurasian disjunctions in Oleaceae. Our results also show an earlier origin of Oleaceae at ~86 Mya and the role of Tropical Asia as a main source of species dispersals., Conclusion: Our new family-wide and extensive phylogenetic tree highlights both the stable relationships within Oleaceae, including the polyphyly of the genus Chionanthus, and the need for further systematic studies within the largest and most undersampled genera of the family (Chionanthus and Jasminum). Increased sampling will also help to fine-tune biogeographical analyses across spatial scales and geological times., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

The ideal approach to Bayesian phylogenetic inference is to estimate all parameters of interest jointly in a single hierarchical model. However, this is often not feasible in practice due to the high computational cost. Instead, phylogenetic pipelines generally consist of sequential analyses, whereby a single point estimate from a given analysis is used as input for the next analysis (e.g., a single multiple sequence alignment is used to estimate a gene tree). In this framework, uncertainty is not propagated from step to step, which can lead to inaccurate or spuriously confident results. Here, we formally develop and test a sequential inference approach for Bayesian phylogenetic inference, which uses importance sampling to generate observations for the next step of an analysis pipeline from the posterior distribution produced in the previous step. Our sequential inference approach presented here not only accounts for uncertainty between analysis steps but also allows for greater flexibility in software choice (and hence model availability) and can be computationally more efficient than the traditional joint inference approach when multiple models are being tested. We show that our sequential inference approach is identical in practice to the joint inference approach only if sufficient information in the data is present (a narrow posterior distribution) and/or sufficiently many important samples are used. Conversely, we show that the common practice of using a single point estimate can be biased, for example, a single phylogeny estimate can transform an unrooted phylogeny into a time-calibrated phylogeny. We demonstrate the theory of sequential Bayesian inference using both a toy example and an empirical case study of divergence-time estimation in insects using a relaxed clock model from transcriptome data. In the empirical example, we estimate 3 posterior distributions of branch lengths from the same data (DNA character matrix with a GTR+Γ+I substitution model, an amino acid data matrix with empirical substitution models, and an amino acid data matrix with the PhyloBayes CAT-GTR model). Finally, we apply 3 different node-calibration strategies and show that divergence time estimates are affected by both the data source and underlying substitution process to estimate branch lengths as well as the node-calibration strategies. Thus, our new sequential Bayesian phylogenetic inference provides the opportunity to efficiently test different approaches for divergence time estimation, including branch-length estimation from other software., (© The Author(s) 2024. Published by Oxford University Press, on behalf of the Society of Systematic Biologists. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Background: Men and women with a migration background comprise an increasing proportion of incident human immunodeficiency virus (HIV) cases across Western Europe., Methods: To characterize sources of transmission in local transmission chains, we used partial HIV consensus sequences with linked demographic and clinical data from the opt-out AIDS Therapy Evaluation in the Netherlands (ATHENA) cohort of people with HIV in the Netherlands and identified phylogenetically and epidemiologically possible HIV transmission pairs in Amsterdam. We interpreted these in the context of estimated infection dates, and quantified population-level sources of transmission to foreign-born and Dutch-born Amsterdam men who have sex with men (MSM) within Amsterdam transmission chains., Results: We estimate that Dutch-born MSM were the predominant sources of infections among all Amsterdam MSM who acquired their infection locally in 2010-2021, and among almost all foreign-born Amsterdam MSM subpopulations. Stratifying by 2-year intervals indicated time trends in transmission dynamics, with a majority of infections originating from foreign-born MSM since 2016, although uncertainty ranges remained wide., Conclusions: Native-born MSM have predominantly driven HIV transmissions in Amsterdam in 2010-2021. However, in the context of rapidly declining incidence in Amsterdam, the contribution from foreign-born MSM living in Amsterdam is increasing, with some evidence that most local transmissions have been from foreign-born Amsterdam MSM since 2016., Competing Interests: Potential conflicts of interest. A. v. S. received funding for managing the ATHENA cohort, supported by a grant from the Dutch Ministry of Health, Welfare and Sport through the Centre for Infectious Disease Control, RIVM; and received grants unrelated to this study from the European Centre for Disease Prevention and Control (ECDC) paid to his institution. P. R. received grants unrelated to this study from Gilead Sciences, ViiV Healthcare, and Merck, paid to his institution. G. d. B. received honoraria to her institution for scientific advisory board participations for Gilead Sciences and speaker fees from Gilead Sciences (2019), Takeda (2018–2022), and ExeVir (2020–current). N. P. received grants unrelated to this study from ECDC and Gilead Sciences Hellas, paid to his institution; and received honoraria for presentations unrelated to this study from Gilead Sciences Hellas. O. R. received grants unrelated to this study from the National Institutes of Health and the Bill & Melinda Gates Foundation, paid to his institution. M. v. d. V. received grants and consulting fees unrelated to this study from ViiV Healthcare, Gilead Sciences, and MSD, paid to his institution, and is an unpaid member of the European AIDS Clinical Society guidelines committee and hepatitis C virus and hepatitis B virus national guideline committee. All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

Variation in coat color is a prominent feature in carnivores, thought to be shaped by environmental factors. As new traits could allow populations to occupy novel niches and habitats, color polymorphism may be maintained by balancing selection. Consequently, color polymorphic species may speciate more rapidly and can give rise to monomorphic daughter species. We thus predicted that, within the Carnivora, (i) speciation rate is higher in polymorphic lineages, (ii) divergence between color polymorphic lineages is more recent, and (iii) within closely related groups, polymorphic lineages are ancestral and monomorphic lineages derived. We also tested whether accelerated speciation rates relate to niche breadth, measured by the number of occupied habitats and range size. We collected data of 48 polymorphic and 192 monomorphic carnivore species, and assessed speciation rates using phylogenetic comparative methods. We found that polymorphic carnivores had higher speciation rates (λ 1  = 0.29, SD = 0.13) than monomorphic species (λ 0  = 0.053, SD = 0.044). Hidden and quantitative state speciation and extinction models inferred that color polymorphism was the main contributing factor, and that niche breadth was not of influence. Therefore, other selective forces than spatial niche segregation, such as predator-prey coevolution, may contribute to color polymorphism in wild carnivores., (© 2024. The Author(s).)

The multigene family of the major histocompatibility complex (MHC) codes for the key antigen-presenting molecules of the vertebrate immune system. In birds, duplicated MHC class II (MHC-II) genes are highly homogenized by concerted evolution, and thus, identification of their orthologous relationships across long evolutionary timescales remains challenging. Relatively low evolutionary rate of avian MHC class IIA genes has been expected to provide a promising avenue to allow such inferences, but availability of MHC-IIA sequences in nonmodel bird species has been limited until recently. Here, taking advantage from accumulating genomic resources, we identified and analyzed MHC-IIA sequences from the most basal lineage of extant birds (Palaeognathae). Conserved region of the MHC-IIA membrane-proximal domain was used to search for orthologous relationships between palaeognath birds and nonavian reptiles. First, analyses of palaeognath sequences revealed the presence of a separate MHC-IIA gene lineage (DAA3) in kiwis, which did not cluster with previously described avian MHC-IIA lineages (DAA1 and DAA2). Next, phylogenetic reconstruction showed that kiwi DAA3 sequences form a single well-supported cluster with turtle MHC-IIA. High similarity of these sequences most likely reflects their remarkable evolutionary conservation and retention of ancient orthologous relationships, which can be traced back to basal archosauromorphs ca. 250 million years ago. Our analyses offer novel insights into macroevolutionary history of the MHC and reinforce the view that rapid accumulation of high-quality genome assemblies across divergent nonmodel species can substantially advance our understanding of gene evolution., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

Background: The phylum Nematoda is incredibly diverse and includes many parasites of humans, livestock, and plants. Peptide-activated G protein-coupled receptors (GPCRs) are central to the regulation of physiology and numerous behaviors, and they represent appealing pharmacological targets for parasite control. Efforts are ongoing to characterize the functions and define the ligands of nematode GPCRs, with already most peptide GPCRs known or predicted in Caenorhabditis elegans. However, comparative analyses of peptide GPCR conservation between C. elegans and other nematode species are limited, and many nematode GPCRs remain orphan. A phylum-wide perspective on peptide GPCR profiles will benefit functional and applied studies of nematode peptide GPCRs., Results: We constructed a pan-phylum resource of C. elegans peptide GPCR orthologs in 125 nematode species using a semi-automated pipeline for analysis of predicted proteome datasets. The peptide GPCR profile varies between nematode species of different phylogenetic clades and multiple C. elegans peptide GPCRs have orthologs across the phylum Nematoda. We identified peptide ligands for two highly conserved orphan receptors, NPR-9 and NPR-16, that belong to the bilaterian galanin/allatostatin A (Gal/AstA) and somatostatin/allatostatin C (SST/AstC) receptor families. The AstA-like NLP-1 peptides activate NPR-9 in cultured cells and are cognate ligands of this receptor in vivo. In addition, we discovered an AstC-type peptide, NLP-99, that activates the AstC-type receptor NPR-16. In our pan-phylum resource, the phylum-wide representation of NPR-9 and NPR-16 resembles that of their cognate ligands more than those of allatostatin-like peptides that do not activate these receptors., Conclusions: The repertoire of C. elegans peptide GPCR orthologs varies across phylogenetic clades and several peptide GPCRs show broad conservation in the phylum Nematoda. Our work functionally characterizes the conserved receptors NPR-9 and NPR-16 as the respective GPCRs for the AstA-like NLP-1 peptides and the AstC-related peptide NLP-99. NLP-1 and NLP-99 are widely conserved in nematodes and their representation matches that of their receptor in most species. These findings demonstrate the conservation of a functional Gal/AstA and SST/AstC signaling system in nematodes. Our dataset of C. elegans peptide GPCR orthologs also lays a foundation for further functional studies of peptide GPCRs in the widely diverse nematode phylum., (© 2024. The Author(s).)

Estimating the statistical robustness of the inferred tree(s) constitutes an integral part of most phylogenetic analyses. Commonly, one computes and assigns a branch support value to each inner branch of the inferred phylogeny. The still most widely used method for calculating branch support on trees inferred under maximum likelihood (ML) is the Standard, nonparametric Felsenstein bootstrap support (SBS). Due to the high computational cost of the SBS, a plethora of methods has been developed to approximate it, for instance, via the rapid bootstrap (RB) algorithm. There have also been attempts to devise faster, alternative support measures, such as the SH-aLRT (Shimodaira-Hasegawa-like approximate likelihood ratio test) or the UltraFast bootstrap 2 (UFBoot2) method. Those faster alternatives exhibit some limitations, such as the need to assess model violations (UFBoot2) or unstable behavior in the low support interval range (SH-aLRT). Here, we present the educated bootstrap guesser (EBG), a machine learning-based tool that predicts SBS branch support values for a given input phylogeny. EBG is on average 9.4 (σ=5.5) times faster than UFBoot2. EBG-based SBS estimates exhibit a median absolute error of 5 when predicting SBS values between 0 and 100. Furthermore, EBG also provides uncertainty measures for all per-branch SBS predictions and thereby allows for a more rigorous and careful interpretation. EBG can, for instance, predict SBS support values on a phylogeny comprising 1,654 SARS-CoV2 genome sequences within 3 h on a mid-class laptop. EBG is available under GNU GPL3., Competing Interests: Conflict of Interest. The authors declare that there are no conflicts of interest regarding the publication of this paper., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

Among cartilaginous fishes, Holocephali represents the species-depauperate, morphologically conservative sister to sharks, rays and skates and the last survivor of a once far greater Palaeozoic and Mesozoic diversity. Currently, holocephalan diversity is concentrated in deep-sea species, suggesting that this lineage might contain relictual diversity that now persists in the ocean depths. However, the relationships of living holocephalans to their extinct relatives and the timescale of their diversification remain unclear. Here, we reconstruct the evolutionary history of holocephalans using comprehensive morphological and DNA sequence datasets. Our results suggest that crown holocephalans entered and diversified in deep (below 1000 m) ocean waters after the Cretaceous-Palaeogene mass extinction, contrasting with the hypothesis that this ecosystem has acted as a refugium of ancient cartilaginous fishes. These invasions were decoupled from the evolution of key features of the holocephalan body plan, including crushing dentition, a single frontal clasper, and holostylic jaw suspension, during the Palaeozoic Era. However, these invasions considerably postdated the appearance of extant holocephalan families 150 million years ago during a major period of biotic turnover in oceans termed the Mesozoic Marine Revolution. These results clarify the origins of living holocephalans as the recent diversification of a single surviving clade among numerous Palaeozoic lineages.

Terrestriality, the adaptation to life on land, is one of the key evolutionary transitions, occurring numerous times across the tree of life. Within Arthropoda, there have been several independent transitions: in hexapods, myriapods, arachnids and isopods. Isopoda is a morphologically diverse order within Crustacea, with species adapted to almost every environment on Earth. The order is divided into 11 suborders with the most speciose, Oniscidea, including terrestrial isopods such as woodlice and sea-slaters. Recent molecular phylogenetic studies have challenged traditional isopod morphological taxonomy, suggesting that several well-accepted suborders, including Oniscidea, may be non-monophyletic. This implies that terrestriality may have evolved multiple times. Current molecular hypotheses, however, are based on limited sequence data. Here, I collate available genome and transcriptome datasets for 36 isopods and four peracarid crustaceans from public sources, generate assemblies and use 970 single-copy orthologues to estimate isopod relationships and divergence times with molecular dating. The resulting phylogenetic analyses support monophyly of terrestrial isopods and suggest conflicting relationships based on nuclear ribosomal RNA sequences may be caused by long-branch attraction. Dating analyses suggest a Permo-Carboniferous origin of isopod terrestriality, much more recently than other terrestrial arthropods.

BackgroundDespite the unprecedented measures implemented globally in early 2020 to prevent the spread of SARS-CoV-2, Sweden, as many other countries, experienced a severe first wave during the COVID-19 pandemic.AimWe investigated the introduction and spread of SARS-CoV-2 into Sweden.MethodsWe analysed stored respiratory specimens (n = 1,979), sampled 7 February-2 April 2020, by PCR for SARS-CoV-2 and sequenced PCR-positive specimens. Sequences generated from newly detected cases and stored positive specimens February-June 2020 (n = 954) were combined with sequences (Sweden: n = 730; other countries: n = 129,913) retrieved from other sources for Nextstrain clade assignment and phylogenetic analyses.ResultsTwelve previously unrecognised SARS-CoV-2 cases were identified: the earliest was sampled on 3 March, 1 week before recognised community transmission. We showed an early influx of clades 20A and 20B from Italy (201/328, 61% of cases exposed abroad) and clades 19A and 20C from Austria (61/328, 19%). Clade 20C dominated the first wave (20C: 908/1,684, 54%; 20B: 438/1,684, 26%; 20A: 263/1,684, 16%), and 800 of 1,684 (48%) Swedish sequences formed a country-specific 20C cluster defined by a spike mutation (G24368T). At the regional level, the proportion of clade 20C sequences correlated with an earlier weighted mean date of COVID-19 deaths.ConclusionCommunity transmission in Sweden started when mitigation efforts still focused on preventing influx. This created a transmission advantage for clade 20C, likely introduced from ongoing cryptic spread in Austria. Therefore, pandemic preparedness should have a comprehensive approach, including capacity for large-scale diagnostics to allow early detection of travel-related cases and community transmission.

Premise: The wheat tribe, Triticeae, has been the subject of molecular phylogenetic analyses for nearly three decades, and extensive phylogenetic conflict has been apparent from the earliest comparisons among DNA-based data sets. While most previous analyses focused primarily on nuclear vs. chloroplast DNA conflict, the present analysis provides a broader picture of conflict among nuclear loci throughout the tribe., Methods: Exon data were generated from over 1000 nuclear loci using targeted sequence capture with custom baits, and nearly complete chloroplast genome sequences were recovered. Phylogenetic conflict was assessed among the trees from the chloroplast genomes, the concatenated nuclear loci, and a series of nuclear-locus subsets guided by Hordeum chromosome gene maps., Results: At the intergeneric level, the analyses collectively revealed a few broadly consistent relationships. However, the prevailing pattern was one of extensive phylogenetic conflict throughout the tribe, among both deep and shallow branches, and with the extent of the conflict varying among data subsets., Conclusions: The results suggest continual introgression or lineage sorting within and among the named lineages of the Triticeae, shaping both deep and shallow relationships in the tribe., (© 2024 The Author(s). American Journal of Botany published by Wiley Periodicals LLC on behalf of Botanical Society of America.)

Background: Human Immunodeficiency virus type 1 (HIV-1) remains a significant global health threat partly due to its ability to develop resistance to anti-retroviral therapies. HIV-1 genotype and drug resistance analysis of the polymerase (pol) sequence is a mainstay of its clinical and public health management. However, as new treatments and resistances evolve, analysis methods must change accordingly. In this study, we outline the development and implementation of a direct whole-genome sequencing approach (dWGS) using probe-capture target-enrichment for HIV-1 genotype and drug resistance analysis., Methods: We implemented dWGS and performed parallel pol Sanger sequencing for clinical samples, followed by comparative genotype and drug-resistance analysis. These HIV-1 WGS sequences were also utilised for a novel partitioned phylogenetic analysis., Results: Optimised nucleic acid extraction and DNAse I treatment significantly increased HIV-1 whole-genome coverage and depth, and improved recovery of high-quality genomes from low viral load clinical samples, enabling routine sequencing of viral loads as low as 1000 copies/mL. Overall, dWGS was robust, accurate and more sensitive for detecting low-frequency variants at drug-resistance sites compared to Sanger sequencing. Analysis of multiple sequence regions improved phylogenetic reconstruction for recombinant HIV-1 sequences compared to analysis of pol sequence alone., Conclusions: These findings demonstrate dWGS enhances HIV-1 drug-resistance analysis by quantitative variant detection and improves reconstruction of HIV-1 phylogenies compared to traditional pol sequencing. This work supports that HIV-1 dWGS is a viable option to replace Sanger sequencing for clinical and public health applications., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Angiostrongylus vasorum is a metastrongylid parasite infecting wild canids and domestic dogs. Its patchy distribution, high pathogenicity and taxonomical classification makes the evolutionary history of A. vasorum intriguing and important to study. First larval stages of A. vasorum were recovered from feces of two grey foxes, Urocyon cinereoargenteus, from Costa Rica. Sequencing and phylogenetic and haplotypic analyses of the ITS2, 18S and cytochrome oxidase subunit 1 (cox1) fragments were performed. Then p- and Nei´s genetic distance, nucleotide substitution rates and species delimitation analyses were conducted with cox1 data of the specimens collected herein and other Angiostrongylus spp. Cophylogenetic congruence and coevolutionary events of Angiostrongylus spp. and their hosts were evaluated using patristic and phenetic distances and maximum parsimony reconciliations. Specimens from Costa Rica clustered in a separate branch from European and Brazilian A. vasorum sequences in the phylogenetic and haplotype network analyses using the ITS2 and cox1 data. In addition, cox1 p-distance of the sequences derived from Costa Rica were up to 8.6 % different to the ones from Europe and Brazil, a finding mirrored in Nei´s genetic distance PCoA. Species delimitation analysis supported a separate group with the sequences from Costa Rica, suggesting that these worms may represent cryptic variants of A. vasorum, a new undescribed taxon or Angiocaulus raillieti, a synonym species of A. vasorum described in Brazil. Moreover, nucleotide substitution rates in A. vasorum were up to six times higher than in the congener Angiostrongylus cantonensis. This finding and the long time elapsed since the last common ancestor between both species may explain the larger diversity in A. vasorum. Finally, cophylogenetic congruence was observed between Angiostrongylus spp. and their hosts, with cospeciation events occurring at deeper taxonomic branching of host order. Altogether, our data suggest that the diversity of the genus Angiostrongylus is larger than expected, since additional species may be circulating in wild canids from the Americas., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Using Illumina NextSeq sequencing and bioinformatics, we identified and characterized thirty-three viral sequences of unsegmented and multipartite viral families in Aedes spp., Culex sp. and Anopheles darlingi female mosquito pools from Porto São Luiz and Pirizal, Alto Pantanal. Seventeen sequences belong to unsegmented viral families, twelve represent putative novel insect-specific viruses (ISVs) within families Chuviridae (3/33; partial genomes) and coding-complete sequences of Xinmoviridae (1/33), Rhabdoviridae (2/33) and Metaviridae (6/33); and five coding-complete sequences of already-known ISVs. Notably, two putative novel rhabdoviruses, Corixo rhabdovirus 1 and 2, were phylogenetically related to Coxipo dielmovirus, but separated from other Alpharhabdovirinae genera, sharing Anopheles spp. as host. Regarding multipartite families, sixteen segments of different putative novel viruses were identified (13 coding-complete segments) within Durnavirales (4/33), Elliovirales (1/33), Hareavirales (3/33) and Reovirales (8/33) orders. Overall, this study describes twenty-eight (28/33) putative novel ISVs and five (5/33) already described viruses using metagenomics approach., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

There are substantial gaps in our empirical knowledge of the effects of chemical exposure on aquatic life that are unlikely to be filled by traditional laboratory toxicity testing alone. One possible alternative of generating new toxicity data is cross-species extrapolation (CSE), a statistical approach in which existing data are used to predict the effect of a chemical on untested species. Some CSE models use relatedness as a predictor of chemical sensitivity, but relatively little is known about how strongly shared evolutionary history influences sensitivity across all chemicals. To address this question, we conducted a survey of phylogenetic signal in the toxicity data from aquatic animal species for a large set of chemicals using a phylogeny inferred from taxonomy. Strong phylogenetic signal was present in just nine of thirty-six toxicity datasets, and there were no clear shared properties among those datasets with strong signal. Strong signal was rare even among chemicals specifically developed to target insects, meaning that these chemicals may be equally lethal to non-target taxa, including chordates. When signal was strong, distinct patterns of sensitivity were evident in the data, which may be informative when assembling toxicity datasets for regulatory use. Although strong signal does not appear to manifest in aquatic toxicity data for most chemicals, we encourage additional phylogenetic evaluations of toxicity data in order to guide the selection of CSE tools and as a means to explore the patterns of chemical sensitivity across the broad diversity of life., (© 2024. The Author(s).)

An aerobic, Gram-stain-negative, catalase-positive, rod-shaped, and motile bacteria, designated as a strain WR6 T was isolated from soil in Republic of Korea. Strain WR6 T grew at temperatures of 10-37°C, at pH of 5.0-9.0, and at NaCl concentrations of 0-3.0% (w/v). Phylogenetic and 16S rRNA gene nucleotide sequence analysis confirmed that strain WR6 T affiliated to the genus Mesorhizobium , with the nearest relative being Mesorhizobium waimense ICMP 19557 T (98.5%). The genome of strain WR6 T was 5,035,462 bp with DNA G+C content of 62.6%. In strain WR6 T , Q-10 was sole ubiquinone; summed feature 8 (C 18:1 ω 7 c and/or C 18:1 ω 6 c ) and C 19:0 cyclo ω 8 c were predominant fatty acids; and diphosphatidylglycerol, phosphatidylglycerol, phosphatidylmethylethanolamine, phosphatidylcholine, and phosphatidylethanolamine were major polar lipids. Based on these polyphasic taxonomic data, strain WR6 T represents a novel species in the genus Mesorhizobium . Accordingly, we propose the name Mesorhizobium koreense sp. nov., with the type strain WR6 T (=KCTC 92695 T =NBRC 116021 T ).

Background: Diplomonads are anaerobic flagellates classified within Metamonada. They contain both host-associated commensals and parasites that reside in the intestinal tracts of animals, including humans (e.g., Giardia intestinalis), as well as free-living representatives that inhabit freshwater and marine anoxic sediments (e.g., Hexamita inflata). The evolutionary trajectories within this group are particularly unusual as the free-living taxa appear to be nested within a clade of host-associated species, suggesting a reversal from host-dependence to a secondarily free-living lifestyle. This is thought to be an exceedingly rare event as parasites often lose genes for metabolic pathways that are essential to a free-living life strategy, as they become increasingly reliant on their host for nutrients and metabolites. To revert to a free-living lifestyle would require the reconstruction of numerous metabolic pathways. All previous studies of diplomonad evolution suffered from either low taxon sampling, low gene sampling, or both, especially among free-living diplomonads, which has weakened the phylogenetic resolution and hindered evolutionary insights into this fascinating transition., Results: We sequenced transcriptomes from 1 host-associated and 13 free-living diplomonad isolates; expanding the genome scale data sampling for diplomonads by roughly threefold. Phylogenomic analyses clearly show that free-living diplomonads form several branches nested within endobiotic species. Moreover, the phylogenetic distribution of genes related to an endobiotic lifestyle suggest their acquisition at the root of diplomonads, while traces of these genes have been identified in free-living diplomonads as well. Based on these results, we propose an evolutionary scenario of ancestral and derived lifestyle transitions across diplomonads., Conclusions: Free-living taxa form several clades nested within endobiotic taxa in our phylogenomic analyses, implying multiple transitions between free-living and endobiotic lifestyles. The evolutionary history of numerous virulence factors corroborates the inference of an endobiotic ancestry of diplomonads, suggesting that there have been several reversals to a free-living lifestyle. Regaining host independence may have been facilitated by a subset of laterally transferred genes. We conclude that the extant diversity of diplomonads has evolved from a non-specialized endobiont, with some taxa becoming highly specialized parasites, others becoming free-living, and some becoming capable of both free-living and endobiotic lifestyles., (© 2024. The Author(s).)

The epidemiology and circulation patterns of various rhinovirus types within populations remains under-explored. We generated 803 VP4/VP2 gene sequences from rhinovirus-positive samples collected from acute respiratory illness (ARI) patients, including both in-patient and outpatient cases, between 1st January and 31st December 2014 from eleven surveillance sites across Kenya and used phylogenetics to characterise virus introductions and spread. RVs were detected throughout the year, with the highest detection rates observed from January to March and June to July. We detected a total of 114 of the 169 currently classified types. Our analysis revealed numerous virus introductions into Kenya characterized by local expansion and extinction, and extensive spatial mixing of types within the country due to the widespread transmission of the virus after an introduction. This work demonstrates that in a single year, the circulation of rhinovirus in Kenya was characterized by substantial genetic diversity, multiple introductions, and extensive geographical spread., (© 2024. The Author(s).)

Zoonotic spillovers of viruses have occurred through the animal trade worldwide. The start of the COVID-19 pandemic was traced epidemiologically to the Huanan Seafood Wholesale Market. Here, we analyze environmental qPCR and sequencing data collected in the Huanan market in early 2020. We demonstrate that market-linked severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genetic diversity is consistent with market emergence and find increased SARS-CoV-2 positivity near and within a wildlife stall. We identify wildlife DNA in all SARS-CoV-2-positive samples from this stall, including species such as civets, bamboo rats, and raccoon dogs, previously identified as possible intermediate hosts. We also detect animal viruses that infect raccoon dogs, civets, and bamboo rats. Combining metagenomic and phylogenetic approaches, we recover genotypes of market animals and compare them with those from farms and other markets. This analysis provides the genetic basis for a shortlist of potential intermediate hosts of SARS-CoV-2 to prioritize for serological and viral sampling., Competing Interests: Declaration of interests J.O.W. and N.M. receive funding from the Centers for Disease Control and Prevention (CDC) through contracts to J.O.W.’s institution unrelated to this research. M.A.S. receives contracts from the US Food & Drug Administration, US Department of Veterans Affairs, and Janssen Research & Development, all outside the scope of this work. R.F.G. is a cofounder of Zalgen Labs, a biotechnology company developing countermeasures for emerging viruses. A.C.-C. is an employee of Cultivarium, a nonprofit organization studying environmental microbes, unrelated to the scope of this work. M.W., A.L.R., J.E.P., A.R., M.A.S., E.C.H., S.A.G., J.O.W., and K.G.A. have received consulting fees and/or provided compensated expert testimony on SARS-CoV-2 and the COVID-19 pandemic. M.P.G.K. was involved in the WHO-convened SARS-CoV-2 origins mission., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Three fish blood flukes (Aporocotylidae Odhner, 1912) infect mullets (Mugiliformes: Mugilidae): Cardicola mugilis Yamaguti, 1970 and Plethorchis acanthus Martin, 1975 infect striped mullet, Mugil cephalus Linnaeus, 1758 in the Central Pacific Ocean (Hawaiian Islands) and Brisbane River (Australia), respectively; Cardicola brasiliensis Knoff & Amato, 1992 infects Lebranche mullet, Mugil liza Valenciennes, 1836 from the Southwestern Atlantic Ocean (Brazil). White mullets were cast-netted from the mouth of Deer River, a coastal saltmarsh of Mobile Bay, in the north-central Gulf of Mexico and examined for blood fluke infections. Specimens of Mugilitrema labowskiae Warren & Bullard n. gen., n. sp. were found infecting the endocardial surface and inter-trabecular spaces of the atrium, ventricle, and bulbous arteriosus. The new genus and species differ from all other aporocotylids by having the combination of two post-caecal testes, a uterus with straight ascending and descending portions, and a common genital pore. The 28S analysis recovered the new species and P. acanthus as sister taxa and Aporocotylidae as monophyletic. Carditis associated with intense infections comprised endocardial hyperplasia, resulting in a thickened cardiac endothelium. Probable dead or deteriorating eggs in the myocardium were encapsulated by granulomas composed of epithelioid histiocytes. Live eggs infected the afferent artery of gill filaments and were associated with varied hyperplasia of the overlying epithelium and haemorrhaging from the afferent artery in high-intensity infections. The new species is the first aporocotylid infecting a mullet from the northwestern Atlantic Ocean and only the second description of demonstrable endocarditis attributed to an adult fish blood fluke infection.

Computer-aided analysis of proteins or nucleic acids seems like a matter of course nowadays; however, the history of Bioinformatics and Computational Biology is quite recent. The advent of high-throughput sequencing has led to the production of "big data", which has also affected the field of virology. The collaboration between the communities of bioinformaticians and virologists already started a few decades ago and it was strongly enhanced by the recent SARS-CoV-2 pandemics. In this article, which is the first in a series on how bioinformatics can enhance virus research, we show that highly useful information is retrievable from selected general and dedicated databases. Indeed, an enormous amount of information-both in terms of nucleotide/protein sequences and their annotation-is deposited in the general databases of international organisations participating in the International Nucleotide Sequence Database Collaboration (INSDC). However, more and more virus-specific databases have been established and are progressively enriched with the contents and features reported in this article. Since viruses are intracellular obligate parasites, a special focus is given to host-pathogen protein-protein interaction databases. Finally, we illustrate several phylogenetic and phylodynamic tools, combining information on algorithms and features with practical information on how to use them and case studies that validate their usefulness. Databases and tools for functional inference will be covered in the next article of this series: Bioinformatics goes viral: II. Sequence-based and structure-based functional analyses for boosting virus research .

Chloroplast (cp) genome sequences have been extensively used for phylogenetic and evolutionary analyses, as many have been sequenced in recent years. Identification of Quercus is challenging because many species overlap phenotypically owing to interspecific hybridization, introgression, and incomplete lineage sorting. Therefore, we wanted to gain a better understanding of this genus at the level of the maternally inherited chloroplast genome. Here, we sequenced, assembled, and annotated the cp genomes of the threatened Quercus marlipoensis (160,995 bp) and Q. kingiana (161,167 bp), and mined these genomes for repeat sequences and codon usage bias. Comparative genomic analyses, phylogenomics, and selection pressure analysis were also performed in these two threatened species along with other species of Quercus. We found that the guanine and cytosine content of the two cp genomes were similar. All 131 annotated genes, including 86 protein-coding genes, 37 transfer RNA genes, and 8 ribosomal RNA genes, had the same order in the two species. A strong A/T bias was detected in the base composition of simple sequence repeats. Among the 59 synonymous codons, the codon usage pattern of the cp genomes in these two species was more inclined toward the A/U ending. Comparative genomic analyses indicated that the cp genomes of Quercus section Ilex are highly conserved. We detected eight highly variable regions that could be used as molecular markers for species identification. The cp genome structure was consistent and different within and among the sections of Quercus. The phylogenetic analysis showed that section Ilex was not monophyletic and was divided into two groups, which were respectively nested with section Cerris and section Cyclobalanopsis. The two threatened species sequenced in this study were grouped into the section Cyclobalanopsis. In conclusion, the analyses of cp genomes of Q. marlipoensis and Q. kingiana promote further study of the taxonomy, phylogeny and evolution of these two threatened species and Quercus., (© 2024. The Author(s).)

Profile mixture models capture distinct biochemical constraints on the amino acid substitution process at different sites in proteins. These models feature a mixture of time-reversible models with a common matrix of exchangeabilities and distinct sets of equilibrium amino acid frequencies known as profiles. Combining the exchangeability matrix with each profile generates the matrix of instantaneous rates of amino acid exchange for that profile. Currently, empirically estimated exchangeability matrices (e.g. the LG matrix) are widely used for phylogenetic inference under profile mixture models. However, these were estimated using a single profile and are unlikely optimal for profile mixture models. Here, we describe the GTRpmix model that allows maximum likelihood estimation of a common exchangeability matrix under any profile mixture model. We show that exchangeability matrices estimated under profile mixture models differ from the LG matrix, dramatically improving model fit and topological estimation accuracy for empirical test cases. Because the GTRpmix model is computationally expensive, we provide two exchangeability matrices estimated from large concatenated phylogenomic-supermatrices to be used for phylogenetic analyses. One, called Eukaryotic Linked Mixture (ELM), is designed for phylogenetic analysis of proteins encoded by nuclear genomes of eukaryotes, and the other, Eukaryotic and Archaeal Linked mixture (EAL), for reconstructing relationships between eukaryotes and Archaea. These matrices, combined with profile mixture models, fit data better and have improved topology estimation relative to the LG matrix combined with the same mixture models. Starting with version 2.3.1, IQ-TREE2 allows users to estimate linked exchangeabilities (i.e. amino acid exchange rates) under profile mixture models., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

Arthropoda represents the most diverse animal phylum, but clarifying the phylogenetic relationships among arthropod taxa remains challenging given the numerous arthropod lineages that diverged over a short period of time. In order to resolve the most controversial aspects of deep arthropod phylogeny, focusing on the Myriapoda, we conducted phylogenetic analyses based on ten super-matrices comprised of 751 to 1,233 orthologous genes across 64 representative arthropod species, including 28 transcriptomes that were newly generated in this study. Our findings provide unambiguous support for the monophyly of the higher arthropod taxa, Chelicerata, Mandibulata, Myriapoda, Pancrustacea, and Hexapoda, while the Crustacea are paraphyletic, with the class Remipedia supported as the lineage most closely related to hexapods. Within the Hexapoda, our results largely affirm previously proposed phylogenetic relationships among deep hexapod lineages, except that the Paraneoptera (Hemiptera, Thysanoptera, and Psocodea) was recovered as a monophyletic lineage in some analyses. The results corroborated the recently proposed phylogenetic framework of the four myriapod classes, wherein Symphyla and Pauropoda, as well as Chilopoda and Diplopoda, are each proposed to be sister taxa. The findings provide important insights into understanding the phylogeny and evolution of arthropods., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

Here, I review the dynamic history of prokaryotic phyla. Following leads set by Darwin, Haeckel and Woese, the concept of phylum has evolved from a group sharing common phenotypes to a set of organisms sharing a common ancestry, with modern taxonomy based on phylogenetic classifications drawn from macromolecular sequences. Phyla came as surprising latecomers to the formalities of prokaryotic nomenclature in 2021. Since then names have been validly published for 46 prokaryotic phyla, replacing some established names with neologisms, prompting criticism and debate within the scientific community. Molecular barcoding enabled phylogenetic analysis of microbial ecosystems without cultivation, leading to the identification of candidate divisions (or phyla) from diverse environments. The introduction of metagenome-assembled genomes marked a significant advance in identifying and classifying uncultured microbial phyla. The lumper-splitter dichotomy has led to disagreements, with experts cautioning against the pressure to create a profusion of new phyla and prominent databases adopting a conservative stance. The Candidatus designation has been widely used to provide provisional status to uncultured prokaryotic taxa, with phyla named under this convention now clearly surpassing those with validly published names. The Genome Taxonomy Database (GTDB) has offered a stable, standardized prokaryotic taxonomy with normalized taxonomic ranks, which has led to both lumping and splitting of pre-existing phyla. The GTDB framework introduced unwieldy alphanumeric placeholder labels, prompting recent publication of over 100 user-friendly Latinate names for unnamed prokaryotic phyla. Most candidate phyla remain 'known unknowns', with limited knowledge of their genomic diversity, ecological roles, or environments. Whether phyla still reflect significant evolutionary and ecological partitions across prokaryotic life remains an area of active debate. However, phyla remain of practical importance for microbiome analyses, particularly in clinical research. Despite potential diminishing returns in discovery of biodiversity, prokaryotic phyla offer extensive research opportunities for microbiologists for the foreseeable future.

Sea anemones (Order Actiniaria) are a diverse group of marine invertebrates ubiquitous across marine ecosystems. Despite their wide distribution and success, a knowledge gap persists in our understanding of their diversity within tropical systems, owed to sampling bias of larger and more charismatic species overshadowing cryptic lineages. This study aims to delineate the sea anemone diversity in Mo'orea (French Polynesia) with the use of a dataset from the Mo'orea Biocode's "BioBlitz" initiative, which prioritized the sampling of more cryptic and understudied taxa. Implementing a target enrichment approach, we integrate 71 newly sequenced samples into an expansive phylogenetic framework and contextualize Mo'orea's diversity within global distribution patterns of sea anemones. Our analysis corroborates the presence of several previously documented sea anemones in French Polynesia and identifies for the first time the occurrence of members of genera Andvakia and Aiptasiomorpha. This research unveils the diverse sea anemone ecosystem in Mo'orea, spotlighting the area's ecological significance and emphasizing the need for continued exploration. Our methodology, encompassing a broad BLAST search coupled with phylogenetic analysis, proved to be a practical and effective approach for overcoming the limitations posed by the lack of comprehensive sequence data for sea anemones. We discuss the merits and limitations of current molecular methodologies and stress the importance of further research into lesser-studied marine organisms like sea anemones. Our work sets a precedent for future phylogenetic studies stemming from BioBlitz endeavors., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Septoria leaf spot is a significant disease affecting cultivated stevia, potentially reducing yields by > 50%. The disease is caused by Septoria steviae , first identified in 1978 in Japan as a new pathogen of stevia. Understanding the origin of S. steviae could clarify how it spread to new production areas. To investigate this, 12 isolates of Septoria sp. were obtained from stevia's native range in the Amambay forests and field plantings in Paraguay from 2018 to 2020. These isolates underwent colony morphology and molecular characterization of Actin , β-Tubulin , Calmodulin , ITS , LSU , RPB2 , and TEF1α loci. GenBank sequences from S. steviae isolates collected in France, Japan, and the United States were included. Multilocus sequence phylogenetic analysis generated a maximum likelihood (ML) tree. The morphological characteristics of Paraguayan isolates were similar to those of previously reported S. steviae type cultures from Japan. The ML analysis showed that Paraguayan isolates formed a monophyletic group with S. steviae isolates from France, Japan, and the United States. During blotter tests, pycnidia and cirri of S. steviae were observed on multiple stevia seed surfaces from different sources. Further characterization confirmed viable pathogenic conidia of S. steviae . This observation suggests that S. steviae could be associated with stevia seed, possibly spreading from the center of origin to other countries. This research is the first to genetically characterize S. steviae from Paraguay and propose its potential spread mechanism from the center of origin to the rest of the world., Competing Interests: The author(s) declare no conflict of interest.

The Eurasian beaver (Castor fiber), native to Hungary, faced local extinction in 1865 and was successfully reintroduced between mid-1980s and 2008. Despite screening programs focusing on animal health during reintroduction in other countries, information about viruses in the Hungarian beaver population remains limited. Polyomaviruses (PyVs) have been identified in various rodents, and have been detected just recently in beavers by us. In this paper we present the full genome analysis of the first PyV detected in Eurasian beaver. The novel PyV was discovered in the kidney tissues of two specimens. The genome is 5244 bp, and contains four genes. Small T-antigen (STAg) and alternative large T ORF (ALTO) genes are directly fused together forming the middle T-antigen (MTAg). VP3 is absent from the genome. Its large T-antigen (LTAg) coding sequence exhibited over 15% genetic divergence from known PyVs, supporting its classification into a new species within the genus Alphapolyomavirus, suggesting to be named Alphapolyomavirus castoris. Phylogenetic analysis, based on the LTAg gene showed, that the beaver PyV forms a distinct clade with primate PyVs within the genus Alphapolyomavirus, separate from other rodent PyVs. Phylogenetic study of the VP1 gene however showed this virus to belong in a distinct clade with the same primate PyVs, and additionally PyVs from rodents and a myocastor, which suggest host virus co-evolution. The virus detection of the euthanized beavers suggests an apathogenic persistent infections. The aquatic lifestyle of beavers may influence virus transmission, warranting further exploration of undiscovered viruses in beavers., Competing Interests: Declaration of competing interest None., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

The aim of this study was to investigate to what extent fragments of the HEV genome could be used for accurate diagnostics and inference of viral population-scale processes. For this, we selected all the published whole genome sequences from the NCBI GenBank and trimmed them to various fragment lengths (ORF1,2,3, ORF1, ORF2, ORF3, 493 nt in ORF2 and 148 nt in ORF2). Each of the fragment lengths was used to infer the richness and diversity of the viral sequence types, typing accuracy, and potential use in phylodynamics. The results obtained from the different fragments were compared. We observed that, generally, the longer the nucleic acid fragment used in typing, the better the accuracy in predicting the viral subtype. However, the dominant HEV subtypes circulating in Europe were relatively well classified even by the 493 nt fragment, with false negative rates as low as 8 in 1000 typed sequences. Most fragments also give comparable results in analyses of population size, albeit with shorter fragments showing a broader 95 % highest posterior density interval and less obvious increase of the viral effective population size. The reconstructed phylogenies of a heterochronous subset indicated a good concordance between all the fragments, with the major clades following similar branching patterns. Furthermore, we have used the HEV sequence data from the Netherlands available in the HEVnet database as a case study for reconstruction of population size changes in the past decades. This data showed that molecular and epidemiological results are concordant and point to an increase in the viral effective population size underlying the observed increase in incidence of acute HEV infection cases. In the absence of whole genome sequencing data, the 493 bp fragment can be used for analyzing HEV strains currently circulating in Europe, as it is informative for describing short term population-scale processes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Despite considerable research efforts in recent years, the deeper phylogenetic relationships among skipper butterflies (Hesperiidae) remain unresolved. This is primarily because of limited sampling, especially within Asian and African lineages. In this study, we consolidated previous data and extensively sampled Asian and African taxa to elucidate the phylogenetic relationships within Hesperiidae. The molecular dataset comprised sequences from two mitochondrial and two nuclear gene regions from 563 species that represented 353 genera. Our analyses revealed seven subfamilies within Hesperiidae: Coeliadinae, Euschemoninae, Eudaminae, Pyrginae, Heteropterinae, Trapezitinae, and Hesperiinae. The systematics of most tribes and genera aligned with those of prior studies. However, notable differences were observed in several tribes and genera. Overall, the position of taxa assigned to incertae sedis in Hesperiinae is largely clarified in this study. Our results strongly support the monophyly of the tribe Tagiadini (Pyrginae), and the systematics of some genera are clarified with comprehensive discussion. We recognize 15 tribes within the subfamily Hesperiinae. Of these, nine tribes are discussed in detail: Aeromachini, Astictopterini, Erionotini, Unkanini (new status), Ancistroidini, Ismini (confirmed status), Plastingini (new status), Gretnini (confirmed status), and Eetionini (confirmed status). We propose four subtribes within Astictopterini: Hypoleucina subtrib.n., Aclerosina, Cupithina, and Astictopterina. Furthermore, we describe a new genus (Hyarotoidesgen.n.) and reinstate two genera (Zeareinst.stat. and Separeinst.stat.) as valid. Additionally, we propose several new combinations: Zea mythecacomb.n.,Sepa bononiacomb.n. & reinst.stat., and Sepa umbrosacomb.n. This study, with extensive sampling of Asian and African taxa, greatly enhances the understanding of the knowledge of the skipper tree of life., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Species in Alternaria sections Infectoriae and Pseudoalternaria are commonly isolated from agricultural crops and a variety of other plant hosts. With the increasing appreciation that species from these two sections are often the dominant taxa recovered from important cereal crops, the need for improved understanding of their biodiversity and taxonomy has grown. Given that morphological characteristics and existing molecular markers are not sufficient for distinguishing among species, we expanded the genomic resources for these sections to support research in biosystematics and species diagnostics. Whole genome assemblies for 22 strains were generated, including the first genomes from section Infectoriae or Pseudoalternaria strains sampled from Canada, which significantly increases the number of publicly released genomes, particularly for section Pseudoalternaria . We performed comprehensive phylogenomic analyses of all available genomes (n = 39) and present the first robust phylogeny for these taxa. The segregation of the two sections was strongly supported by genomewide data, and multiple lineages were detected within each section. We then provide an overview of the biosystematics of these groups by analyzing two standard molecular markers from the largest sample of section Infectoriae and Pseudoalternaria strains studied to date. The patterns of relative diversity suggest that, in many cases, multiple species described based on minor morphological differences may actually represent different strains of the same species. A list of candidate loci for development into new informative molecular markers, which are diagnostic for sections and lineages, was created from analyses of phylogenetic signals from individual genes across the entire genome.

Nine viruses of the bean common mosaic virus subgroup of potyviruses are major international crop pathogens, but their phylogenetically closest relatives from non-crop plants have mostly been found only in SE Asia and Oceania, which is thus likely to be their "centre of emergence". We have compared over 700 of the complete genomic ORFs of the crop pandemic and the non-crop viruses in various ways. Only one-third of crop virus genomes are non-recombinant, but more than half the non-crop virus genomes are. Four of the viruses were from crops domesticated in the Old World (Africa to SE Asia), and the other five were from New World crops. There was a temporal signal in only three of the crop virus datasets, but it confirmed that the most recent common ancestors of all the crop viruses were before inter-continental marine trade started after 1492 CE, whereas all the crown clusters of the phylogenies are from after that date. The non-crop virus datasets are genetically more diverse than those of the crop viruses, and Tajima's D analyses showed that their populations were contracting, and only one of the crop viruses had a significantly expanding population. dN/dS analyses showed that most of the genes and codons in all the viruses were under significant negative selection, and the few that were under significant positive selection were mostly in the PIPO-encoding region of the P3 protein, or the PIPO protein itself. Interestingly, more positively selected codons were found in non-crop than in crop viruses, and, as the hosts of the former were taxonomically more diverse than the latter, this may indicate that the positively selected codons are involved in host range determination; AlphaFold3 modelling was used to investigate this possibility.

Background: Emerging zoonotic diseases arise from cross-species transmission events between wild or domesticated animals and humans, with bats being one of the major reservoirs of zoonotic viruses. Viral metagenomics has led to the discovery of many viruses, but efforts have mainly been focused on some areas of the world and on certain viral families., Methods: We set out to describe full-length genomes of new picorna-like viruses by collecting feces from hundreds of bats captured in different regions of Spain. Viral sequences were obtained by high-throughput Illumina sequencing and analyzed phylogenetically to classify them in the context of known viruses. Linear discriminant analysis (LDA) was performed to infer likely hosts based on genome composition., Results: We found five complete or nearly complete genomes belonging to the family Picornaviridae, including a new species of the subfamily Ensavirinae. LDA suggested that these were true vertebrate viruses, rather than viruses from the bat diet. Some of these viruses were related to picornaviruses previously found in other bat species from distant geographical regions. We also found a calhevirus genome that most likely belongs to a proposed new family within the order Picornavirales, and for which genome composition analysis suggested a plant host., Conclusions: Our findings describe new picorna-like viral species and variants circulating in the Iberian Peninsula, illustrate the wide geographical distribution and interspecies transmissibility of picornaviruses, and suggest new hosts for calheviruses., (© 2024. The Author(s).)

The Streptophyta emerged about a billion years ago. Nowadays, this branch of the green lineage is most famous for one of its clades, the land plants (Embryophyta). Although Embryophyta make up the major share of species numbers in Streptophyta, there is a diversity of probably >5000 species of streptophyte algae that form a paraphyletic grade next to land plants. Here, we focus on the deep divergences that gave rise to the diversity of streptophytes, hence particularly on the streptophyte algae. Phylogenomic efforts have not only clarified the position of streptophyte algae relative to land plants, but recent efforts have also begun to unravel the relationships and major radiations within streptophyte algal diversity. We illustrate how new phylogenomic perspectives have changed our view on the evolutionary emergence of key traits, such as intricate signalling networks that are intertwined with multicellular growth and the chemodiverse hotbed from which they emerged. These traits are key for the biology of land plants but were bequeathed from their algal progenitors., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Annals of Botany Company.)

Range contraction and expansion from glaciation have led to genetic divergence that may be particularly pronounced in fossorial species with low dispersal. The plains pocket gopher (Geomys bursarius) is a fossorial species that ranges widely across North America but has a poorly understood phylogeny. We used mitogenomes (14,996 base pairs) from 56 individuals across seven subspecies, plus two outgroup species, to assess genetic divergence from minimum spanning trees, measure genetic distances, and infer phylogenetic trees using BEAST. We found G. b. wisconsinensis was monophyletic with recent divergence. Further assessment is needed for G. b. major because it was paraphyletic and exhibited inconsistent groupings with other clades. Importantly, we identified G. b. illinoensis as being genetically distinct and monophyletic likely due to a unique colonization event eastward across the Mississippi River. Because G. b. illinoensis faces continued pressures from niche reduction and habitat loss, we recommend that G. b. illinoensis be considered an evolutionary significant unit warranting conservation actions to promote connectivity and restore suitable habitat. Such conservation efforts should benefit other grassland species including those originating from clades west of the Mississippi River that may also be evolutionary significant units., (© The American Genetic Association. 2024.)

Background: Pseudomonas juntendi is a newly identified opportunistic pathogen, of which we have limited understanding. P. juntendi strains are often multidrug resistant, which complicates clinical management of infection., Methods: A strain of Pseudomonas juntendi (strain L4326) isolated from feces was characterized by MALDI-TOF-MS and Average Nucleotide Identity BLAST. This strain was further subject to whole-genome sequencing and Maximum Likelihood phylogenetic analysis. The strain was phenotypically characterized by antimicrobial susceptibility testing and conjugation assays., Results: We have isolated the novel P. juntendi strain L4236, which was multidrug resistant, but retained sensitivity to amikacin. L4236 harbored a megaplasmid that encoded bla OXA-1 and a novel bla IMP-1 resistance gene variant. P. juntendi strain L4236 was phylogenetically related to P. juntendi strain SAMN30525517., Conclusion: A rare P. juntendi strain was isolated from human feces in southern China with a megaplasmid coharboring bla IMP-1-like and bla OXA-1 . Antimicrobial selection pressures may have driven acquisition of drug-resistance gene mutations and carriage of the megaplasmid., (© 2024. The Author(s).)

There are a staggering number of publicly available bacterial genome sequences (at writing, 2.0 million assemblies in NCBI's GenBank alone), and the deposition rate continues to increase. This wealth of data begs for phylogenetic analyses to place these sequences within an evolutionary context. A phylogenetic placement not only aids in taxonomic classification but informs the evolution of novel phenotypes, targets of selection, and horizontal gene transfer. Building trees from multi-gene codon alignments is a laborious task that requires bioinformatic expertise, rigorous curation of orthologs, and heavy computation. Compounding the problem is the lack of tools that can streamline these processes for building trees from large-scale genomic data. Here we present OrthoPhyl, which takes bacterial genome assemblies and reconstructs trees from whole genome codon alignments. The analysis pipeline can analyze an arbitrarily large number of input genomes (>1200 tested here) by identifying a diversity-spanning subset of assemblies and using these genomes to build gene models to infer orthologs in the full dataset. To illustrate the versatility of OrthoPhyl, we show three use cases: E. coli/Shigella, Brucella/Ochrobactrum and the order Rickettsiales. We compare trees generated with OrthoPhyl to trees generated with kSNP3 and GToTree along with published trees using alternative methods. We show that OrthoPhyl trees are consistent with other methods while incorporating more data, allowing for greater numbers of input genomes, and more flexibility of analysis., Competing Interests: Conflicts of interest The author(s) declare no conflict of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Genetics Society of America.)

Bayesian phylogenetic inference is powerful but computationally intensive. Researchers may find themselves with two phylogenetic posteriors on overlapping data sets and may wish to approximate a combined result without having to re-run potentially expensive Markov chains on the combined data set. This raises the question: given overlapping subsets of a set of taxa (e.g. species or virus samples), and given posterior distributions on phylogenetic tree topologies for each of these taxon sets, how can we optimize a probability distribution on phylogenetic tree topologies for the entire taxon set? In this paper we develop a variational approach to this problem and demonstrate its effectiveness. Specifically, we develop an algorithm to find a suitable support of the variational tree topology distribution on the entire taxon set, as well as a gradient-descent algorithm to minimize the divergence from the restrictions of the variational distribution to each of the given per-subset probability distributions, in an effort to approximate the posterior distribution on the entire taxon set., (© 2024. The Author(s).)

Aim: We aim to determine the evolutionary origins and population genetics of mallard-like ducks of Oceania, greater Indonesia, and the Philippines., Location: Oceania, greater Indonesia, and the Philippines., Taxon: Mallard (Anas platyrhynchos), Pacific black duck (A. superciliosa spp.), and Philippine duck (A. luzonica) METHODS: Thousands of nuclear ddRAD-seq loci and the mitochondrial DNA control region were assayed across individuals representative of each species' range. We assessed population structure and phylogenetic relationships, as well as estimated demographic histories to reconstruct the biogeographical history of each species., Results: Philippine and Pacific black ducks represent unique genetic lineages that diverged from the mallard 1-2 million years ago. We find no support for the Philippine duck representing a hybrid species as once posited; however, their low levels of genetic diversity requires further attention. We find a lack of substructure among Philippine ducks. However, we found pronounced differentiation between subspecies of Pacific black ducks, especially between A. s. superciliosa from New Zealand and A. s. rogersi from Australia, Papua New Guinea, and Timor-Leste, Indonesia. Anas superciliosa pelewensis gave mixed results; individuals from the Solomon Islands were differentiated from the other subspecies, but those from the island of Aunu'u, American Samoa, were genetically more similar to A. s. rogersi than A. s. pelewensis samples from the Solomon Islands. Finally, we find limited evidence of interspecific gene flow at evolutionary scales, and mallard introgression among contemporary samples., Main Conclusions: Mallard-like ducks radiated across Oceania, greater Indonesia, and the Philippines within the last 2 million years. Only the Pacific black duck showed unique sub-structuring that largely followed known sub-species ranges, except for A. s. pelewensis. We posit that the high interrelatedness among Solomon Island samples suggests that their genetic distinctiveness may simply be the result of high levels of genetic drift. In contrast, we conclude that mainland Australian Pacific black ducks were the most likely source for the recent colonization of American Samoa. As a result, our findings suggest that either the A. s. pelewensis subspecies designations and/or its geographical range may require re-evaluation. Continued re-evaluation of evolutionary and taxonomic relationships is necessary when attempting to reconstruct and understand biogeographical histories, with important implications towards any attempts to implement conservation strategies., (Published by Elsevier Inc.)

Rhodiola, belonging to the Crassulaceae family, is a perennial herbaceous plant genus. There are about 90 Rhodiola species worldwide, some of which have been reported to have medicinal properties. Rhodiola sachalinensis is a perennial medicinal herb within this genus and, in the present study, its chloroplast genome was sequenced, assembled, annotated and compared with 24 other Rhodiola species. The results obtained show that the chloroplast genome of R. sachalinensis is 151 595 bp long and has a CG content of 37.7%. The inverted repeats (IR) region of the Rhodiola chloroplast genome is the most conserved region, with the main differences being observed in the ycf1 and ndhF genes at the IRb-small single copy boundary, and rps19 and trnH genes at the IRa-large single copy boundary. Phylogenetic analysis showed that Rhodiola species form two major clades, and species with recorded medicinal properties, clustered together in one branch except for R. dumulosa. Within the genus, R. sachalinensis is most closely related to Rhodiola rosea, although comparative analyses showed that only R. sachalinensis and Rhodiola subopposita contained the psbZ gene, which encodes a highly conserved protein subunit of the Photosystem II core complex. Overall, the present study contributes to the understanding of the chloroplast genome of Rhodiola species, and provides a theoretical basis for the study of their genetic diversity and possible use as medicinal plants., (© 2024 The Author(s). FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Last year, we published research using phylogenetic comparative methods (PCMs) to reveal no phylogenetic evidence for elevated lineage-level extinction rates in angiosperms across K-Pg (Thompson JB, Ramírez-Barahona S. 2023 No phylogenetic evidence for angiosperm mass extinction at the Cretaceous-Palaeogene (K-Pg) boundary. Biol. Lett. 19 , 20230314. (doi:10.1098/rsbl.2023.0314)), results that are in step with the global angiosperm fossil record. In a critique of our paper (Hagen ER. 2024 A critique of Thompson and Ramírez-Barahona (2023) or: how I learned to stop worrying and love the fossil record. EcoEvoRxiv. (doi:10.32942/X2631W)), simulation work is presented to argue we erred in our methodological choices and interpretations, and that we should have deferred to fossil evidence. In our opinion, underlying this critique are poor methodological choices on simulations and philosophical problems surrounding the definition of a mass extinction event, which leads to incorrect interpretations of both the fossil record and PCMs. We further argue that deferring to one source of evidence in favour of the other shuts the door to important evolutionary and philosophical questions.

Constraint is a fundamental concept in evolutionary theory. Morphology and ecology both are limited by functional, historical and developmental factors to a subset of the theoretical range species could occupy. Cat-like carnivorans (Feliformia) offer a unique opportunity to investigate phenotypic constraint, as several feliform clades are purported to be limited to generalized ecomorphological roles, while others possess extremely specialized durophagous (bone-crushing) and sabretooth morphology. We investigated the evolutionary history of feliforms by considering their phylogeny, morphological disparity and rates of evolution. We recover results that show a mosaic pattern exists in the degree of morphological disparity per anatomical region per clade and ecology. Non-hypercarnivores, such as viverrids (civets and genets), Malagasy euplerids and lophocyonids (extinct hypocarnivores), have the greatest dental disparity, while hypercarnivores (felids, nimravids, many hyaenids) have the lowest dental disparity but highest cranial and mandibular disparity (excluding dentition). However, high disparity is not necessarily associated with high rates of evolution, but instead with ecological radiations. We reveal that relationships between specialization and disparity are not as simple as past research has concluded. Instead, morphological disparity results from an anatomical mosaic of evolution, where different ecologies correlate with and likely channel unique patterns/combinations of disparity per anatomical partition.

Onychophora are cryptic, soil-dwelling invertebrates known for their biogeographic affinities, diversity of reproductive modes, close phylogenetic relationship to arthropods, and peculiar prey capture mechanism. The 216 valid species of Onychophora are grouped into two families - Peripatopsidae and Peripatidae - and apart from a few relationships among major lineages within these two families, a stable phylogenetic backbone for the phylum has yet to be resolved. This has hindered our understanding of onychophoran biogeographic patterns, evolutionary history, and systematics. Neopatida, the Neotropical clade of peripatids, has proved particularly difficult, with recalcitrant nodes and low resolution, potentially due to rapid radiation of the group during the Cretaceous. Previous studies have had to compromise between number of loci and number of taxa due to limitations of Sanger sequencing and phylotranscriptomics, respectively. Additionally, aspects of their genome size and structure have made molecular phylogenetics difficult and data matrices have been affected by missing data. To address these issues, we leveraged recent, published transcriptomes and the first high quality genome for the phylum and designed a high affinity ultraconserved element (UCE) probe set for Onychophora. This new probe set, consisting of ∼ 20,000 probes that target 1,465 loci across both families, has high locus recovery and phylogenetic utility. Phylogenetic analyses recovered the monophyly of major clades of Onychophora and revealed a novel lineage from the Neotropics that challenges our current understanding of onychophoran biogeographic endemicity. This new resource could drastically increase the power of molecular datasets and potentially allow access to genomic scale data from archival museum specimens to further tackle the issues exasperating onychophoran systematics., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Bovine betacoronavirus (BoCoV) is a pneumoenteric pathogen of cattle that is closely related to human coronavirus OC43. Vaccines are administered to protect against diseases caused by BoCoV, but knowledge gaps exist with regard to correlates of protection and the effect of immune evasion on driving evolution. In this study, immune epitopes were mapped onto BoCoV structural proteins, including spike and haemagglutinin esterase (HE), and then supported with targeted gene sequencing of Irish clinical isolates and selective pressure analysis. Increased prevalence of diversifying selection and amino acid changes in some mapped immune epitopes suggests that immune escape is selecting for non-synonymous mutations arising in these regions. Selection analysis and sequencing provided increased support for neutralising antibody (nAb) epitopes compared to others, suggesting that nAbs are an important arm of the immune response to BoCoV. Phylogenetic analysis of spike and HE sequences showed that Irish isolates from this study were in the European clade, except for one HE sequence that sat in the Asian/American clade, while the spike gene of this sample was in the European clade. Recombination between a European and an Asian/American isolate would give rise to such a sequence. This study has gathered evidence suggesting that pressure to evade the nAb response is contributing to BoCoV evolution.

Four species of the genus Wrangelia are presently known from the western Atlantic Ocean: W. argus, W. bicuspidata, W. penicillata, and W. gordoniae, with the first three historically being reported from Bermuda. Morphological and molecular barcode (COI-5P) and phylogenetic analyses used in this study (SSU, LSU, rbcL) indicated eight species groupings of Wrangelia in Bermuda, excluding two of the historically recognized species, retaining only W. argus while adding seven new species, of which six are formally described. What had been historically reported as W. penicillata from Bermuda was shown to be distinct from Mediterranean Sea specimens (type locality) and was shown to be a mixture of W. hesperia sp. nov. and W. incrassata sp. nov. Along with these two, three other new species (W. laxa sp. nov., W. ryancraigii sp. nov., and W. secundiramea sp. nov.) have complete rhizoidal cortication tightly covering axial cells of indeterminate axes below the apices, distinguishing them from the two local incompletely corticated congeners W. argus and W. abscondita sp. nov., the latter a morphologically cryptic sister species with W. bicuspidata from the Caribbean Sea. Only one of the new species, W. ryancraigii, has thus far been observed in the mesophotic zone off the Bermuda platform, and it is morphologically cryptic with the euphotic zone's W. laxa., (© 2024 Phycological Society of America.)

The evolution of animals and their gut symbionts is a complex phenomenon, obscured by lability and diversity. In social organisms, transmission of symbionts among relatives may yield systems with more stable associations. Here, we study the history of a social insect symbiosis involving cephalotine ants and their extracellular gut bacteria, which come predominantly from host-specialized lineages. We perform multi-locus phylogenetics for symbionts from nine bacterial orders, and map prior amplicon sequence data to lineage-assigned symbiont genomes, studying distributions of rigorously defined symbionts across 20 host species. Based on monophyly and additional hypothesis testing, we estimate that these specialized gut bacteria belong to 18 distinct lineages, of which 15 have been successfully isolated and cultured. Several symbiont lineages showed evidence for domestication events that occurred later in cephalotine evolutionary history, and only one lineage was ubiquitously detected in all 20 host species and 48 colonies sampled with amplicon 16S rRNA sequencing. We found evidence for phylogenetically constrained distributions in four symbionts, suggesting historical or genetic impacts on community composition. Two lineages showed evidence for frequent intra-lineage co-infections, highlighting the potential for niche divergence after initial domestication. Nearly all symbionts showed evidence for occasional host switching, but four may, more often, co-diversify with their hosts. Through our further assessment of symbiont localization and genomic functional profiles, we demonstrate distinct niches for symbionts with shared evolutionary histories, prompting further questions on the forces underlying the evolution of hosts and their gut microbiomes., (© 2024 John Wiley & Sons Ltd.)

Gene duplication followed by nucleotide differentiation is one of the simplest mechanisms to develop new functions for genes. However, the evolutionary processes underlying the divergence of multigene families remain controversial. We used multigene families found within the diversity of toxic proteins in centipede venom to test two hypotheses related to venom evolution: the two-speed mode of venom evolution and the rapid accumulation of variation in exposed residues (RAVER) model. The two-speed mode of venom evolution proposes that different types of selection impact ancient and younger venomous lineages with negative selection being the predominant form in ancient lineages and positive selection being the dominant form in younger lineages. The RAVER hypothesis proposes that, instead of different types of selection acting on different ages of venomous lineages, the different types of selection will selectively contribute to amino acid variation based on whether the residue is exposed to the solvent where it can potentially interact directly with toxin targets. This hypothesis parallels the longstanding understanding of protein evolution that suggests that residues found within the structural or active regions of the protein will be under negative or purifying selection, and residues that do not form part of these areas will be more prone to positive selection. To test these two hypotheses, we compared the venom of 26 centipedes from the order Scolopendromorpha from six currently recognized species from across North America using both transcriptomics and proteomics. We first estimated their phylogenetic relationships and uncovered paraphyly among the genus Scolopendra and evidence for cryptic diversity among currently recognized species. Using our phylogeny, we then characterized the diverse venom components from across the identified clades using a combination of transcriptomics and proteomics. We conducted selection-based analyses in the context of predicted three-dimensional properties of the venom proteins and found support for both hypotheses. Consistent with the two-speed hypothesis, we found a prevalence of negative selection across all proteins. Consistent with the RAVER hypothesis, we found evidence of positive selection on solvent-exposed residues, with structural and less-exposed residues showing stronger signal for negative selection. Through the use of phylogenetics, transcriptomics, proteomics, and selection-based analyses, we were able to describe the evolution of venom from an ancient venomous lineage and support principles of protein evolution that directly relate to multigene family evolution., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)