Molecular Phylogenetics and Evolution

We provide the first comprehensive analysis of the origin of two enigmatic Satyrinae genera of uncertain affinities. Calisto, the only Satyrinae genus from the West Indies and endemic to these islands, has resisted numerous attempts at phylogenetic placement, regardless of the data type or methods used. Llorenteana, a monotypic genus from northwestern Mexico, has never been included in a molecular phylogenetic study, and past authors have placed it in five different genera and subtribes. We used mostly published genomic data, but also newly sequenced whole genome data from museum specimens and old DNA extracts, extracted BUSCO genes and prepared several datasets. These datasets differed in the degree of heterogeneity and saturation, the number of nucleotide positions used (all positions or only the first two), and were analyzed as nucleotides or as amino acids. We employed several methods for phylogenetic reconstruction using both partitioned and mixture models, as well as ASTRAL, and we inferred divergence times and ancestral areas of origin for Calisto and Llorenteana. The phylogenetic placement of Calisto varied among datasets when we used partitioned models and ASTRAL; however, most datasets resulted in the same relationships under mixture models. Our results suggest that Calisto is part of a clade of Old World origin that colonized the New World from north to south, thus sharing ancestry with Nearctic taxa. Llorenteana constitutes one of the earliest splits within the Euptychiina, a subtribe of Neotropical origin, but descending together with the Pronophilina from Nearctic ancestors. We propose the recognition of Erebiina stat. rev. as the only subtribe comprising the former Calistina syn. nov., Callerebiina syn. nov, Maniolina syn. nov., and Ypthimina syn. nov.

The temperate rainforests of the Pacific Northwest of North America harbor many endemic taxa whose evolutionary histories have been shaped by major climatic and geologic events. The enigmatic taildropper slugs (genus Prophysaon) are one example, notable for their ability to autonomize their tails to escape predators. Despite extensive work uncovering the evolutionary history of individual lineages, relationships among the nine recognized species of Prophysaon remain poorly understood due to insufficient molecular data. To address this, we collected transcriptomes for six of the nine currently accepted species of Prophysaon. Using these data, we were able to resolve species relationships, calling into question the existing subgeneric classification based on morphology. We also detected undescribed phenotypic diversity within the P. andersonii--P. foliolatum species complex, with molecular data supporting the distinctness of two phenotypically distinct populations from Washington. Finally, our transcriptomic data suggest a moderate role of introgression in shaping the evolutionary history of Prophysaon. Here, we synonymize the subgenus Mimetarion with nominotypical Prophysaon. Future work should further investigate whether the undescribed diversity detected here represents species level differentiation.

AO_SCPLOWBSTRACTC_SCPLOWThis study reconstructs the phylogeny of an expansive set of Neotropical leaf beetles in the subfamily Chrysomelinae. From 33 species in the genus Platyphora Gistel, and an additional 37 species representing 16 beetle genera, five genes, three nuclear, and two mitochondrial, were sequenced and used to obtain a well-supported molecular phylogeny using both Bayesian and Maximum Likelihood. The subtribes Chrysomelina and Doryphorina (sensu Daccordi 1982) were monophyletic, while the genus Platyphora was polyphyletic. The genus Leptinotarsa Chevrolat is confirmed to be distinct from Stilodes Chevrolat. Host plant family was recorded for both adults and larvae using direct observations where possible. Ancestral host plant use was reconstructed using Bayesian trait analyses. A complicated history of host plant switches among a restricted set of plant families is revealed: In the paraphyletic Platyphora, one clade that includes Proseicela and Leptinotarsa had two switches from Asclepiadiodeae to Solanaceae, one switch to Moraceae, and one switch to Malpighiaceae, another Platyphora clade had switches between Asteraceae and Rauvolfioideae, and from Rauvolfioideae to Asclepiadiodeae, with other members of the same clade feeding on Boraginaceae and Convolvulaceae. All species included in the clade containing Tritaenia and Stilodes fed on Malpighiaceae, and all species included in the Cosmogramma and Calligrapha clade fed on Malvaceae.

Diversisporales comprises species with worldwide distribution that produce glomoid, otosporoid, or tricisporoid spores. The recent reorganization of the order by Oehl et al. (2016) recognizes two families, Diversisporaceae and Corymbiglomeraceae, comprising one and five genera, respectively. Several Glomeromycotan specimens collected in northern and southeastern Mexico and in French Polynesian atolls were characterized using both morphological and molecular analyses. Phylogenetic inference revealed that they represent new members in the Diversisporales, supporting the reorganization of the genus Redeckera into three independent lineages: Albocarpum gen. nov., with A. arenaceum sp. nov., A. leptohyphum sp. nov., and A. fulvum comb. nov., Pulvinocarpum pulvinatum gen. et comb. nov., and Redeckera, which retains five species, including R. varelae sp. nov. In addition, we described Melanocarpum mexicanum gen. et sp. nov. and Diversispora papillosa sp. nov. A broader phylogeny, based on eDNA sequences and representative of Diversisporales species, including the newly described taxa, further supported the split of Redeckera and suggested three additional clades likely corresponding to a new family and two new genera, awaiting the discovery of representative morphospecies to be formally described. Using eDNA sequences metadata, the occurrences of the newly described taxa were mapped, allowing to recognize distribution patterns, mostly in the pantropical zone, distinguish widespread and rare species, and suggest possible endemisms. Finally, the coexistence of species forming large sporocarps (A. fulvum and A. leptohyphum) alongside species forming spores in loose aggregates (A. arenaceum), prompted us to propose a possible sporulation dimorphism in Albocarpum, an argument previously raised to explain the nested placement of Corymbiglomus and Paracorymbiglomus within the Redeckera clade.

AO_SCPLOWBSTRACTC_SCPLOWSpecies represent a fundamental unit of biodiversity in evolutionary biology, but the nature of the speciation continuum and inadequate sampling of organisms with broad distributions provide substantial challenges to species delimitation. The Pacific Treefrog complex (Pseudacris regilla sensu lato) is an iconic but systematically poorly understood group of chorus frogs inhabiting a vast portion of western North America. Current studies tentatively recognize three species in this complex (P. hypochondriaca, P. regilla, P. sierra), but disagreement remains among morphological, mitochondrial, and nuclear genetic data. In this study, we used thorough geographic sampling and thousands of nuclear loci to clarify the phylogenetic relationships and divergence history of P. regilla s.l. lineages and recommend a new taxonomic arrangement. We inferred recent divergence with gene flow between P. regilla and P. sierra, topological inconsistencies, and genealogical divergence indices that place P. regilla and P. sierra firmly in the "gray zone" of speciation. Pseudacris hypochondriaca diverged over 0.5 Ma without gene flow until recent secondary contact with the "north" (P. regilla + P. sierra) lineage. Based on inferences from our genomic data and recently published acoustic signal differentiation, we propose a two species taxonomy for this complex, recognizing the "north" lineage as P. regilla. Our study shows how extensive geographic sampling, high-throughput sequencing, and multiple analytical approaches can resolve systematic uncertainties in challenging species complexes.

Abstract/SummaryHere we untangle the systematics of the Asiatic white-eye complex (Zosterops spp.) to better understand the early stages of a recent island radiation. We adopt an integrative approach involving allelic data, genome-scale single nucleotide polymorphisms (SNPs), and museum-based morphometrics coupled with a comprehensive sampling to provide the most holistic understanding of the group to date. The island lineages of Asiatic white-eyes across Indonesia, the Philippines, East Asia, the adjacent oceanic islands of the Western Pacific underwent a deep split separating Zosterops everetti and Z. nigrorum in the Phillippines from a very rapid radiation including Z. japonicus, Z. meyeni, and Z. montanus in the Philippines, Japan, and Indonesia. Z. nigrorum catarmanensis on Camiguin South in the Philippines was found to be nested within Z. montanus and a species limit between Z. nigrorum populations on Panay and Luzon was strongly supported. Phylogenetic splits and population structure were detected within the clade containing Z. japonicus, Z. meyeni, and Z. montanus. A well-supported split separates a northern group including Northern Philippines Z. montanus subspecies, Z. meyeni, and Z. japonicus from the southerly Z. montanus taxa. This creates a paraphyletic Z. montanus. However, based on speciation rates within the broader Asiatic white-eye clade this break likely does not yet represent evolutionarily independent species lineages. Morphological evolution is taking place within the Asiatic white-eyes especially within the robust, large-billed subspecies of Z. japonicus on the oceanic islands of Japan and in the newly identified yellow-morph of Z. montanus on Camiguin South.

Incilius marmoreus inhabits an extensive range along the Pacific Coast of Mexico and a smaller allopatric region in the State of Veracruz, exhibiting an unusual distribution among herpetofauna. Gunther (1901) classified the Pacific coastal toads Bufo argillaceus and B. lateralis as conspecific with Incilius [Bufo] marmoreus, which has its type locality in Veracruz. Here, we adopt a multidisciplinary approach to reevaluate the phylogeography and taxonomy of I. marmoreus by gathering and analyzing morphological data and conducting phylogenetic and population genetic analyses from genome-wide SNP data. Our results uphold the current taxonomy by concurring with Gunther (1901). Our phylogenetic and population genetic analyses suggest that I. marmoreus from Veracruz are closely related to those from Oaxaca whilst coalescent analyses recovered a north-south split along the Pacific Coast estimated to have occurred [~]0.86 Mya followed by a shallow east-west split in the southern lineage that separates the Pacific coastal populations and the allopatric population in Veracruz [~]0.33 Mya. This species displays marked morphological and genetic diversity throughout its range, but this variation appears to be consistent with gene flow across contiguous populations rather than the existence of independent evolutionary lineages. The processes leading to the geographic isolation of the population on the coast of Veracruz remain uncertain, but we hypothesize that climatic and vegetation changes in the Late Pleistocene may have played a role.

Haemosporida is a diverse order of vector-borne apicomplexan parasites infecting terrestrial vertebrates worldwide, including humans, but the evolutionary relationships among its genera remain unresolved. The phylogenetic placement of two bat-restricted genera, Nycteria and Polychromophilus, both of which lack erythrocytic schizogony, has varied across studies depending on taxon sampling and marker choice. To address this problem, an expanded dataset of near-complete mitochondrial (mtDNA) genomes together with nine nuclear loci were analyzed. Phylogenetic analyses of mtDNA recovered Nycteria and Polychromophilus as a strongly supported monophyletic clade. In contrast, analyses based only on the three mitochondrial coding genes (CDS) or a reduced nuclear dataset failed to recover their monophyly and showed low support and extensive topological conflict at deeper nodes. These results indicate that near-complete mitochondrial genomes recover phylogenetic signal that is not captured by reduced mitochondrial coding sequences or partial nuclear datasets. Molecular dating analyses further showed that divergence estimates for a putative Nycteria-Polychromophilus clade are compatible with the proposed times for bats diversification, and consistent with the broader haemosporidian timescale. When the Nycteria-Polychromophilus clade was incorporated as a calibration prior, divergence-time estimates became more precise without altering the overall evolutionary timeframe. Substantial mitochondrial gene-order rearrangements in a distinct Nycteria lineage were confirmed, highlighting structural divergence within this bat-associated group. In addition, heterogeneity in rates across mtDNA haemosporidian lineages was observed. Together, these findings support the existence of a distinct bat-associated clade whose deeper placement and evolutionary significance should be tested with broader phylogenomic sampling. Author SummaryMalaria parasites belong to a diverse group of organisms that infect many kinds of vertebrates, including birds, reptiles, and mammals (such as humans). Understanding how these parasites are related to each other is important for explaining how key biological traits have evolved. However, the relationships among major groups of haemosporidian parasites, including malaria parasites, remain unclear, particularly for those infecting bats. In this study, we focused on two groups of bat parasites, Nycteria and Polychromophilus, which share unusual biological features. The inferred evolutionary relationships of these two genera to other haemosporidians have been inconsistent across previous studies. By analyzing near-complete mitochondrial genomes, we found strong evidence that these two groups descended from a common evolutionary ancestor. In contrast, smaller datasets including nuclear genes failed to recover this relationship and produced conflicting results, suggesting that they lack sufficient information to resolve deep evolutionary relationships. We also found that this bat-associated lineage likely originated around the same time as early bats. In addition, we identified structural changes in the mitochondrial genome of one lineage, highlighting its evolutionary distinctiveness. Together, our results suggest that bats host a unique group of malaria parasites and demonstrate that more complete genetic data are essential for resolving their evolutionary history.

Macromiidae is a widely distributed lineage of libelluloid dragonflies with a largely allopatric genus-level distribution across the Holarctic, Afrotropical, Australasian, and Indo-Malayan regions. Previous studies involving this family have been complicated by morphological convergence and limited phylogenetic sampling. Here, we present the most densely sampled phylogenetic framework for Macromiidae to date, using Anchored Hybrid Enrichment data from 62 of the 125 described species. Our sampling represents all four genera and major geographic regions, including Libelluloid and Cordulegastrid outgroups. Maximum likelihood recovered three major lineages: Epophthalmia, Phyllomacromia, and Macromia sensu lato, with Epophthalmia strongly supported as sister to Phyllomacromia. Didymops was not recovered as monophyletic and was placed within Macromia, although deeper relationships within the Macromia complex showed some gene tree discordance. We additionally scored seven male genitalic characters and reconstructed their evolution across a dated phylogeny. We revealed that these traits varied heavily in phylogenetic signal, with some characters supporting the major clades and others showing high degree of homoplasy. Fossil-calibrated divergence time estimation placed the crown origin of Macromiidae in the late Oligocene (24 Ma), with other major intrafamilial divergences concentrated in the Miocene. Historical biogeographic reconstructions consistently supported Afrotropical origins for Phyllomacromia, Indo-Malayan centered ancestry for Epophthalmia, and a multi-region history for Macromia + Didymops spanning Indo-Malayan, Australasian, and Nearctic regions. Habitat reconstructions favored lentic ancestry for Macromiidae, and diversification rate variation was best explained by trait-independent models rather than lentic/lotic habitat association.

The phyla making up the major animal clade of Spiralia have been clear since the advent of molecular phylogenetics; the relationships between these spiralian phyla have not. The lack of consensus over the relationships between these important animal phyla might be a clue implying their emergence in an explosive radiation. Focusing on the five largest spiralian phyla (Annelida, Brachiopoda, Mollusca, Nemertea and Platyhelminthes) and using two phylogenomic datasets, we have applied site-bootstrapping and taxon-jackknifing to explore this example of taxonomic instability. Analyses on the 105 possible rooted trees relating them showed that interphylum branches are very short. Preference for rooting Spiralia on Platyhelminthes is a long-branch artefact. Most analyses on the 15 unrooted trees showed a preference for the same topology but the support over other solutions was non significant. We conclude that the spiralian phyla emerged in rapid succession resulting in a difficult to resolve radiation. The deep history we infer for Spiralia has wide ranging implications for our interpretation of Cambrian fossils and for the evolution of traits such as biomineralization, segmentation and larvae. Impact StatementAnalyses of two independent phylogenomic datasets suggest an explosive radiation at the origin of Spiralia, with implications for understanding the groups evolutionary history.

Understanding evolutionary relationships in hyperdiverse plant groups remains a major challenge in systematics. The orchid genus Bulbophyllum, the second largest genus of flowering plants, represents an exceptional example of phylogenetic and morphological complexity. Relationships, particularly within the species-rich Asian clade, have remained poorly resolved due to extensive morphological variation and limited resolution in previous phylogenetic studies. Here, we reconstructed phylogenetic relationships using 63 plastid genes from 355 specimens representing 322 species and 65 of the 97 recognised sections of Bulbophyllum. Our analyses confirmed that the genus comprises five major evolutionary lineages comprised of species predominantly from Australasia, Madagascar, Continental Africa, Neotropics, and Asia. We provide the first robust phylogenetic evidence for a dichotomous split within the Asian clade into two well-supported lineages: the Asian-Malesian clade and the Malesian-Papuasian clade, with the latter containing a strongly supported Papuasian subclade. Additionally, this study supports the monophyly of several currently recognised sections while clarifying relationships in previously problematic groups. This study provides the most comprehensive plastid-based phylogenomic framework for Bulbophyllum to date and establishes a foundation for future taxonomic revision and integrative analyses of diversification and trait evolution within this hyperdiverse genus.

Understanding the phylogenetic relationships among eukaryotic lineages is essential for tracing the evolution of key phenotypic traits and inferring the nature of the Last Eukaryotic Common Ancestor. While phylogenomic analyses have clustered eukaryotic taxa into several well-supported major supergroups, the relationships among them remain largely uncertain. Phylogenetic signal erosion over deep time and limited available taxon sampling are among the possible causes. However, most previous studies rely on variations of the same core protein dataset, hence containing the same potential systematic biases. Here, we reconstructed the eukaryotic Tree of Life using a largely independent, marker-rich dataset derived from highly conserved Benchmarking Universal Single-Copy Orthologs. Unlike previous collections, our 277-marker supermatrix minimizes ribosomal protein representation and shares less than 25% overlap with previous datasets. State-of-the-art analyses of this dataset confirm most eukaryotic supergroups previously observed, but suggest different positions for some lineages. Notably, Telonemia clusters with Haptophyta rather than SAR (Stramenopiles-Alveolata-Rhizaria), and Ancyromonadida and Malawimonadida form a monophyletic group at the base of the Opimoda. Our results highlight the importance of analyzing independent phylogenomic datasets and support the hypothesis that extant eukaryotic diversity encompasses a small number of high-rank, supergroup lineages.

BackgroundPhylogenomic studies frequently reveal widespread gene tree discordance, primarily arising from incomplete lineage sorting (ILS) and hybridization and/or introgression. Disentangling these processes is especially challenging in rapidly radiating lineages. The genus Oryza, with its rapid diversification and multiple genome types, exemplifies this pervasive phylogenetic incongruence. We integrated multiple genomic datasets including whole-genome resequencing, transcriptomes, and published genomes from diploid Oryza species. Concatenation and multispecies coalescent analyses recovered a robust, congruent species tree, placing the FF and GG genome groups as a monophyletic basal clade, followed by successive divergence of the EE, CC, BB, and AA lineages, a topology differing from some prior hypotheses. ResultsTo assess the sources of discordance, we employed a suite of complementary phylogenomic methods. Quantifying introgression via Branch Lengths (QuIBL)-based model comparisons suggested that [~]74.17% of gene tree-species tree discordance is better explained by post-speciation introgression, whereas only [~]15.56% is consistent with ILS alone. Phylogenetic networks (PhyloNet) and allele-sharing statistics (D-statistics, f-branch) corroborated these results, indicating widespread historical introgression both within and between genome groups. Furthermore, genome-wide scans using the fdM statistic localized introgressed genomic regions, which showed reduced interspecific divergence and were enriched for genes involved in stress responses and metabolism. ConclusionsTaken together, our results demonstrate that historical introgression, not ILS, is the dominant force shaping phylogenetic discordance in diploid Oryza. The integrative phylogenomic framework implemented here, which quantifies the contributions of introgression versus ILS and maps the genomic footprint of gene flow, provides a replicable strategy for resolving complex evolutionary histories in other rapidly radiating lineages.

AbstractThe rate of evolution of a single morphological character is not homogeneous across the phylogeny and this rate heterogeneity varies between morphological characters. However, traditional models of morphological character evolution often assume that all characters evolve according to a time-homogeneous Markov process, which applies uniformly across the entire phylogeny. While models incorporating amongcharacter rate variation alleviate the assumption of the same rate for all characters, they still fail to address lineage-specific rate variation for individual characters. The covarion model, originally developed for molecular data to model the invariability of some sites for parts of the phylogeny, provides a promising framework for addressing this issue in morphological phylogenetics. In this study, we extend the covarion model in RevBayes to morphological character evolution, which we call the covariomorph model, and apply it to a diverse range of morphological datasets. Our covariomorph model utilizes multiple rate categories derived from a discretized probability distribution, which scales rate matrices accordingly. Characters are allowed to evolve within any of these rate categories, with the possibility of switching between rate categories during the evolutionary process. We verified our implementation of the covariomorph model with the help of simulations. Additionally, we examined 164 empirical datasets, finding patterns of rate heterogeneity compatible with covarion-like dynamics in approximately half of them. Upon further examination of two focal datasets that exhibited covarion-like rate variation, we found that the covariomorph model provides a more nuanced approach to incorporate rate variation across lineages, significantly affecting the resulting tree topology and branch lengths compared to traditional models. The observed sensitivity of branch lengths to model choice underscores potential implications of this approach for divergence time estimation and evolutionary rate calculations. By accounting for lineageand character-specific rate shifts, the covariomorph model offers a robust framework to improve the accuracy of morphological phylogenetic inference.

Ancestral state reconstruction is a classical problem of broad relevance in phylogenetics. Likelihood-based methods for reconstructing ancestral states under discrete character models, such as Markov models, have proven extremely useful, but only work so long as the assumed model yields a tractable likelihood function. Unfortunately, extending a simple but tractable phylogenetic model to possess new, but biologically realistic, properties often results in an intractable likelihood, preventing its use in standard modeling tasks, including ancestral state reconstruction. The rapid advancement of deep learning offers a potential alternative to likelihood-based inference of ancestral states, particularly for models with intractable likelihoods. In this study, we modify the phylogenetic deep learning software O_SCPLOWPHYDDLEC_SCPLOW to conduct ancestral state reconstruction. We evaluate O_SCPLOWPHYDDLEC_SCPLOWs performance under various methodological and modeling conditions, while comparing to Bayesian inference when possible. For simple models and small trees, its performance resembles the performance of Bayesian inference, but worsens as tree size increases. While O_SCPLOWPHYDDLEC_SCPLOW still performs adequately for more complex models, such as speciation and extinction models, the estimates differ more from Bayesian inference in comparison with simpler models. Lastly, we use O_SCPLOWPHYDDLEC_SCPLOW to infer ancestral states for two empirical datasets, one of the ancestral ranges of a subclade of the genus Liolaemus and ancestral locations for sequences from the 2014 Sierra Leone Ebola virus disease outbreak.

The genus Pyrrhura (Psittacidae: Arini) is one of the most diverse groups of Neotropical parrots. Its species are charismatic, widely kept as pets, and frequently bred outside their native ranges. Yet, nearly half are currently listed as threatened by the IUCN within their natural distributions. Conservation assessments and population estimates often depend on the validity of accepted taxonomic boundaries. However, despite previous systematic efforts, the evolutionary relationships among and within many Pyrrhura species remain poorly resolved, largely due to a recent and rapid radiation. Here, we generated whole-genome sequences for all currently recognized Pyrrhura species, including multiple intraspecific taxa, to reconstruct a robust nuclear phylogeny under the multi-species coalescent model, alongside the most comprehensive mitogenome-based phylogeny of the genus to date. Although both phylogenies supported the monophyly of most currently accepted species, we identified several instances of mito-nuclear discordance, particularly involving the placement of early-diverging lineages, which are best explained by incomplete lineage sorting and historical gene flow. Additionally, we detected three distinct captive lineages that do not cluster with any known wild populations, suggesting substantial overlooked genetic diversity in the worlds captive populations. Ancestral range reconstructions indicate multiple and relatively recent colonization events into the northern and central Andes, likely associated with the uplift of the Andes and the emergence of new ecological niches. Together, our results reveal a complex evolutionary history in Pyrrhura, shaped by rapid radiations, incomplete lineage sorting, and gene flow. We show that integrating nuclear and mitochondrial data with broad geographic and taxonomic sampling, including captive individuals, can uncover overlooked genetic diversity and help to resolve long-standing systematic uncertainties. Finally, we show that several topological discrepancies among previous studies can be attributed to differences in sampling strategies, particularly within the most polytypic Pyrrhura species.

Partitioned and mixture models are widely employed in Maximum Likelihood phylogenetic analyses of large genomic datasets. Comparing the fit of the two types of models has been challenging, because standard information-theoretic approaches cannot be applied. Mixture models are increasingly popular for the analysis of amino acid datasets and can lead to different conclusions compared to partitioned models. This raises an important question - which type of model tends to perform better? Susko et al. (2026) recently introduced the marginal Akaike information criterion (mAIC), which allows mixture models and partitioned models to be directly compared for the first time. Here, we use the mAIC and a range of other approaches to compare the fit of mixture and partitioned models across a diverse set of empirical datasets. We show that mixture models are universally favoured on amino acid datasets. This has important implications for interpreting empirical analyses and suggests that continued development of mixture models is an important avenue for future research.

Ophiostomatoids are an ecological group of microfungi that commonly associate with bark and ambrosia beetles. As well as being insect symbionts, they play significant ecological roles as plant pathogens, and include species responsible for major forest tree diseases. Despite their ecological similarities, ophiostomatoids are distributed across two quite distantly related orders, the Microascales and Ophiostomatales. Historically, these fungi were considered a single natural group; however, molecular studies have revealed their independent origins and convergent ecological strategies. Previous phylogenetic studies of these fungi have typically focused on resolving taxonomic issues or understanding individual lifestyles, such as beetle-cultivated ambrosia lineages or vascular wilt pathogens. As a result, we lack a comprehensive phylogenetic framework that integrates dense species-level sampling with ecological data across both orders. Such frameworks are essential for understanding the broader phylogenetic and ecological context in which key fungal lifestyles have evolved. Here, we assembled and analysed all available sequence data for the Microascales and Ophiostomatales from seven widely used fungal marker loci to reconstruct a densely sampled phylogeny for each order. We evaluated locus performance and showed that whilst individual loci fail to resolve many taxa, concatenated datasets produce robust, well-supported topologies consistent with published genomic studies. By mapping ecological traits onto these trees, we show that lifestyle diversity and beetle associations are much more variable in the Microascales than in the Ophiostomatales, despite comparable species richness. Presenting both orders together provides a unique comparative perspective on the ecology and evolution of ophiostomatoids. As metabarcoding datasets of ophiostomatoids become increasingly common, this integrative framework can offer a valuable resource for environmental sequence identification and investigating fungal lifestyle switches, which in turn can support future biodiversity and ecology studies.

Species delimitation is a fundamental challenge in systematic biology, particularly for geographically variable taxa with hierarchical population structure and gene flow. Migration-aware coalescent models provide a powerful framework for investigating lineage divergence and accurately defining species boundaries. In this study, we combine statistical evaluations of gene flow with phylogenetic and population structure analyses to delimit species of fence lizards within the Sceloporus undulatus complex, a group characterized by extensive population subdivision, mitochondrial DNA introgression, and nuclear gene flow. We find that the undulatus complex exhibits uneven variation in genetic, morphological, and bioclimatic traits, resulting in variable distinctiveness among groups. In some cases, species boundaries are recognized by clear genetic discontinuities without gene flow. In others, shallow divergence, paraphyly, and gene flow produce leaky boundaries and fuzzy species limits. Mitochondrial introgression is extensive and concentrated at species boundaries, whereas nuclear gene flow occurs between only a few species and at much lower levels than within species. Neither within-species populations or species are substantially diverged across morphology or bioclimatic space, highlighting the limited utility of these traits for diagnosing species in this group. By integrating estimates of gene flow with phylogenetic and population structure analyses, this study provides a robust and biologically meaningful revised taxonomic framework for the undulatus complex that identifies independently evolving lineages as species.

In Toxoplasma gondii, microneme proteins (MICs) are secreted components of the apical complex that play central roles in motility, host cell attachment, and invasion. Because proteins at the host-parasite interface are often predicted to evolve rapidly, MICs have been suggested as candidates for adaptive diversification. We tested this expectation using comparative analyses of three relatively understudied microneme proteins, MIC13, MIC12, and MIC16. Coding sequences were assembled from GenBank and ToxoDB, aligned by translation, and analyzed using maximum-likelihood phylogenetics, codon-based tests of selection, and predicted protein structure. MIC13 was represented by 51 sequences, MIC12 by 30, and MIC16 by 34, spanning multiple T. gondii haplogroups and including Hammondia hammondi and Neospora caninum as outgroups. All three genes were highly conserved among T. gondii strains, but their phylogenetic trees were topologically incongruent, indicating that individual MICs do not recover a single shared strain history. Contrary to expectation, no positively selected codons were detected in any gene. Instead, purifying selection was detected at one site in MIC13 and 15 sites in MIC12, while no significant codon-specific selection was detected in MIC16. Several constrained MIC12 sites overlapped annotated EGF and calcium-binding EGF-like domains, consistent with structural conservation of extracellular adhesion modules. AlphaFold prediction of MIC13 supported two sialic acid-binding micronemal adhesive repeat regions, but the single constrained MIC13 site did not overlap these motifs. Together, these results indicate that MIC13, MIC12, and MIC16 are shaped more by sequence conservation and heterogeneous gene histories than by strong recurrent positive selection. These findings refine expectations for microneme evolution in T. gondii and highlight conserved domains that may be important for parasite invasion and future functional study.