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Abstract Much of our understanding of the history of life hinges upon time calibration, the process of assigning absolute times to cladogenetic events. Bayesian approaches to time‐scaling phylogenetic trees have dramatically grown in complexity, and depend today upon numerous methodological choices. Arriving at objective justifications for all of these is difficult and time‐consuming. Thus, divergence times are routinely inferred under only one or a handful of parametric conditions, often times chosen arbitrarily. Progress towards building robust biological timescales necessitates the development of better methods to visualize and quantify the sensitivity of results to these decisions. Here, we present an R package that assists in this endeavour through the use of chronospaces, that is, graphical representations summarizing variation in the node ages contained in time‐calibrated trees. We further test this approach by estimating divergence times for three empirical datasets—spanning widely differing evolutionary timeframes—using the software PhyloBayes. Our results reveal large differences in the impact of many common methodological decisions, with the choice of clock (uncorrelated vs autocorrelated) and loci having strong effects on inferred ages. Other decisions have comparatively minor consequences, including the use of the computationally intensive site‐heterogeneous model CAT‐GTR, whose effect might only be discernible for exceedingly old divergences (e.g. the deepest eukaryote nodes). The package chronospace implements a range of graphical and analytical tools that assist in the exploration of sensitivity and the prioritization of computational resources in the inference of divergence times.

ABSTRACT Understanding the functions of mitochondrial genomes is crucial for studies related to the evolution of genomes, phylogenomics, and species identification. For the first time, complete mitogenome of Potamalpheops sp. a non‐snapping shrimp has been successfully sequenced and characterized from Taiwan that belongs to the Decapoda order, Crustacea class, and Caridea infraorder. This study involved analysis of nucleotide composition, codon usage, gene ordering, evolutionary selection pressure, and comparative mitogenomics. The mitogenome of Potamalpheops sp. is 16,605 base pairs in length and consists of standard set of 37 genes found in metazoans. The gene rearrangements in the mitochondrial genome of this species shows extensive rearrangements comparing to the typical pattern found in pancrustaceans mitogenomes. Therefore, it could be concluded that gene rearrangements most likely happen only in the caridea infraorder. The current investigation discovered transposition of the tRNA and rRNA genes along with reversal in strands in the tRNAs. No other Alpheidae mitochondrial genome that has been investigated thus far has revealed this pattern. All 13 protein coding genes in the mitochondrial genomes of superfamily Alphoidea exhibited Ka/Ks values lower than 1, according to the ratios of nonsynonymous and synonymous substitutions rates. This suggests that a strong purifying selection had taken place. The maximum likelihood tree consisting of 46 mitogenomes of infraorder Caridea along with outgroups, revealed the existence of Potamalpheops sp. in the family Alpheidae and it formed a monophyletic group along with Palaemonoidea and Alpheoidea superfamily.

Abstract Premise Target sequence capture (Hyb‐Seq) is a cost‐effective sequencing strategy that employs RNA probes to enrich for specific genomic sequences. By targeting conserved low‐copy orthologs, Hyb‐Seq enables efficient phylogenomic investigations. Here, we present Asparagaceae1726—a Hyb‐Seq probe set targeting 1726 low‐copy nuclear genes for phylogenomics in the angiosperm family Asparagaceae—which will aid the often‐challenging delineation and resolution of evolutionary relationships within Asparagaceae. Methods Here we describe and validate the Asparagaceae1726 probe set (https://github.com/bentzpc/Asparagaceae1726) in six of the seven subfamilies of Asparagaceae. We perform phylogenomic analyses with these 1726 loci and evaluate how inclusion of paralogs and bycatch plastome sequences can enhance phylogenomic inference with target‐enriched data sets. Results We recovered at least 82% of target orthologs from all sampled taxa, and phylogenomic analyses resulted in strong support for all subfamilial relationships. Additionally, topology and branch support were congruent between analyses with and without inclusion of target paralogs, suggesting that paralogs had limited effect on phylogenomic inference. Discussion Asparagaceae1726 is effective across the family and enables the generation of robust data sets for phylogenomics of any Asparagaceae taxon. Asparagaceae1726 establishes a standardized set of loci for phylogenomic analysis in Asparagaceae, which we hope will be widely used for extensible and reproducible investigations of diversification in the family.

Abstract Phylogenetic inference of polyploid species is the first step towards understanding their patterns of diversification. In this paper, we review the challenges and limitations of inferring species relationships of polyploid plants using traditional phylogenetic sequencing approaches, as well as the mischaracterization of the species tree from single or multiple gene trees. We provide a roadmap to infer interspecific relationships among polyploid lineages by comparing and evaluating the application of current phylogenetic, phylogenomic, transcriptomic, and whole‐genome approaches using different sequencing platforms. For polyploid species tree reconstruction, we assess the following criteria: (1) the amount of prior information or tools required to capture the genetic region(s) of interest; (2) the probability of recovering homeologs for polyploid species; and (3) the time efficiency of downstream data analysis. Moreover, we discuss bioinformatic pipelines that can reconstruct networks of polyploid species relationships. In summary, although current phylogenomic approaches have improved our understanding of reticulate species relationships in polyploid‐rich genera, the difficulties of recovering reliable orthologous genes and sorting all homeologous copies for allopolyploids remain a challenge. In the future, assembled long‐read sequencing data will assist the recovery and identification of multiple gene copies, which can be particularly useful for reconstructing the multiple independent origins of polyploids.

Abstract Insect pests cause tremendous impact to agriculture worldwide. Species identification is crucial for implementing appropriate measures of pest control but can be challenging in closely related species. True fruit flies of the genus Anastrepha Schiner (Diptera: Tephritidae) include some of the most serious agricultural pests in the Americas, with the Anastrepha fraterculus (Wiedemann) complex being one of the most important due to its extreme polyphagy and wide distribution across most of the New World tropics and subtropics. The eight morphotypes described for this complex as well as other closely related species are classified in the fraterculus species group, whose evolutionary relationships are unresolved due to incomplete lineage sorting and introgression. We performed multifaceted phylogenomic approaches using thousands of genes to unravel the evolutionary relationships within the A. fraterculus complex to provide a baseline for molecular diagnosis of these pests. We used a methodology that accommodates variable sources of data (transcriptome, genome, and whole‐genome shotgun sequencing) and developed a tool to align and filter orthologs, generating reliable datasets for phylogenetic studies. We inferred 3031 gene trees that displayed high levels of discordance. Nevertheless, the topologies of the inferred coalescent species trees were consistent across methods and datasets, except for one lineage in the A. fraterculus complex. Furthermore, network analysis indicated introgression across lineages in the fraterculus group. We present a robust phylogeny of the group that provides insights into the intricate patterns of evolution of the A. fraterculus complex supporting the hypothesis that this complex is an assemblage of closely related cryptic lineages that have evolved under interspecific gene flow. Despite this complex evolutionary scenario, our subsampling analysis revealed that a set of as few as 80 loci has a similar phylogenetic resolution as the genome‐scale dataset, offering a foundation to develop more efficient diagnostic tools in this species group.

Abstract Transcriptome sequencing technologies have revolutionized the field of phylogenomics by facilitating the identification of homologous genes for species without whole genome sequences. To infer complex evolution relationship among eukaryotes, it is essential to obtain complete sequences of protein‐coding genes to provide informative mutations. However, transcriptomes in eukaryotes often consist of a large number of duplicated genes and alternative isoforms, bringing great challenges for developing effective tools to obtain complete coding gene sequences. Here, we present a net‐flow based assembler TransMCL, which aims to assemble fragmented transcripts into complete sequences while eliminating redundant isoforms during homologue clustering. By employing Markov clustering strategies and homologous gene guidance, TransMCL can accurately assemble genes for multiple organisms in an affordable time frame, making it well‐suited for phylogenomic studies based on transcriptomic data. Our results demonstrate that TransMCL can assemble 89.95%–92.95% of the total expressed genes into near‐complete transcripts on benchmark plant/animal datasets. Furthermore, applying TransMCL to multiple transcriptomes in a single run enhances the completeness of genes, even in the absence of guidance homologues from closely related species. These findings highlight the potential of TransMCL in phylogenomic studies, enabling the comprehensive characterization of gene families at the whole genome scale. By overcoming the challenges of complex gene contents in eukaryotes, TransMCL can significantly enhance our understanding of evolution of gene families across species.

Abstract Premise A probe set was previously designed to target 384 nuclear loci in the Melastomataceae family; however, when trying to use it, we encountered several practical and conceptual problems, such as the presence of sequences in reverse complement, intronic regions with stop codons, and other issues. This raised concerns regarding the use of this probe set for sequence recovery in Melastomataceae. Methods In order to correct these issues, we cleaned the Melastomataceae probe set, extended it with additional sequences, and compared its performance with the original version. Results The final probe set targets 396 putative nuclear loci represented by 6009 template sequences. The probe set has been made available, along with details on the cleaning process, for reproducibility. We show that the new probe set performs better than the original version in terms of sequence recovery. Discussion This updated, extended, and cleaned probe set will improve the availability of phylogenomic resources across the Melastomataceae family. It is fully compatible with sequence recovery and extraction pipelines. The cleaning process can also be applied to any plant‐targeting probe set that would need to be cleaned or updated if new genomic resources for the targeted taxa become available.

Abstract Premise The genus Calceolaria (Calceolariaceae) is emblematic of the Andes, is hypothesized to have originated as a recent, rapid radiation, and has important taxonomic needs. Additionally, the genus is a model for the study of specialized pollination systems, as its flowers are nectarless and many offer floral oils as a pollination reward collected by specialist bees. Despite their evolutionary and ecological significance, obtaining a resolved phylogeny for the group has proved difficult. To address this challenge, we present a new bait set for targeted sequencing of nuclear loci in Calceolariaceae and close relatives. Methods We developed a bioinformatic workflow to use incomplete, low‐coverage genomes of 10 Calceolaria species to identify single‐copy loci suitable for phylogenetic studies and design baits for targeted sequencing. Results Our approach resulted in the identification of 809 single‐copy loci (733 noncoding and 76 coding regions) and the development of 39,937 baits, which we validated in silico (10 specimens) and in vitro (29 Calceolariaceae and six outgroups). In both cases, the data allowed us to recover robust phylogenetic estimates. Discussion Our results demonstrate the appropriateness of the bait set for sequencing recent and historic specimens of Calceolariaceae and close relatives, and open new doors for further investigation of the evolutionary history of this hyperdiverse genus.

Abstract We made an in‐depth review of historical studies of the cupressophyte conifer genus Cephalotaxus Siebold & Zucc. with an emphasis on its systematic position. We suggest that the systematic position of the genus is better understood using an integrative approach, so the evolution of phenetic characters is discussed within the context of recent phylogenomics. We propose that the genus should be classified as a separate family Cephalotaxaceae belonging to the clade consisting of Cupressaceae, Cephalotaxaceae, and Taxaceae; the family Cephalotaxaceae is sister to the Taxaceae but not nested within the Taxaceae and is characterized by a unique set of characters including morphology, anatomy, embryology, and chemistry. The family Cephalotaxaceae shows transitional characters between the Cupressaceae and the Taxaceae; the family possesses female cones with a primary cone axis bearing 5–8 pairs of decussate bracts, which is similar to the typical female cones of the Cupressaceae, on the one hand, and may have given rise to the reduced female cone of the Taxaceae with one terminal ovule partially or completely enclosed in a fleshy aril. In parallel, the compound male cone of the Cephalotaxaceae evolved into the seemingly “simple” male cones of the Taxaceae by means of reduction, elimination, and fusion.

Abstract Premise The HybPiper pipeline has become one of the most widely used tools for the assembly of target capture data for phylogenomic analysis. After the production of locus sequences and before phylogenetic analysis, the identification of paralogs is a critical step for ensuring the accurate inference of evolutionary relationships. Algorithmic approaches using gene tree topologies for the inference of ortholog groups are computationally efficient and broadly applicable to non‐model organisms, especially in the absence of a known species tree. Methods and Results We containerized and expanded the functionality of both HybPiper and a pipeline for the inference of ortholog groups, providing novel options for the treatment of target capture sequence data, and allowing seamless use of the outputs of the former as inputs for the latter. The Singularity container presented here includes all dependencies, and the corresponding pipelines (hybpiper‐nf and paragone‐nf, respectively) are implemented via two Nextflow scripts for easier deployment and to vastly reduce the number of commands required for their use. Conclusions The hybpiper‐nf and paragone‐nf pipelines are easily installed and provide a user‐friendly experience and robust results to the phylogenetic community. They are used by the Australian Angiosperm Tree of Life project. The pipelines are available at https://github.com/chrisjackson-pellicle/hybpiper-nf and https://github.com/chrisjackson-pellicle/paragone-nf.

Abstract We aimed to infer ancestral area and historical colonization of Lobelia columnaris in the sky islands of Bioko and Cameroon through dated phylogeny using chloroplast genomes. Specifically, we aim to answer the following questions: (1) What are the phylogenetic relationships among Bioko Island and Cameroon populations? (2) Are the older populations found in the older sky islands? We assembled novel plastomes from 20 individuals of L. columnaris from 5 mountain systems. The plastome data were explored with phylogenetic analyses using Maximum Likelihood and Bayesian Inference. The populations of L. columnaris have a monophyletic origin, subdivided into three plastomes‐geographic clades. The plastid phylogenomic results and age of the sky islands indicate that L. columnaris colonized first along with the Cameroon Volcanic Line's young sky islands of Bioko. The crown group (1.54 Ma) split the population in Bioko and mainland Cameroon. It is possible that Bioko was the ancestral area and likely isolated during cold and dry conditions in forest refugia. Presumably, the colonization history occurred during the middle‐late Pleistocene from South Bioko's young sky island to North Bioko and the northern old sky islands in Cameroon. Furthermore, the central depression with lowland forest between North and South Bioko is a current geographic barrier that keeps separating the populations of Bioko from each other. Also, the shallow sea channel keeps isolated the populations of Bioko and the mainland populations. The Pleistocene climatic oscillations led to the divergence of the Cameroon and Bioko populations into three clades. L. columnaris colonized the older sky islands in mainland Cameroon after establishment in Bioko's younger sky islands. Contrary to expectations, the biogeography history was an inverse progression with respect to the age of the Afromontane sky islands.

Abstract Recovering phylogenetic relationships in lineages experiencing intense diversification has always been a persistent challenge in evolutionary studies, including in Gentiana section Chondrophyllae sensu lato (s.l.). Indeed, this subcosmopolitan taxon encompasses more than 180 mostly annual species distributed around the world. We sequenced and assembled 22 new plastomes representing 21 species in section Chondrophyllae s.l. In addition to previously released plastome data, our study includes all main lineages within the section. We reconstructed their phylogenetic relationships based on protein‐coding genes and recombinant DNA (rDNA) cistron sequences, and then investigated plastome structural evolution as well as divergence time. Despite an admittedly humble species cover overall, we recovered a well‐supported phylogenetic tree based on plastome data, and found significant discordance between phylogenetic relationships and taxonomic treatments. Our results show that G. capitata and G. leucomelaena diverged early within the section, which is then further divided into two clades. The divergence time estimation showed that section Chondrophyllae s.l. evolved in the second half of the Oligocene. We found that section Chondrophyllae s.l. had the smallest average plastome size (128 KB) in tribe Gentianeae (Gentianaceae), with frequent gene and sequence losses such as the ndh complex and its flanking regions. In addition, we detected both expansion and contraction of the inverted repeat (IR) regions. Our study suggests that plastome degradation parallels the diversification of this group, and illustrates the strong discordance between phylogenetic relationships and taxonomic treatments, which now need to be carefully revised.

Abstract The relative roles of rivers versus refugia in shaping the high levels of species diversity in tropical rainforests have been widely debated for decades. Only recently has it become possible to take an integrative approach to test predictions derived from these hypotheses using genomic sequencing and paleo‐species distribution modeling. Herein, we tested the predictions of the classic river, refuge, and river‐refuge hypotheses on diversification in the arboreal sub‐Saharan African snake genus Toxicodryas. We used dated phylogeographic inferences, population clustering analyses, demographic model selection, and paleo‐distribution modeling to conduct a phylogenomic and historical demographic analysis of this genus. Our results revealed significant population genetic structure within both Toxicodryas species, corresponding geographically to river barriers and divergence times from the mid‐Miocene to Pliocene. Our demographic analyses supported the interpretation that rivers are indications of strong barriers to gene flow among populations since their divergence. Additionally, we found no support for a major contraction of suitable habitat during the last glacial maximum, allowing us to reject both the refuge and river‐refuge hypotheses in favor of the river‐barrier hypothesis. Based on conservative interpretations of our species delimitation analyses with the Sanger and ddRAD data sets, two new cryptic species are identified from east‐central Africa. This study highlights the complexity of diversification dynamics in the African tropics and the advantages of integrative approaches to studying speciation in tropical regions.

Abstract Phylogenetic inference and species delimitation can be challenging in taxonomic groups that have recently radiated and where introgression produces conflicting gene trees, especially when species delimitation has traditionally relied on mitochondrial data and color pattern. Chromodoris, a genus of colorful and toxic nudibranch in the Indo‐Pacific, has been shown to have extraordinary cryptic diversity and mimicry, and has recently radiated, ultimately complicating species delimitation. In these cases, additional genome‐wide data can help improve phylogenetic resolution and provide important insights about evolutionary history. Here, we employ a transcriptome‐based exon capture approach to resolve Chromodoris phylogeny with data from 2,925 exons and 1,630 genes, derived from 15 nudibranch transcriptomes. We show that some previously identified mimics instead show mitonuclear discordance, likely deriving from introgression or mitochondrial capture, but we confirm one “pure” mimic in Western Australia. Sister–species relationships and species‐level entities were recovered with high support in both concatenated maximum likelihood (ML) and summary coalescent phylogenies, but the ML topologies were highly variable while the coalescent topologies were consistent across datasets. Our work also demonstrates the broad phylogenetic utility of 149 genes that were previously identified from eupulmonate gastropods. This study is one of the first to (a) demonstrate the efficacy of exon capture for recovering relationships among recently radiated invertebrate taxa, (b) employ genome‐wide nuclear markers to test mimicry hypotheses in nudibranchs and (c) provide evidence for introgression and mitochondrial capture in nudibranchs.

Abstract The tips in the tree of life serve as foci for conservation and management, yet clear delimitations are masked by inherent variance at the species–population interface. Analyses using thousands of nuclear loci can potentially sort inconsistencies, yet standard categories applied to this parsing are themselves potentially conflicting and/or subjective [e.g., DPS (distinct population segments); DUs (Diagnosable Units‐Canada); MUs (management units); SSP (subspecies); ESUs (Evolutionarily Significant Units); and UIEUs (uniquely identified evolutionary units)]. One potential solution for consistent categorization is to create a comparative framework by accumulating statistical results from independent studies and evaluating congruence among data sets. Our study illustrates this approach in speckled dace (Leuciscidae: Rhinichthys osculus) endemic to two basins (Owens and Amargosa) in the Death Valley ecosystem. These fish persist in the Mojave Desert as isolated Plio‐Pleistocene relicts and are of conservation concern, but lack formal taxonomic descriptions/designations. Double digest RAD (ddRAD) methods identified 14,355 SNP loci across 10 populations (N = 140). Species delimitation analyses [multispecies coalescent (MSC) and unsupervised machine learning (UML)] delineated four putative ESUs. FST outlier loci (N = 106) were juxtaposed to uncover the potential for localized adaptations. We detected one hybrid population that resulted from upstream reconnection of habitat following contemporary pluvial periods, whereas remaining populations represent relics of ancient tectonism within geographically isolated springs and groundwater‐fed streams. Our study offers three salient conclusions: a blueprint for a multifaceted delimitation of conservation units; a proposed mechanism by which criteria for intraspecific biodiversity can be potentially standardized; and a strong argument for the proactive management of critically endangered Death Valley ecosystem fishes.

Abstract Native grasslands are one of the most endangered ecosystems in North America. In this study, we examined the ecological and evolutionary roles of endangered and threatened (e/t) grasses by establishing robust evolutionary relationships with other nonthreatened native and introduced grass species of the community. We hypothesized that the phylogenomic distribution of e/t species of grasses in Illinois would be phylogenetically clustered because closely related species would be vulnerable to the same threats and have similar requirements for survival. This study presents the first time a phylogeny based on complete plastome DNA of Poaceae was analyzed by phylogenetic diversity analysis. To avoid the disturbance of e/t populations, DNA was extracted from herbarium specimens. Next‐generation sequencing (NGS) techniques were used to sequence DNA of plastid genomes (plastomes). The resulting phylogenomic tree was analyzed by phylogenetic diversity metrics. The extracted DNA successfully produced complete plastomes demonstrating that herbarium material is a practical source of DNA for genomic studies. The phylogenomic tree was strongly supported and defined Dichanthelium as a separate clade from Panicum. The phylogenetic metrics revealed phylogenetic clustering of e/t species, confirming our hypothesis.

Abstract The delimitation of species boundaries, particularly those obscured by reticulation, is a critical step in contemporary biodiversity assessment. It is especially relevant for conservation and management of indigenous fishes in western North America, represented herein by two species with dissimilar life histories codistributed in the highly modified Colorado River (i.e., flannelmouth sucker, Catostomus latipinnis; bluehead sucker, C. (Pantosteus) discobolus). To quantify phylogenomic patterns and examine proposed taxonomic revisions, we first employed double‐digest restriction site‐associated DNA sequencing (ddRAD), yielding 39,755 unlinked SNPs across 139 samples. These were subsequently evaluated with multiple analytical approaches and by contrasting life history data. Three phylogenetic methods and a Bayesian assignment test highlighted similar phylogenomic patterns in each, but with considerable difference in presumed times of divergence. Three lineages were detected in bluehead sucker, supporting elevation of C. (P.) virescens to species status and recognizing C. (P.) discobolus yarrowi (Zuni bluehead sucker) as a discrete entity. Admixture in the latter necessitated a reevaluation of its contemporary and historic distributions, underscoring how biodiversity identification can be confounded by complex evolutionary histories. In addition, we defined three separate flannelmouth sucker lineages as ESUs (evolutionarily significant units), given limited phenotypic and genetic differentiation, contemporary isolation, and lack of concordance (per the genealogical concordance component of the phylogenetic species concept). Introgression was diagnosed in both species, with the Little Colorado and Virgin rivers in particular. Our diagnostic methods, and the agreement of our SNPs with previous morphological, enzymatic, and mitochondrial work, allowed us to partition complex evolutionary histories into requisite components, such as isolation versus secondary contact.

Abstract Bacillus anthracis, the causative agent of anthrax, is a considerable global health threat affecting wildlife, livestock, and the general public. In this study, whole‐genome sequence analysis of over 350 B. anthracis isolates was used to establish a new high‐resolution global genotyping framework that is both biogeographically informative and compatible with multiple genomic assays. The data presented in this study shed new light on the diverse global dissemination of this species and indicate that many lineages may be uniquely suited to the geographic regions in which they are found. In addition, we demonstrate that plasmid genomic structure for this species is largely consistent with chromosomal population structure, suggesting vertical inheritance in this bacterium has contributed to its evolutionary persistence. This classification methodology is the first based on population genomic structure for this species and has potential use for local and broader institutions seeking to understand both disease outbreak origins and recent introductions. In addition, we provide access to a newly developed genotyping script as well as the full whole‐genome sequence analyses output for this study, allowing future studies to rapidly employ and append their data in the context of this global collection. This framework may act as a powerful tool for public health agencies, wildlife disease laboratories, and researchers seeking to utilize and expand this classification scheme for further investigations into B. anthracis evolution.

Abstract Selection of appropriate genetic markers to quantify phylogenetic diversity is crucial for community ecology studies. Yet, systematic evaluation of marker genes for this purpose is scarcely done. Recently, the combined effort of phycologists has produced a rich plastid genome resource with taxonomic representation spanning all of the major lineages of the red algae (Rhodophyta). In this proof‐of‐concept study, we leveraged this resource by developing and applying a phylogenomic strategy to seek candidate plastid markers suitable for phylogenetic community analysis. We ranked the core genes of 107 published plastid genomes based on various sequence‐derived properties and their tree distance to plastid genome phylogenies. The resulting ranking revealed that the most widely used marker, rbcL, is not necessarily the optimal marker, while other promising markers might have been overlooked. We designed and tested PCR primers for several candidate marker genes, and successfully amplified one of them, rpoC1, in a taxonomically broad set of red algal specimens. We suggest that our general marker identification methodology and the rpoC1 primers will be useful to the phycological community for investigating the biodiversity and community ecology of the red algae.

Abstract Dusky Salamanders (genus Desmognathus) currently comprise only 22 described, extant species. However, recent mitochondrial and nuclear estimates indicate the presence of up to 49 candidate species based on ecogeographic sampling. Previous studies also suggest a complex history of hybridization between these lineages. Studies in other groups suggest that disregarding admixture may affect both phylogenetic inference and clustering‐based species delimitation. With a dataset comprising 233 Anchored Hybrid Enrichment (AHE) loci sequenced for 896 Desmognathus specimens from all 49 candidate species, we test three hypotheses regarding (i) species‐level diversity, (ii) hybridization and admixture, and (iii) misleading phylogenetic inference. Using phylogenetic and population‐clustering analyses considering gene flow, we find support for at least 47 candidate species in the phylogenomic dataset, some of which are newly characterized here while others represent combinations of previously named lineages that are collapsed in the current dataset. Within these, we observe significant phylogeographic structure, with up to 64 total geographic genetic lineages, many of which hybridize either narrowly at contact zones or extensively across ecological gradients. We find strong support for both recent admixture between terminal lineages and ancient hybridization across internal branches. This signal appears to distort concatenated phylogenetic inference, wherein more heavily admixed terminal specimens occupy apparently artifactual early‐diverging topological positions, occasionally to the extent of forming false clades of intermediate hybrids. Additional geographic and genetic sampling and more robust computational approaches will be needed to clarify taxonomy, and to reconstruct a network topology to display evolutionary relationships in a manner that is consistent with their complex history of reticulation.

The Diphyllatea (CRuMs) are heterotrophic protists currently divided into three distinct clades (Diphy I-III). Diphy I are biflagellates in the genus Diphylleia, whereas Diphy II and III represent cryptic clades comprising Collodictyon-type quadriflagellates that were recently distinguished based on rRNA gene phylogenies. Here, we isolated Diphyllatea from freshwater crater lakes on two South Pacific islands and generated high-quality transcriptomes from species representing each clade, including the first transcriptomic data from Diphy III. Phylogenomic analyses support the separation of Diphy II and III, while transcriptome completeness highlights the utility of these data for future studies. Lastly, we discuss the biogeography and ecology of Diphyllatea on these remote islands., (© 2024 The Author(s). Journal of Eukaryotic Microbiology published by Wiley Periodicals LLC on behalf of International Society of Protistologists.)

Premise: Cleomaceae is an important model clade for studies of evolutionary processes including genome evolution, floral form diversification, and photosynthetic pathway evolution. Diversification and divergence patterns in Cleomaceae remain tangled as research has been restricted by its worldwide distribution, limited genetic sampling and species coverage, and a lack of definitive fossil calibration points., Methods: We used target sequence capture and the Angiosperms353 probe set to perform a phylogenetic study of Cleomaceae. We estimated divergence times and biogeographic analyses to explore the origin and diversification of the family. Seed morphology across extant taxa was documented with multifocal image-stacking techniques and morphological characters were extracted, analyzed, and compared to fossil records., Results: We recovered a well-supported and resolved phylogenetic tree of Cleomaceae generic relationships that includes 236 (~86%) species. We identified 11 principal clades and confidently placed Cleomella as sister to the rest of the family. Our analyses suggested that Cleomaceae and Brassicaceae diverged ~56 mya, and Cleomaceae began to diversify ~53 mya in the Palearctic and Africa. Multiple transatlantic disjunct distributions were identified. Seeds were imaged from 218 (~80%) species in the family and compared to all known fossil species., Conclusions: Our results represent the most comprehensive phylogenetic study of Cleomaceae to date. We identified transatlantic disjunctions and proposed explanations for these patterns, most likely either long-distance dispersals or contractions in latitudinal distributions caused by climate change over geological timescales. We found that seed morphology varied considerably but mostly mirrored generic relationships., (© 2024 The Author(s). American Journal of Botany published by Wiley Periodicals LLC on behalf of Botanical Society of America.)

Premise: Reticulate evolution, often accompanied by polyploidy, is prevalent in plants, and particularly in the ferns. Resolving the resulting non-bifurcating histories remains a major challenge for plant phylogenetics. Here, we present a phylogenomic investigation into the complex evolutionary history of the vining ferns, Lygodium (Lygodiaceae, Schizaeales)., Methods: Using a targeted enrichment approach with the GoFlag 408 flagellate land plant probe set, we generated large nuclear and plastid sequence datasets for nearly all taxa in the genus and constructed the most comprehensive phylogeny of the family to date using concatenated maximum likelihood and coalescence approaches. We integrated this phylogeny with cytological and spore data to explore karyotype evolution and generate hypotheses about the origins of putative polyploids and hybrids., Results: Our data and analyses support the origins of several putative allopolyploids (e.g., L. cubense, L. heterodoxum) and hybrids (e.g., L. ×fayae) and also highlight the potential prevalence of autopolyploidy in this clade (e.g., L. articulatum, L. flexuosum, and L. longifolium)., Conclusions: Our robust phylogenetic framework provides valuable insights into dynamic reticulate evolution in this clade and demonstrates the utility of target-capture data for resolving these complex relationships., (© 2024 The Author(s). American Journal of Botany published by Wiley Periodicals LLC on behalf of Botanical Society of America.)

Premise: The Caryophyllaceae (the carnation family) have undergone multiple transitions into colder climates and convergence on cushion plant adaptation, indicating that they may provide a natural system for cold adaptation research. Previous research has suggested that putative ancient whole-genome duplications (WGDs) are correlated with niche shifts into colder climates across the Caryophyllales. Here, we explored the genomic changes potentially involved in one of these discovered shifts in the Caryophyllaceae., Methods: We constructed a data set combining 26 newly generated transcriptomes with 45 published transcriptomes, including 11 cushion plant species across seven genera. With this data set, we inferred a dated phylogeny for the Caryophyllaceae and mapped ancient WGDs and gene duplications onto the phylogeny. We also examined functional groups enriched for gene duplications related to the climatic shift., Results: The ASTRAL topology was mostly congruent with the current consensus of relationships within the family. We inferred 15 putative ancient WGDs in the family, including eight that have not been previously published. The oldest ancient WGD (ca. 64.4-56.7 million years ago), WGD1, was found to be associated with a shift into colder climates by previous research. Gene regions associated with ubiquitination were overrepresented in gene duplications retained after WGD1 and those convergently retained by cushion plants in Colobanthus and Eremogone, along with other functional annotations., Conclusions: Gene family expansions induced by ancient WGDs may have contributed to the shifts to cold climatic niches in the Caryophyllaceae. Transcriptomic data are crucial resources that help unravel heterogeneity in deep-time evolutionary patterns in plants., (© 2024 The Author(s). American Journal of Botany published by Wiley Periodicals LLC on behalf of Botanical Society of America.)

Dinoflagellates are an abundant and diverse group of protists representing a wealth of unique biology and ecology. While many dinoflagellates are photosynthetic or mixotrophic, many taxa are heterotrophs, often with complex feeding strategies. Compared to their photosynthetic counterparts, heterotrophic dinoflagellates remain understudied, as they are difficult to culture. One exception, a long-cultured isolate originally classified as Amphidinium but recently reclassified as Oxytoxum, has been the subject of a number of feeding, growth, and chemosensory studies. This lineage was recently determined to be closely related to Prorocentrum using phylogenetics of ribosomal RNA gene sequences, but the exact nature of this relationship remains unresolved. Using transcriptomes sequenced from culture and three single cells from the environment, we produce a robust phylogeny of 242 genes, revealing Oxytoxum is likely sister to the Prorocentrum clade, rather than nested within it. Molecular investigations uncover evidence of a reduced, nonphotosynthetic plastid and proteorhodopsin, a photoactive proton pump acquired horizontally from bacteria. We describe the ultrastructure of O. lohmannii, including densely packed trichocysts, and a new type of mucocyst. We observe that O. lohmannii feeds preferentially on cryptophytes using myzocytosis, but can also feed on various phytoflagellates using conventional phagocytosis. O. lohmannii is amenable to culture, providing an opportunity to better study heterotrophic dinoflagellate biology and feeding ecology., (© 2024 The Author(s). Journal of Eukaryotic Microbiology published by Wiley Periodicals LLC on behalf of International Society of Protistologists.)

Premise: Leafless, heterotrophic plants are prime examples of organismal modification, the genomic consequences of which have received considerable interest. In particular, plastid genomes (plastomes) are being sequenced at a high rate, allowing continual refinement of conceptual models of reductive evolution in heterotrophs. However, numerous sampling gaps exist, hindering the ability to conduct comprehensive phylogenomic analyses in these plants., Methods: Using floral tissue from an herbarium specimen, we sequenced and analyzed the plastome of Degranvillea dermaptera, a rarely collected, leafless orchid species from South America about which little is known, including its phylogenetic affinities., Results: The plastome is the most reduced of those sequenced among the orchid subfamily Orchidoideae. In Degranvillea, it has lost the majority of genes found in leafy autotrophic species, is structurally rearranged, and has similar gene content to the most reduced plastomes among the orchids. We found strong evidence for the placement of Degranvillea within the subtribe Spiranthinae using models that explicitly account for heterotachy, or lineage-specific evolutionary rate variation over time. We further found evidence of relaxed selection on several genes and of correlations among substitution rates and several other "traits" of the plastome among leafless members of orchid subfamily Orchidoideae., Conclusions: Our findings advance knowledge on the phylogenetic relationships and paths of plastid genome evolution among the orchids, which have experienced more independent transitions to heterotrophy than any other plant family. This study demonstrates the importance of herbarium collections in comparative genomics of poorly known species of conservation concern., (© 2024 Botanical Society of America.)

Premise: The huge diversity of Salix subgenus Chamaetia/Vetrix clade in North America and the lack of phylogenetic resolution within this clade has presented a difficult but fascinating challenge for taxonomists to resolve. Here we tested the existing taxonomic classification with molecular tools., Methods: In this study, 132 samples representing 46 species from 22 described sections of shrub willows from the United States and Canada were analyzed and combined with 67 samples from Eurasia. The ploidy levels of the samples were determined using flow cytometry and nQuire. Sequences were produced using a RAD sequencing approach and subsequently analyzed with ipyrad, then used for phylogenetic reconstructions (RAxML, SplitsTree), dating analyses (BEAST, SNAPPER), and character evolution analyses of 14 selected morphological traits (Mesquite)., Results: The RAD sequencing approach allowed the production of a well-resolved phylogeny of shrub willows. The resulting tree showed an exclusively North American (NA) clade in sister position to a Eurasian clade, which included some North American endemics. The NA clade began to diversify in the Miocene. Polyploid species appeared in each observed clade. Character evolution analyses revealed that adaptive traits such as habit and adaxial nectaries evolved multiple times independently., Conclusions: The diversity in shrub willows was shaped by an evolutionary radiation in North America. Most species were monophyletic, but the existing sectional classification could not be supported by molecular data. Nevertheless, monophyletic lineages share several morphological characters, which might be useful in the revision of the taxonomic classification of shrub willows., (© 2024 The Author(s). American Journal of Botany published by Wiley Periodicals LLC on behalf of Botanical Society of America.)

Premise Researchers adopting target‐enrichment approaches often struggle with the decision of whether to use universal or lineage‐specific probe sets. To circumvent this quandary, we investigate the efficacy of a simultaneous enrichment by combining universal probes and lineage‐specific probes in a single hybridization reaction, to benefit from the qualities of both probe sets with little added cost or effort. Methods and Results Using 26 Brassicaceae libraries and standard enrichment protocols, we compare results from three independent data sets. A large average fraction of reads mapping to the Angiosperms353 (24–31%) and Brassicaceae (35–59%) targets resulted in a sizable reconstruction of loci for each target set (x̄ ≥ 70%). Conclusions High levels of enrichment and locus reconstruction for the two target sets demonstrate that the sampling of genomic regions can be easily extended through the combination of probe sets in single enrichment reactions. We hope that these findings will facilitate the production of expanded data sets that answer individual research questions and simultaneously allow wider applications by the research community as a whole.

Premise Understanding relationships among orchid species and populations is of critical importance for orchid conservation. Target sequence capture has become a standard method for extracting hundreds of orthologous loci for phylogenomics. Up‐front cost and time associated with design of bait sets makes this method prohibitively expensive for many researchers. Therefore, we designed a target capture kit to reliably sequence hundreds of orthologous loci across orchid lineages. Methods We designed an Orchidaceae target capture bait set for 963 single‐copy genes identified in published orchid genome sequences. The bait set was tested on 28 orchid species, with representatives of the subfamilies Cypripedioideae, Orchidoideae, and Epidendroideae. Results Between 1,518,041 and 87,946,590 paired‐end 150‐base reads were generated for target‐enriched genomic libraries. We assembled an average of 812 genes per library for Epidendroideae species and a mean of 501 genes for species in the subfamilies Orchidoideae and Cypripedioideae. Furthermore, libraries had on average 107 of the 254 genes that are included in the Angiosperms353 bait set, allowing for direct comparison of studies using either bait set. Discussion The Orchidaceae963 kit will enable greater accessibility and utility of next‐generation sequencing for orchid systematics, population genetics, and identification in the illegal orchid trade.

Premise Phylogenetic studies in the Compositae are challenging due to the sheer size of the family and the challenges they pose for molecular tools, ranging from the genomic impact of polyploid events to their very conserved plastid genomes. The search for better molecular tools for phylogenetic studies led to the development of the family‐specific Compositae1061 probe set, as well as the universal Angiosperms353 probe set designed for all flowering plants. In this study, we evaluate the extent to which data generated using the family‐specific kit and those obtained with the universal kit can be merged for downstream analyses. Methods We used comparative methods to verify the presence of shared loci between probe sets. Using two sets of eight samples sequenced with Compositae1061 and Angiosperms353, we ran phylogenetic analyses with and without loci flagged as paralogs, a gene tree discordance analysis, and a complementary phylogenetic analysis mixing samples from both sample sets. Results Our results show that the Compositae1061 kit provides an average of 721 loci, with 9–46% of them presenting paralogs, while the Angiosperms353 set yields an average of 287 loci, which are less affected by paralogy. Analyses mixing samples from both sets showed that the presence of 30 shared loci in the probe sets allows the combination of data generated in different ways. Discussion Combining data generated using different probe sets opens up the possibility of collaborative efforts and shared data within the synantherological community.

Abstract Eleven of eighteen Society Island Partula species endemic to the Windward Island subgroup (Moorea and Tahiti) have been extirpated by an ill‐advised biological control program. The conservation status of this critically endangered tree snail radiation is of considerable import, but is clouded by taxonomic uncertainty due to the extensive lack of congruence among species designations, diagnostic morphologies, and molecular markers. Using a combination of museum, captive, and remnant wild snails, we obtained the first high‐resolution nuclear genomic perspective of the evolutionary relationships and survival of fourteen Windward Island Partula species, totaling 93 specimens. We analyzed ~1,607–28,194 nuclear genomic loci collected with the double digest restriction‐site associated sequencing method. Results from phylogenomic trees, species estimation, and population assignment tests yielded monophyly of the Windward Island subgroup. Within this group, two well‐supported clades encompassing five species complexes were recovered. Clade 1 was restricted to Tahiti and contained two species complexes: “P. affinis” (three species) and “P. otaheitana” (five species). Clade 2 occurred on Moorea and on Tahiti and consisted of three species complexes: one Tahitian, “P. clara/P. hyalina”; the other two, “P. taeniata” (three species) and “P. suturalis” (six species), Moorean. Our genomic results largely corroborated previous mitochondrial DNA survival estimates for Moorea and Tahiti, with all five species complexes having members surviving in captivity and/or as remnant wild populations, although the details vary in each case. Continued, proactive conservation and management may yet ensure a phylogenetically representative survival of the fabled Partula species of Moorea and Tahiti.

Abstract Targeted capture and enrichment approaches have proven effective for phylogenetic study. Ultraconserved elements (UCEs) in particular have exhibited great utility for phylogenomic analyses, with the software package phyluce being among the most utilized pipelines for UCE phylogenomics, including probe design. Despite the success of UCEs, it is becoming increasing apparent that diverse lineages require probe sets tailored to focal taxa in order to improve locus recovery. However, factors affecting probe design and methods for optimizing probe sets to focal taxa remain underexplored. Here, we use newly available beetle (Coleoptera) genomic resources to investigate factors affecting UCE probe set design using phyluce. In particular, we explore the effects of stringency during initial design steps, as well as base genome choice on resulting probe sets and locus recovery. We found that both base genome choice and initial bait design stringency parameters greatly alter the number of resultant probes included in final probe sets and strongly affect the number of loci detected and recovered during in silico testing of these probe sets. In addition, we identify attributes of base genomes that correlated with high performance in probe design. Ultimately, we provide a recommended workflow for using Phyluce to design an optimized UCE probe set that will work across a targeted lineage, and use our findings to develop a new, open‐source UCE probe set for beetles of the suborder Adephaga.

Premise Large phylogenetic data sets have often been restricted to small numbers of loci from GenBank, and a vetted sampling‐to‐sequencing phylogenomic protocol scaling to thousands of species is not yet available. Here, we report a high‐throughput collections‐based approach that empowers researchers to explore more branches of the tree of life with numerous loci. Methods We developed an integrated Specimen‐to‐Laboratory Information Management System (SLIMS), connecting sampling and wet lab efforts with progress tracking at each stage. Using unique identifiers encoded in QR codes and a taxonomic database, a research team can sample herbarium specimens, efficiently record the sampling event, and capture specimen images. After sampling in herbaria, images are uploaded to a citizen science platform for metadata generation, and tissue samples are moved through a simple, high‐throughput, plate‐based herbarium DNA extraction and sequencing protocol. Results We applied this sampling‐to‐sequencing workflow to ~15,000 species, producing for the first time a data set with ~50% taxonomic representation of the “nitrogen‐fixing clade” of angiosperms. Discussion The approach we present is appropriate at any taxonomic scale and is extensible to other collection types. The widespread use of large‐scale sampling strategies repositions herbaria as accessible but largely untapped resources for broad taxonomic sampling with thousands of species.

PREMISE New sequencing technologies facilitate the generation of large‐scale molecular data sets for constructing the plant tree of life. We describe a new probe set for target enrichment sequencing to generate nuclear sequence data to build phylogenetic trees with any flagellate land plants, including hornworts, liverworts, mosses, lycophytes, ferns, and all gymnosperms. METHODS We leveraged existing transcriptome and genome sequence data to design the GoFlag 451 probes, a set of 56,989 probes for target enrichment sequencing of 451 exons that are found in 248 single‐copy or low‐copy nuclear genes across flagellate plant lineages. RESULTS Our results indicate that target enrichment using the GoFlag451 probe set can provide large nuclear data sets that can be used to resolve relationships among both distantly and closely related taxa across the flagellate land plants. We also describe the GoFlag 408 probes, an optimized probe set covering 408 of the 451 exons from the GoFlag 451 probe set that is commercialized by RAPiD Genomics. CONCLUSIONS A target enrichment approach using the new probe set provides a relatively low‐cost solution to obtain large‐scale nuclear sequence data for inferring phylogenetic relationships across flagellate land plants.

Premise: Phylogenetic approaches can provide valuable insights on how and when a biome emerged and developed using its structuring species. In this context, Brachystegia Benth, a dominant genus of trees in miombo woodlands, appears as a key witness of the history of the largest woodland and savanna biome of Africa., Methods: We reconstructed the evolutionary history of the genus using targeted-enrichment sequencing on 60 Brachystegia specimens for a nearly complete species sampling. Phylogenomic inferences used supermatrix (RAxML-NG) and summary-method (ASTRAL-III) approaches. Conflicts between species and gene trees were assessed, and the phylogeny was time-calibrated in BEAST. Introgression between species was explored using Phylonet., Results: The phylogenies were globally congruent regardless of the method used. Most of the species were recovered as monophyletic, unlike previous plastid phylogenetic reconstructions where lineages were shared among geographically close individuals independently of species identity. Still, most of the individual gene trees had low levels of phylogenetic information and, when informative, were mostly in conflict with the reconstructed species trees. These results suggest incomplete lineage sorting and/or reticulate evolution, which was supported by network analyses. The BEAST analysis supported a Pliocene origin for current Brachystegia lineages, with most of the diversification events dated to the Pliocene-Pleistocene., Conclusions: These results suggest a recent origin of species of the miombo, congruently with their spatial expansion documented from plastid data. Brachystegia species appear to behave potentially as a syngameon, a group of interfertile but still relatively well-delineated species, an aspect that deserves further investigations., (© 2024 Botanical Society of America.)

Premise Putatively single‐copy nuclear (SCN) loci, which are identified using genomic resources of closely related species, are ideal for phylogenomic inference. However, suitable genomic resources are not available for many clades, including Melastomataceae. We introduce a versatile approach to identify SCN loci for clades with few genomic resources and use it to develop probes for target enrichment in the distantly related Memecylon and Tibouchina (Melastomataceae). Methods We present a two‐tiered pipeline. First, we identified putatively SCN loci using MarkerMiner and transcriptomes from distantly related species in Melastomataceae. Published loci and genes of functional significance were then added (384 total loci). Second, using HybPiper, we retrieved 689 homologous template sequences for these loci using genome‐skimming data from within the focal clades. Results We sequenced 193 loci common to Memecylon and Tibouchina. Probes designed from 56 template sequences successfully targeted sequences in both clades. Probes designed from genome‐skimming data within a focal clade were more successful than probes designed from other sources. Discussion Our pipeline successfully identified and targeted SCN loci in Memecylon and Tibouchina, enabling phylogenomic studies in both clades and potentially across Melastomataceae. This pipeline could be easily applied to other clades with few genomic resources.

During the course of independent studies in Europe, North America, and Africa, seven yeast strains were isolated from insect frass, decaying wood, tree flux, and olive oil sediment. Phylogenetic analysis of two barcoding DNA regions (internal transcribed spacer and the D1/D2 domain of the LSU rRNA gene) revealed that they belong to two closely related undescribed species distinct from all genera in the family Debaryomycetaceae. For reliable taxonomic placement the genomes of four strains of the two novel species and six type strains of closely related species were sequenced. Orthologous genes from 54 genomes of representatives of the Pichiomycetes and 23 outgroup taxa were concatenated to construct a fully supported phylogenetic tree. Consistent with the assumptions, we found that the two new species belong to a novel genus. In addition, the delimitation of the novel species was supported by genetic distance calculations from average nucleotide identity (ANI) and digital DNA:DNA hybridization (dDDH) values. The physiological characterization of the novel species was generally consistent with their genomic content. All strains had two alleles encoding secretory lipase in either two or three copies depending on the species. However, lipolytic activity was detected only in strains with three copies of the secretory lipase gene. Nevertheless, lipolytic activity might be related to their association with the insect gut. Based on these results, formal descriptions of the new genus Rasporella gen. nov. and of two new species Rasporella dianae sp. nov. (holotype UCDFST 68-643 T , MycoBank no.: 850238) and Rasporella oleae sp. nov. (holotype ZIM 2471 T , MycoBank no.: 850126) are provided., (© 2023 The Authors. Yeast published by John Wiley & Sons Ltd.)

Premise: Although Boechera (Boechereae, Brassicaceae) has become a plant model system for both ecological genomics and evolutionary biology, all previous phylogenetic studies have had limited success in resolving species relationships within the genus. The recent effective application of sequence data from target enrichment approaches to resolve the evolutionary relationships of several other challenging plant groups prompted us to investigate their usefulness in Boechera and Boechereae., Methods: To resolve the phylogeny of Boechera and closely related genera, we utilized the Hybpiper pipeline to analyze two combined bait sets: Angiosperms353, with broad applicability across flowering plants; and a Brassicaceae-specific bait set designed for use in the mustard family. Relationships for 101 samples representing 81 currently recognized species were inferred from a total of 1114 low-copy nuclear genes using both supermatrix and species coalescence methods., Results: Our analyses resulted in a well-resolved and highly supported phylogeny of the tribe Boechereae. Boechereae is divided into two major clades, one comprising all western North American species of Boechera, the other encompassing the eight other genera of the tribe. Our understanding of relationships within Boechera is enhanced by the recognition of three core clades that are further subdivided into robust regional species complexes., Conclusions: This study presents the first broadly sampled, well-resolved phylogeny for most known sexual diploid Boechera. This effort provides the foundation for a new phylogenetically informed taxonomy of Boechera that is crucial for its continued use as a model system., (© 2023 Botanical Society of America.)