Placing new sequences onto reference phylogenies is increasingly used for analyzing environmental samples, especially microbiomes. Existing placement methods assume that query sequences have evolved under specific models directly on the reference phylogeny. For example, they assume single-gene data (e.g., 16S rRNA amplicons) have evolved under the GTR model on a gene tree. Placement, however, often has a more ambitious goal: extending a (genome-wide) species tree given data from individual genes without knowing the evolutionary model. Addressing this challenging problem requires new directions. Here, we introduce Deep-learning Enabled Phylogenetic Placement (DEPP), an algorithm that learns to extend species trees using single genes without pre-specified models. In simulations and on real data, we show that DEPP can match the accuracy of model-based methods without any prior knowledge of the model. We also show that DEPP can update the multi-locus microbial tree-of-life with single genes with high accuracy. We further demonstrate that DEPP can combine 16S and metagenomic data onto a single tree, enabling community structure analyses that take advantage of both sources of data.

Phylogenetics has long relied on the use of orthologs, or genes related through speciation events, to infer species relationships. However, identifying orthologs is difficult because gene duplication can obscure relationships among genes. Researchers have been particularly concerned with the insidious effects of pseudoorthologs-duplicated genes that are mistaken for orthologs because they are present in a single copy in each sampled species. Because gene tree topologies of pseudoorthologs may differ from the species tree topology, they have often been invoked as the cause of counterintuitive results in phylogenetics. Despite these perceived problems, no previous work has calculated the probabilities of pseudoortholog topologies or has been able to circumscribe the regions of parameter space in which pseudoorthologs are most likely to occur. Here, we introduce a model for calculating the probabilities and branch lengths of orthologs and pseudoorthologs, including concordant and discordant pseudoortholog topologies, on a rooted three-taxon species tree. We show that the probability of orthologs is high relative to the probability of pseudoorthologs across reasonable regions of parameter space. Furthermore, the probabilities of the two discordant topologies are equal and never exceed that of the concordant topology, generally being much lower. We describe the species tree topologies most prone to generating pseudoorthologs, finding that they are likely to present problems to phylogenetic inference irrespective of the presence of pseudoorthologs. Overall, our results suggest that pseudoorthologs are unlikely to mislead inferences of species relationships under the biological scenarios considered here.[Birth-death model; orthologs; paralogs; phylogenetics.].

Widely used approaches for extracting phylogenetic information from aligned sets of molecular sequences rely upon probabilistic models of nucleotide substitution or amino-acid replacement. The phylogenetic information that can be extracted depends on the number of columns in the sequence alignment and will be decreased when the alignment contains gaps due to insertion or deletion events. Motivated by the measurement of information loss, we suggest assessment of the effective sequence length (ESL) of an aligned data set. The ESL can differ from the actual number of columns in a sequence alignment because of the presence of alignment gaps. Furthermore, the estimation of phylogenetic information is affected by model misspecification. Inevitably, the actual process of molecular evolution differs from the probabilistic models employed to describe this process. This disparity means the amount of phylogenetic information in an actual sequence alignment will differ from the amount in a simulated data set of equal size, which motivated us to develop a new test for model adequacy. Via theory and empirical data analysis, we show how to disentangle the effects of gaps and model misspecification. By comparing the Fisher information of actual and simulated sequences, we identify which alignment sites and tree branches are most affected by gaps and model misspecification. [Fisher information; gaps; insertion; deletion; indel; model adequacy; goodness-of-fit test; sequence alignment.].

Introgression is an important biological process affecting at least 10% of the extant species in the animal kingdom. Introgression significantly impacts inference of phylogenetic species relationships where a strictly binary tree model cannot adequately explain reticulate net-like species relationships. Here, we use phylogenomic approaches to understand patterns of introgression along the evolutionary history of a unique, nonmodel insect system: dragonflies and damselflies (Odonata). We demonstrate that introgression is a pervasive evolutionary force across various taxonomic levels within Odonata. In particular, we show that the morphologically "intermediate" species of Anisozygoptera (one of the three primary suborders within Odonata besides Zygoptera and Anisoptera), which retain phenotypic characteristics of the other two suborders, experienced high levels of introgression likely coming from zygopteran genomes. Additionally, we find evidence for multiple cases of deep inter-superfamilial ancestral introgression. [Gene flow; Odonata; phylogenomics; reticulate evolution.].

Estimating time-dependent rates of speciation and extinction from dated phylogenetic trees of extant species (timetrees), and determining how and why they vary, is key to understanding how ecological and evolutionary processes shape biodiversity. Due to an increasing availability of phylogenetic trees, a growing number of process-based methods relying on the birth-death model have been developed in the last decade to address a variety of questions in macroevolution. However, this methodological progress has regularly been criticized such that one may wonder how reliable the estimations of speciation and extinction rates are. In particular, using lineages-through-time (LTT) plots, a recent study has shown that there are an infinite number of equally likely diversification scenarios that can generate any timetree. This has led to questioning whether or not diversification rates should be estimated at all. Here, we summarize, clarify, and highlight technical considerations on recent findings regarding the capacity of models to disentangle diversification histories. Using simulations, we illustrate the characteristics of newly proposed "pulled rates" and their utility. We recognize that the recent findings are a step forward in understanding the behavior of macroevolutionary modeling, but they in no way suggest we should abandon diversification modeling altogether. On the contrary, the study of macroevolution using phylogenetic trees has never been more exciting and promising than today. We still face important limitations in regard to data availability and methods, but by acknowledging them we can better target our joint efforts as a scientific community. [Birth-death models; extinction; phylogenetics; speciation.].

The historical signal in nucleotide sequences becomes eroded over time by substitutions occurring repeatedly at the same sites. This phenomenon, known as substitution saturation, is recognized as one of the primary obstacles to deep-time phylogenetic inference using genome-scale data sets. We present a new test of substitution saturation and demonstrate its performance in simulated and empirical data. For some of the 36 empirical phylogenomic data sets that we examined, we detect substitution saturation in around 50% of loci. We found that saturation tends to be flagged as problematic in loci with highly discordant phylogenetic signals across sites. Within each data set, the loci with smaller numbers of informative sites are more likely to be flagged as containing problematic levels of saturation. The entropy saturation test proposed here is sensitive to high evolutionary rates relative to the evolutionary timeframe, while also being sensitive to several factors known to mislead phylogenetic inference, including short internal branches relative to external branches, short nucleotide sequences, and tree imbalance. Our study demonstrates that excluding loci with substitution saturation can be an effective means of mitigating the negative impact of multiple substitutions on phylogenetic inferences. [Phylogenetic model performance; phylogenomics; substitution model; substitution saturation; test statistics.].

Species tree inference from gene family trees is a significant problem in computational biology. However, gene tree heterogeneity, which can be caused by several factors including gene duplication and loss, makes the estimation of species trees very challenging. While there have been several species tree estimation methods introduced in recent years to specifically address gene tree heterogeneity due to gene duplication and loss (such as DupTree, FastMulRFS, ASTRAL-Pro, and SpeciesRax), many incur high cost in terms of both running time and memory. We introduce a new approach, DISCO, that decomposes the multi-copy gene family trees into many single copy trees, which allows for methods previously designed for species tree inference in a single copy gene tree context to be used. We prove that using DISCO with ASTRAL (i.e., ASTRAL-DISCO) is statistically consistent under the GDL model, provided that ASTRAL-Pro correctly roots and tags each gene family tree. We evaluate DISCO paired with different methods for estimating species trees from single copy genes (e.g., ASTRAL, ASTRID, and IQ-TREE) under a wide range of model conditions, and establish that high accuracy can be obtained even when ASTRAL-Pro is not able to correctly roots and tags the gene family trees. We also compare results using MI, an alternative decomposition strategy from Yang Y. and Smith S.A. (2014), and find that DISCO provides better accuracy, most likely as a result of covering more of the gene family tree leafset in the output decomposition. [Concatenation analysis; gene duplication and loss; species tree inference; summary method.].

Phylogenetic networks provide a powerful framework for modeling and analyzing reticulate evolutionary histories. While polyploidy has been shown to be prevalent not only in plants but also in other groups of eukaryotic species, most work done thus far on phylogenetic network inference assumes diploid hybridization. These inference methods have been applied, with varying degrees of success, to data sets with polyploid species, even though polyploidy violates the mathematical assumptions underlying these methods. Statistical methods were developed recently for handling specific types of polyploids and so were parsimony methods that could handle polyploidy more generally yet while excluding processes such as incomplete lineage sorting. In this article, we introduce a new method for inferring most parsimonious phylogenetic networks on data that include polyploid species. Taking gene tree topologies as input, the method seeks a phylogenetic network that minimizes deep coalescences while accounting for polyploidy. We demonstrate the performance of the method on both simulated and biological data. The inference method as well as a method for evaluating evolutionary hypotheses in the form of phylogenetic networks are implemented and publicly available in the PhyloNet software package. [Incomplete lineage sorting; minimizing deep coalescences; multilabeled trees; multispecies network coalescent; phylogenetic networks; polyploidy.].

Integrative taxonomy is central to modern taxonomy and systematic biology, including behavior, niche preference, distribution, morphological analysis, and DNA barcoding. However, decades of use demonstrate that these methods can face challenges when used in isolation, for instance, potential misidentifications due to phenotypic plasticity for morphological methods, and incorrect identifications because of introgression, incomplete lineage sorting, and horizontal gene transfer for DNA barcoding. Although researchers have advocated the use of integrative taxonomy, few detailed algorithms have been proposed. Here, we develop a convolutional neural network method (morphology-molecule network [MMNet]) that integrates morphological and molecular data for species identification. The newly proposed method (MMNet) worked better than four currently available alternative methods when tested with 10 independent data sets representing varying genetic diversity from different taxa. High accuracies were achieved for all groups, including beetles (98.1% of 123 species), butterflies (98.8% of 24 species), fishes (96.3% of 214 species), and moths (96.4% of 150 total species). Further, MMNet demonstrated a high degree of accuracy ($>$98%) in four data sets including closely related species from the same genus. The average accuracy of two modest subgenomic (single nucleotide polymorphism) data sets, comprising eight putative subspecies respectively, is 90%. Additional tests show that the success rate of species identification under this method most strongly depends on the amount of training data, and is robust to sequence length and image size. Analyses on the contribution of different data types (image vs. gene) indicate that both morphological and genetic data are important to the model, and that genetic data contribute slightly more. The approaches developed here serve as a foundation for the future integration of multimodal information for integrative taxonomy, such as image, audio, video, 3D scanning, and biosensor data, to characterize organisms more comprehensively as a basis for improved investigation, monitoring, and conservation of biodiversity. [Convolutional neural network; deep learning; integrative taxonomy; single nucleotide polymorphism; species identification.].

Mountain systems harbor a substantial fraction of global biodiversity and, thus, provide excellent opportunities to study rapid diversification and to understand the historical processes underlying the assembly of biodiversity hotspots. The rich biodiversity in mountains is widely regarded as having arisen under the influence of geological and climatic processes as well as the complex interactions among them. However, the relative contribution of geology and climate in driving species radiation is seldom explored. Here, we studied the evolutionary radiation of Oreocharis (Gesneriaceae), which has diversified extensively throughout East Asia, especially within the Hengduan Mountains (HDM), using transcriptomic data and a time calibrated phylogeny for 88% (111/126) of all species of the genus. In particular, we applied phylogenetic reconstructions to evaluate the extent of incomplete lineage sorting accompanying the early and rapid radiation in the genus. We then fit macroevolutionary models to explore its spatial and diversification dynamics in Oreocharis and applied explicit birth-death models to investigate the effects of past environmental changes on its diversification. Evidence from 574 orthologous loci suggest that Oreocharis underwent an impressive early burst of speciation starting ca. 12 Ma in the Miocene, followed by a drastic decline in speciation toward the present. Although we found no evidence for a shift in diversification rate across the phylogeny of Oreocharis, we showed a difference in diversification dynamics between the HDM and non-HDM lineages, with higher diversification rates in the HDM. The diversification dynamic of Oreocharis is most likely positively associated with temperature-dependent speciation and dependency on the Asian monsoons. We suggest that the warm and humid climate of the mid-Miocene was probably the primary driver of the rapid diversification in Oreocharis, while mountain building of the HDM might have indirectly affected species diversification of the HDM lineage. This study highlights the importance of past climatic changes, combined with mountain building, in creating strong environmental heterogeneity and driving diversification of mountain plants, and suggests that the biodiversity in the HDM cannot directly be attributed to mountain uplift, contrary to many recent speculations.[East Asian monsoons; environmental heterogeneity; Hengduan Mountains; incomplete lineage sorting; Oreocharis; past climate change; rapid diversification; transcriptome.].

Whole genome sequences are beginning to revolutionize our understanding of phylogenetic relationships. Yet, even whole genome sequences can fail to resolve the evolutionary history of the most rapidly radiating lineages, where incomplete lineage sorting, standing genetic variation, introgression, and other factors obscure the phylogenetic history of the group. To overcome such challenges, one emerging strategy is to integrate results across different methods. Most such approaches have been implemented on reduced representation genomic data sets, but whole genomes should provide the maximum possible evidence approach. Here, we test the ability of single nucleotide polymorphisms extracted from whole genome resequencing data, implemented in an integrative genomic approach, to resolve key nodes in the phylogeny of the mbuna, rock-dwelling cichlid fishes of Lake Malaŵi, which epitomize the phylogenetic intractability that often accompanies explosive lineage diversification. This monophyletic radiation has diversified at an unparalleled rate into several hundred species in less than 2 million years. Using an array of phylogenomic methods, we consistently recovered four major clades of mbuna, but a large basal polytomy among them. Although introgression between clades apparently contributed to the challenge of phylogenetic reconstruction, reduction of the data set to nonintrogressed sites still did not help to resolve the basal polytomy. On the other hand, relationships among six congeneric species pairs were resolved without ambiguity, even in one case where existing data led us to predict that resolution would be difficult. We conclude that the bursts of diversification at the earliest stages of the mbuna radiation may be phylogenetically unresolvable, but other regions of the tree are phylogenetically clearly supported. Integration of multiple phylogenomic approaches will continue to increase confidence in relationships inferred from these and other whole-genome data sets. [Incomplete lineage sorting; introgression; linkage disequilibrium; multispecies coalescence; rapid radiation; soft polytomy.].

Compared to other regions, the drivers of diversification in Africa are poorly understood. We studied a radiation of insects with over 100 species occurring in a wide range of habitats across the Afrotropics to investigate the fundamental evolutionary processes and geological events that generate and maintain patterns of species richness on the continent. By investigating the evolutionary history of Bicyclus butterflies within a phylogenetic framework, we inferred the group's origin at the Oligo-Miocene boundary from ancestors in the Congolian rainforests of central Africa. Abrupt climatic fluctuations during the Miocene (ca. 19-17 Ma) likely fragmented ancestral populations, resulting in at least eight early-divergent lineages. Only one of these lineages appears to have diversified during the drastic climate and biome changes of the early Miocene, radiating into the largest group of extant species. The other seven lineages diversified in forest ecosystems during the late Miocene and Pleistocene when climatic conditions were more favorable-warmer and wetter. Our results suggest changing Neogene climate, uplift of eastern African orogens, and biotic interactions have had different effects on the various subclades of Bicyclus, producing one of the most spectacular butterfly radiations in Africa. [Afrotropics; biodiversity; biome; biotic interactions; Court Jester; extinction; grasslands; paleoclimates; Red Queen; refugia forests; dependent-diversification; speciation.].

Reticulation, caused by hybridization and allopolyploidization, is considered an important and frequent phenomenon in the evolution of numerous plant lineages. Although both processes represent important driving forces of evolution, they are mostly ignored in phylogenetic studies involving a large number of species. Indeed only a scattering of methods exists to recover a comprehensive reticulated evolutionary history for a broad taxon sampling. Among these methods, comparisons of topologies obtained from plastid markers with those from a few nuclear sequences are favored, even though they restrict in-depth studies of hybridization and polyploidization. The genus Rosa encompasses c. 150 species widely distributed throughout the northern hemisphere and represents a challenging taxonomic group in which hybridization and polyploidization are prominent. Our main objective was to develop a general framework that would take patterns of reticulation into account in the study of the phylogenetic relationships among Rosa species. Using amplicon sequencing, we targeted allele variation in the nuclear genome as well as haploid sequences in the chloroplast genome. We successfully recovered robust plastid and nuclear phylogenies and performed in-depth tests for several scenarios of hybridization using a maximum pseudo-likelihood approach on taxon subsets. Our diploid-first approach followed by hybrid and polyploid grafting resolved most of the evolutionary relationships among Rosa subgenera, sections, and selected species. Based on these results, we provide new directions for a future revision of the infrageneric classification in Rosa. The stepwise strategy proposed here can be used to reconstruct the phylogenetic relationships of other challenging taxonomic groups with large numbers of hybrid and polyploid taxa. [Amplicon sequencing; interspecific hybridization; polyploid detection; reticulate evolution.].

Establishing an evolutionary timeline is fundamental for tackling a great variety of topics in evolutionary biology, including the reconstruction of patterns of historical biogeography, coevolution, and diversification. However, the tree of life is pruned by extinction and molecular data cannot be gathered for extinct lineages. Until recently methodological challenges have prevented the application of tip-dating Bayesian approaches in morphology-based fossil-only data sets. Herein, we present a morphological data set for a group of cricetid rodents to which we apply an array of methods fairly new in paleontology that can be used by paleontologists for the analysis of entirely extinct clades. We compare the tree topologies obtained by traditional parsimony, time-calibrated, and noncalibrated Bayesian inference phylogenetic approaches and calculate stratigraphic congruence indices for each. Bayesian tip-dated clock methods outperform parsimony in the case of our data set, which includes highly homoplastic morphological characters. Regardless, all three topologies support the monophyly of Megacricetodontinae, Democricetodontinae, and Cricetodontinae. Dispersal and speciation events inferred through Bayesian Binary Markov chain Monte Carlo and biodiversity analyses provide evidence for a correlation between biogeographic events, climatic changes, and diversification in cricetids. [Bayesian tip-dating; Cricetidae; Miocene; morphological clock; paleobiodiversity; paleobiogeography; paleoecology; parsimony; STRAP.].

Gene flow and reticulation are increasingly recognized as important processes in the diversification of many taxonomic groups. With the increasing ease of collecting genomic data and the development of multispecies coalescent network approaches, such reticulations can be accounted for when inferring phylogeny and diversification. Caribbean Anolis lizards are a classic example of an adaptive radiation in which species have independently radiated on the islands of the Greater Antilles into the same ecomorph classes. Within the Jamaican radiation at least one species, Anolis opalinus, has been documented to be polyphyletic in its mitochondrial DNA, which could be the result of an ancient reticulation event or incomplete lineage sorting (ILS). Here, we generate mtDNA and genotyping-by-sequencing (GBS) data and implement gene tree, species tree, and multispecies coalescent network methods to infer the diversification of this group. Our mtDNA gene tree recovers the same relationships previously inferred for this group, which is strikingly different from the species tree inferred from our GBS data. Posterior predictive simulations suggest that our genomic data violate commonly adopted assumptions of the multispecies coalescent model (MSCM), so we use network approaches to infer phylogenetic relationships. The inferred network topology contains a reticulation event but does not explain the mtDNA polyphyly observed in this group; however, coalescent simulations suggest that the observed mtDNA topology is likely the result of past introgression. How common a signature of gene flow and reticulation is across the radiation of Anolis is unknown; however, the reticulation events that we demonstrate here may have allowed for adaptive evolution, as has been suggested in other, more recent, adaptive radiations. [Adaptive radiation; hybridization; introgression; multispecies network coalescent; posterior predictive simulation.].

A potential shortcoming of concatenation methods for species tree estimation is their failure to account for incomplete lineage sorting. Coalescent methods address this problem but make various assumptions that, if violated, can result in worse performance than concatenation. Given the challenges of analyzing DNA sequences with both concatenation and coalescent methods, retroelement insertions (RIs) have emerged as powerful phylogenomic markers for species tree estimation. Here, we show that two recently proposed quartet-based methods, SDPquartets and ASTRAL_BP, are statistically consistent estimators of the unrooted species tree topology under the coalescent when RIs follow a neutral infinite-sites model of mutation and the expected number of new RIs per generation is constant across the species tree. The accuracy of these (and other) methods for inferring species trees from RIs has yet to be assessed on simulated data sets, where the true species tree topology is known. Therefore, we evaluated eight methods given RIs simulated from four model species trees, all of which have short branches and at least three of which are in the anomaly zone. In our simulation study, ASTRAL_BP and SDPquartets always recovered the correct species tree topology when given a sufficiently large number of RIs, as predicted. A distance-based method (ASTRID_BP) and Dollo parsimony also performed well in recovering the species tree topology. In contrast, unordered, polymorphism, and Camin-Sokal parsimony (as well as an approach based on MDC) typically fail to recover the correct species tree topology in anomaly zone situations with more than four ingroup taxa. Of the methods studied, only ASTRAL_BP automatically estimates internal branch lengths (in coalescent units) and support values (i.e., local posterior probabilities). We examined the accuracy of branch length estimation, finding that estimated lengths were accurate for short branches but upwardly biased otherwise. This led us to derive the maximum likelihood (branch length) estimate for when RIs are given as input instead of binary gene trees; this corrected formula produced accurate estimates of branch lengths in our simulation study provided that a sufficiently large number of RIs were given as input. Lastly, we evaluated the impact of data quantity on species tree estimation by repeating the above experiments with input sizes varying from 100 to 100,000 parsimony-informative RIs. We found that, when given just 1000 parsimony-informative RIs as input, ASTRAL_BP successfully reconstructed major clades (i.e., clades separated by branches $>0.3$ coalescent units) with high support and identified rapid radiations (i.e., shorter connected branches), although not their precise branching order. The local posterior probability was effective for controlling false positive branches in these scenarios. [Coalescence; incomplete lineage sorting; Laurasiatheria; Palaeognathae; parsimony; polymorphism parsimony; retroelement insertions; species trees; transposon.].

Erratum in Syst Biol. 2022 Feb 09;:

Planning for the protection of species often involves difficult choices about which species to prioritize, given constrained resources. One way of prioritizing species is to consider their "evolutionary distinctiveness", i.e. their relative evolutionary isolation on a phylogenetic tree. Several evolutionary isolation metrics or phylogenetic diversity indices have been introduced in the literature, among them the so-called Fair Proportion index (also known as the "evolutionary distinctiveness" score). This index apportions the total diversity of a tree among all leaves, thereby providing a simple prioritization criterion for conservation. Here, we focus on the prioritization order obtained from the Fair Proportion index and analyze the effects of species extinction on this ranking. More precisely, we analyze the extent to which the ranking order may change when some species go extinct and the Fair Proportion index is re-computed for the remaining taxa. We show that for each phylogenetic tree, there are edge lengths such that the extinction of one leaf per cherry completely reverses the ranking. Moreover, we show that even if only the lowest ranked species goes extinct, the ranking order may drastically change. We end by analyzing the effects of these two extinction scenarios (extinction of the lowest ranked species and extinction of one leaf per cherry) for a collection of empirical and simulated trees. In both cases, we can observe significant changes in the prioritization orders, highlighting the empirical relevance of our theoretical findings.

Phylogenetic reconstruction using concatenated loci ("phylogenomics" or "supermatrix phylogeny") is a powerful tool for solving evolutionary splits that are poorly resolved in single gene/protein trees (SGTs). However, recent phylogenomic attempts to resolve the eukaryote root have yielded conflicting results, along with claims of various artefacts hidden in the data. We have investigated these conflicts using two new methods for assessing phylogenetic conflict. ConJak uses whole marker (gene or protein) jackknifing to assess deviation from a central mean for each individual sequence, while ConWin uses a sliding window to screen for incongruent protein fragments (mosaics). Both methods allow selective masking of individual sequences or sequence fragments in order to minimize missing data, an important consideration for resolving deep splits with limited data. Analyses focused on a set of 76 eukaryotic proteins of bacterial-ancestry previously used in various combinations to assess the branching order among the three major divisions of eukaryotes: Amorphea (mainly animals, fungi and Amoebozoa), Diaphoretickes (most other well-known eukaryotes and nearly all algae) and Excavata, represented here by Discoba (Jakobida, Heterolobosea, and Euglenozoa). ConJak analyses found strong outliers to be concentrated in under-sampled lineages, while ConWin analyses of Discoba, the most under-sampled of the major lineages, detected potentially incongruent fragments scattered throughout. Phylogenetic analyses of the full data using an LG-gamma model support a Discoba sister scenario (neozoan-excavate root), which rises to 99-100% bootstrap support with data masked according to either protocol. However, analyses with two site-specific (CAT) mixture models yielded widely inconsistent results and a striking sensitivity to missing data. The neozoan-excavate root places Amorphea and Diaphoretickes as more closely related to each other than either is to Discoba, a fundamental relationship that should remain unaffected by additional taxa.