SEGUL: Ultrafast, memory-efficient and mobile-friendly software for manipulating and summarizing phylogenomic datasets.

Phylogenetic studies now routinely require manipulating and summarizing thousands of data files. For most of these tasks, currently available software requires considerable computing resources and substantial knowledge of command-line applications. We develop an ultrafast and memory-efficient software, SEGUL, that performs common phylogenomic dataset manipulations and calculates statistics summarizing essential data features. Our software is available as standalone command-line interface (CLI) and graphical user interface (GUI) applications, and as a library for Rust, R and Python, with possible support of other languages. The CLI and library versions run native on Windows, Linux and macOS, including Apple ARM Macs. The GUI version extends support to include mobile iOS, iPadOS and Android operating systems. SEGUL leverages the high performance of the Rust programming language to offer fast execution times and low memory footprints regardless of dataset size and platform choice. The inclusion of a GUI minimizes bioinformatics barriers to phylogenomics while SEGUL's efficiency reduces economic barriers by allowing analysis on inexpensive hardware. Our support for mobile operating systems further enables teaching phylogenomics where access to computing power is limited.

Specimen collection is essential for modern science.

Natural history museums are vital repositories of specimens, samples and data that inform about the natural world; this Formal Comment revisits a Perspective that advocated for the adoption of compassionate collection practices, querying whether it will ever be possible to completely do away with whole animal specimen collection.

Molecular evolution of male reproduction across species with highly divergent sperm morphology in diverse murine rodents.

Sperm competition can drive rapid evolution of male reproductive traits, but it remains unclear how variation in sperm competition intensity shapes phenotypic and molecular diversity across clades. Old World mice and rats (subfamily Murinae) comprise a rapid radiation and exhibit incredible diversity in sperm morphology and production. We combined phenotype and sequence data to model the evolution of reproductive traits and genes across 78 murine species. We identified several shifts towards smaller relative testes mass, a trait reflective of reduced sperm competition. Several sperm traits were associated with relative testes mass, suggesting that mating system evolution likely selects for convergent traits related to sperm competitive ability. Molecular evolutionary rates of spermatogenesis proteins also correlated with relative testes mass, but in an unexpected direction. We predicted that sperm competition would result in rapid divergence among species with large relative testes mass, but instead found that many spermatogenesis genes evolve more rapidly in species with smaller relative testes mass due to relaxed purifying selection. While some reproductive genes evolved under positive selection, relaxed selection played a greater role underlying rapid evolution in small testes species. Our work demonstrates that sexual selection can impose strong purifying selection shaping the evolution of male reproduction.

Predicting Species Boundaries and Assessing Undescribed Diversity in Pneumocystis, an Obligate Lung Symbiont.

Far more biodiversity exists in Fungi than has been described, or could be described in several lifetimes, given current rates of species discovery. Although this problem is widespread taxonomically, our knowledge of animal-associated fungi is especially lacking. Fungi in the genus Pneumocystis are obligate inhabitants of mammal lungs, and they have been detected in a phylogenetically diverse array of species representing many major mammal lineages. The hypothesis that Pneumocystis cospeciate with their mammalian hosts suggests that thousands of Pneumocystis species may exist, potentially equal to the number of mammal species. However, only six species have been described, and the true correspondence of Pneumocystis diversity to host species boundaries is unclear. Here, we use molecular species delimitation to estimate the boundaries of Pneumocystis species sampled from 55 mammal species representing eight orders. Our results suggest that Pneumocystis species often colonize several closely related mammals, especially those in the same genus. Using the newly estimated ratio of fungal to host diversity, we estimate ≈4600 to 6250 Pneumocystis species inhabit the 6495 currently recognized extant mammal species. Additionally, we review the literature and find that only 240 (~3.7%) mammal species have been screened for Pneumocystis, and many detected Pneumocystis lineages are not represented by any genetic data. Although crude, our findings challenge the dominant perspective of strict specificity of Pneumocystis to their mammal hosts and highlight an abundance of undescribed diversity.

Molecular data from the holotype of the enigmatic Bornean Black Shrew, Suncusater Medway, 1965 (Soricidae, Crocidurinae), place it in the genus Palawanosorex.

Although Borneo has received more attention from biologists than most other islands in the Malay Archipelago, many questions regarding the systematic relationships of Bornean mammals remain. Using next-generation sequencing technology, we obtained mitochondrial DNA sequences from the holotype of Suncusater, the only known specimen of this shrew. Several shrews collected recently in Sarawak are closely aligned, both morphologically and mitochondrially, with the holotype of S.ater. Phylogenetic analyses of mitochondrial sequences indicate that the S.ater holotype and new Sarawak specimens do not belong to the genus Suncus, but instead are most closely related to Palawanosorexmuscorum. Until now Palawanosorex has been known only from the neighboring Philippine island of Palawan. Additional sequences from nuclear ultra-conserved elements from the new Sarawak specimens strongly support a sister relationship to P.muscorum. We therefore transfer ater to Palawanosorex. The new specimens demonstrate that P.ater is more widespread in northern Borneo than previously recorded. Continued sampling of Bornean mammal diversity and reexamination of type material are critical in understanding the evolutionary history of the biologically rich Malay Archipelago.

Molecules and fossils tell distinct yet complementary stories of mammal diversification.

Reconstructing the tempo at which biodiversity arose is a fundamental goal of evolutionary biologists, yet the relative merits of evolutionary-rate estimates are debated based on whether they are derived from the fossil record or time-calibrated phylogenies (timetrees) of living species. Extinct lineages unsampled in timetrees are known to "pull" speciation rates downward, but the temporal scale at which this bias matters is unclear. To investigate this problem, we compare mammalian diversification-rate signatures in a credible set of molecular timetrees (n = 5,911 species, ∼70% from DNA) to those in fossil genus durations (n = 5,320). We use fossil extinction rates to correct or "push" the timetree-based (pulled) speciation-rate estimates, finding a surge of speciation during the Paleocene (∼66-56 million years ago, Ma) between the Cretaceous-Paleogene (K-Pg) boundary and the Paleocene-Eocene Thermal Maximum (PETM). However, about two-thirds of the K-Pg-to-PETM originating taxa did not leave modern descendants, indicating that this rate signature is likely undetectable from extant lineages alone. For groups without substantial fossil records, thankfully all is not lost. Pushed and pulled speciation rates converge starting ∼10 Ma and are equal at the present day when recent evolutionary processes can be estimated without bias using species-specific "tip" rates of speciation. Clade-wide moments of tip rates also enable enriched inference, as the skewness of tip rates is shown to approximate a clade's extent of past diversification-rate shifts. Molecular timetrees need fossil-correction to address deep-time questions, but they are sufficient for shallower time questions where extinctions are fewer.

Molecular Evolution of Ecological Specialisation: Genomic Insights from the Diversification of Murine Rodents.

Adaptive radiations are characterized by the diversification and ecological differentiation of species, and replicated cases of this process provide natural experiments for understanding the repeatability and pace of molecular evolution. During adaptive radiation, genes related to ecological specialization may be subject to recurrent positive directional selection. However, it is not clear to what extent patterns of lineage-specific ecological specialization (including phenotypic convergence) are correlated with shared signatures of molecular evolution. To test this, we sequenced whole exomes from a phylogenetically dispersed sample of 38 murine rodent species, a group characterized by multiple, nested adaptive radiations comprising extensive ecological and phenotypic diversity. We found that genes associated with immunity, reproduction, diet, digestion, and taste have been subject to pervasive positive selection during the diversification of murine rodents. We also found a significant correlation between genome-wide positive selection and dietary specialization, with a higher proportion of positively selected codon sites in derived dietary forms (i.e., carnivores and herbivores) than in ancestral forms (i.e., omnivores). Despite striking convergent evolution of skull morphology and dentition in two distantly related worm-eating specialists, we did not detect more genes with shared signatures of positive or relaxed selection than in a nonconvergent species comparison. Although a small number of the genes we detected can be incidentally linked to craniofacial morphology or diet, protein-coding regions are unlikely to be the primary genetic basis of this complex convergent phenotype. Our results suggest a link between positive selection and derived ecological phenotypes, and highlight specific genes and general functional categories that may have played an integral role in the extensive and rapid diversification of murine rodents.

Locomotory mode transitions alter phenotypic evolution and lineage diversification in an ecologically rich clade of mammals.

The relationship between organismal function and form is a cornerstone of biology because functional diversity is key to generating and maintaining ecological diversity. Morphological changes often occur in unison with behavioral or ecological transitions, and this process may foster diversification, but alternately could trap a species on an adaptive peak. We estimated the most comprehensive phylogenetic hypothesis of Murinae, a young (∼15 million years) and diverse (∼700 species) clade of mammals. We then tested for correlated evolution among four morphological traits with potential links to locomotor modes (Arboreal, General, Terrestrial, and Amphibious), then investigated the effects of locomotion on morphological and lineage diversification. We found unique combinations of trait values for each locomotor mode, including strong covariance between the tail and hindfoot lengths of specialized Arboreal and ecologically flexible General species. Low diversification rates and long branch lengths suggest that specialized lineages represent stable evolutionary "cul-de-sacs." General species, characterized by the classic "rat-like" body plan and broad locomotor abilities, have narrow optimal trait values and slow phenotypic evolution, but high lineage diversification rates. Our findings suggest that versatile, generalist forms act as seeds of species diversity and morphological specialization, which together build ecologically diverse radiations.

Convergent evolution of olfactory and thermoregulatory capacities in small amphibious mammals.

Olfaction and thermoregulation are key functions for mammals. The former is critical to feeding, mating, and predator avoidance behaviors, while the latter is essential for homeothermy. Aquatic and amphibious mammals face olfactory and thermoregulatory challenges not generally encountered by terrestrial species. In mammals, the nasal cavity houses a bony system supporting soft tissues and sensory organs implicated in either olfactory or thermoregulatory functions. It is hypothesized that to cope with aquatic environments, amphibious mammals have expanded their thermoregulatory capacity at the expense of their olfactory system. We investigated the evolutionary history of this potential trade-off using a comparative dataset of three-dimensional (3D) CT scans of 189 skulls, capturing 17 independent transitions from a strictly terrestrial to an amphibious lifestyle across small mammals (Afrosoricida, Eulipotyphla, and Rodentia). We identified rapid and repeated loss of olfactory capacities synchronously associated with gains in thermoregulatory capacity in amphibious taxa sampled from across mammalian phylogenetic diversity. Evolutionary models further reveal that these convergences result from faster rates of turbinal bone evolution and release of selective constraints on the thermoregulatory-olfaction trade-off in amphibious species. Lastly, we demonstrated that traits related to vital functions evolved faster to the optimum compared to traits that are not related to vital functions.

Inferring the mammal tree: Species-level sets of phylogenies for questions in ecology, evolution, and conservation.

Big, time-scaled phylogenies are fundamental to connecting evolutionary processes to modern biodiversity patterns. Yet inferring reliable phylogenetic trees for thousands of species involves numerous trade-offs that have limited their utility to comparative biologists. To establish a robust evolutionary timescale for all approximately 6,000 living species of mammals, we developed credible sets of trees that capture root-to-tip uncertainty in topology and divergence times. Our "backbone-and-patch" approach to tree building applies a newly assembled 31-gene supermatrix to two levels of Bayesian inference: (1) backbone relationships and ages among major lineages, using fossil node or tip dating, and (2) species-level "patch" phylogenies with nonoverlapping in-groups that each correspond to one representative lineage in the backbone. Species unsampled for DNA are either excluded ("DNA-only" trees) or imputed within taxonomic constraints using branch lengths drawn from local birth-death models ("completed" trees). Joining time-scaled patches to backbones results in species-level trees of extant Mammalia with all branches estimated under the same modeling framework, thereby facilitating rate comparisons among lineages as disparate as marsupials and placentals. We compare our phylogenetic trees to previous estimates of mammal-wide phylogeny and divergence times, finding that (1) node ages are broadly concordant among studies, and (2) recent (tip-level) rates of speciation are estimated more accurately in our study than in previous "supertree" approaches, in which unresolved nodes led to branch-length artifacts. Credible sets of mammalian phylogenetic history are now available for download at http://vertlife.org/phylosubsets, enabling investigations of long-standing questions in comparative biology.

A phylogenomic rodent tree reveals the repeated evolution of masseter architectures.

Understanding the number of times a trait has evolved is a necessary foundation for comprehending its potential relationships with selective regimes, developmental constraints and evolutionary diversification. Rodents make up over 40% of extant mammalian species, and their ecological and evolutionary success has been partially attributed to the increase in biting efficiency that resulted from a forward shift of one or two portions of the masseter muscle from the zygomatic arch onto the rostrum. This forward shift has occurred in three discrete ways, but the number of times it has occurred has never been explicitly quantified. We estimated an ultrametric phylogeny, the first to include all rodent families, using thousands of ultraconserved elements. We examined support for evolutionary relationships among the five rodent suborders and then incorporated relevant fossils, fitted models of character evolution, and used stochastic character mapping to determine that a portion of the masseter muscle has moved forward onto the rostrum at least seven times (with one reversal) during the approximately 70 Myr history of rodents. Combined, the repeated evolution of this key innovation, its increasing prevalence through time, and the species diversity of clades with this character underscores the adaptive value of improved biting efficiency and the relative ease with which some advantageous traits arise.

Convergent evolution of an extreme dietary specialisation, the olfactory system of worm-eating rodents.

Turbinal bones are key components of the mammalian rostrum that contribute to three critical functions: (1) homeothermy, (2) water conservation and (3) olfaction. With over 700 extant species, murine rodents (Murinae) are the most species-rich mammalian subfamily, with most of that diversity residing in the Indo-Australian Archipelago. Their evolutionary history includes several cases of putative, but untested ecomorphological convergence, especially with traits related to diet. Among the most spectacular rodent ecomorphs are the vermivores which independently evolved in several island systems. We used 3D CT-scans (N = 87) of murine turbinal bones to quantify olfactory capacities as well as heat or water conservation adaptations. We obtained similar results from an existing 2D complexity method and two new 3D methodologies that quantify bone complexity. Using comparative phylogenetic methods, we identified a significant convergent signal in the rostral morphology within the highly specialised vermivores. Vermivorous species have significantly larger and more complex olfactory turbinals than do carnivores and omnivores. Increased olfactory capacities may be a major adaptive feature facilitating rats' capacity to prey on elusive earthworms. The narrow snout that characterises vermivores exhibits significantly reduced respiratory turbinals, which may reduce their heat and water conservation capacities.

Geographic isolation and elevational gradients promote diversification in an endemic shrew on Sulawesi.

Phylogeographic research on endemic primates and amphibians inhabiting the Indonesian island of Sulawesi revealed the existence of seven areas of endemism (AoEs). Here, we use phylogenetic and population genetic analyses of one mitochondrial gene and 15 nuclear loci to assess geographic patterns of genetic partitioning in a shrew (Crocidura elongata) that is endemic to Sulawesi, but occurs across the island. We uncover substantial genetic diversity in this species both between and within AoEs, but we also identify close relationships between populations residing in different AoEs. One of the earliest divergences within C. elongata distinguishes a high-elevation clade from low-elevation clades. In addition, on one mountain, we observe three distinct genetic groups from low, middle, and high elevations, suggesting divergence along a single elevational gradient. In general, our results show that C. elongata, like several other Sulawesi endemic taxa, harbors extensive genetic diversity. This diversity is structured in part by known AoE boundaries, but also by elevational gradients and geographic isolation within AoEs.

Investigating Difficult Nodes in the Placental Mammal Tree with Expanded Taxon Sampling and Thousands of Ultraconserved Elements.

The phylogeny of eutherian mammals contains some of the most recalcitrant nodes in the tetrapod tree of life. We combined comprehensive taxon and character sampling to explore three of the most debated interordinal relationships among placental mammals. We performed in silico extraction of ultraconserved element loci from 72 published genomes and invitro enrichment and sequencing of ultraconserved elements from 28 additional mammals, resulting in alignments of 3,787 loci. We analyzed these data using concatenated and multispecies coalescent phylogenetic approaches, topological tests, and exploration of support among individual loci to identify the root of Eutheria and the sister groups of tree shrews (Scandentia) and horses (Perissodactyla). Individual loci provided weak, but often consistent support for topological hypotheses. Although many gene trees lacked accepted species-tree relationships, summary coalescent topologies were largely consistent with inferences from concatenation. At the root of Eutheria, we identified consistent support for a sister relationship between Xenarthra and Afrotheria (i.e., Atlantogenata). At the other nodes of interest, support was less consistent. We suggest Scandentia is the sister of Primatomorpha (Euarchonta), but we failed to reject a sister relationship between Scandentia and Glires. Similarly, we suggest Perissodactyla is sister to Cetartiodactyla (Euungulata), but a sister relationship between Perissodactyla and Chiroptera remains plausible.

Local endemism and within-island diversification of shrews illustrate the importance of speciation in building Sundaland mammal diversity.

Island systems are important models for evolutionary biology because they provide convenient, discrete biogeographic units of study. Continental islands with a history of intermittent dry land connections confound the discrete definitions of islands and have led zoologists to predict (1) little differentiation of terrestrial organisms among continental shelf islands and (2) extinction, rather than speciation, to be the main cause of differences in community composition among islands. However, few continental island systems have been subjected to well-sampled phylogeographic studies, leaving these biogeographic assumptions of connectivity largely untested. We analyzed nine unlinked loci from shrews of the genus Crocidura from seven mountains and two lowland localities on the Sundaic continental shelf islands of Sumatra and Java. Coalescent species delimitation strongly supported all currently recognized Crocidura species from Sumatra (six species) and Java (five species), as well as one undescribed species endemic to each island. We find that nearly all species of Crocidura in the region are endemic to a single island and several of these have their closest relative(s) on the same island. Intra-island genetic divergence among allopatric, conspecific populations is often substantial, perhaps indicating species-level diversity remains underestimated. One recent (Pleistocene) speciation event generated two morphologically distinct, syntopic species on Java, further highlighting the prevalence of within-island diversification. Our results suggest that both between- and within-island speciation processes generated local endemism in Sundaland, supplementing the traditional view that the region's fauna is relictual and primarily governed by extinction. This article is protected by copyright. All rights reserved.

Repeated evolution of carnivory among Indo-Australian rodents.

Convergent evolution, often observed in island archipelagos, provides compelling evidence for the importance of natural selection as a generator of species and ecological diversity. The Indo-Australian Archipelago (IAA) is the world's largest island system and encompasses distinct biogeographic units, including the Asian (Sunda) and Australian (Sahul) continental shelves, which together bracket the oceanic archipelagos of the Philippines and Wallacea. Each of these biogeographic units houses numerous endemic rodents in the family Muridae. Carnivorous murids, that is those that feed on animals, have evolved independently in Sunda, Sulawesi (part of Wallacea), the Philippines, and Sahul, but the number of origins of carnivory among IAA murids is unknown. We conducted a comprehensive phylogenetic analysis of carnivorous murids of the IAA, combined with estimates of ancestral states for broad diet categories (herbivore, omnivore, and carnivore) and geographic ranges. These analyses demonstrate that carnivory evolved independently four times after overwater colonization, including in situ origins on the Philippines, Sulawesi, and Sahul. In each biogeographic unit the origin of carnivory was followed by evolution of more specialized carnivorous ecomorphs such as vermivores, insectivores, and amphibious rats.

The Challenges of Resolving a Rapid, Recent Radiation: Empirical and Simulated Phylogenomics of Philippine Shrews.

Phylogenetic relationships in recent, rapid radiations can be difficult to resolve due to incomplete lineage sorting and reliance on genetic markers that evolve slowly relative to the rate of speciation. By incorporating hundreds to thousands of unlinked loci, phylogenomic analyses have the potential to mitigate these difficulties. Here, we attempt to resolve phylogenetic relationships among eight shrew species (genus Crocidura) from the Philippines, a phylogenetic problem that has proven intractable with small (< 10 loci) data sets. We sequenced hundreds of ultraconserved elements and whole mitochondrial genomes in these species and estimated phylogenies using concatenation, summary coalescent, and hierarchical coalescent methods. The concatenated approach recovered a maximally supported and fully resolved tree. In contrast, the coalescent-based approaches produced similar topologies, but each had several poorly supported nodes. Using simulations, we demonstrate that the concatenated tree could be positively misleading. Our simulations also show that the tree shape we tend to infer, which involves a series of short internal branches, is difficult to resolve, even if substitution models are known and multiple individuals per species are sampled. As such, the low support we obtained for backbone relationships in our coalescent-based inferences reflects a real and appropriate lack of certainty. Our results illuminate the challenges of estimating a bifurcating tree in a rapid and recent radiation, providing a rare empirical example of a nearly simultaneous series of speciation events in a terrestrial animal lineage as it spreads across an oceanic archipelago.

Conservation genetics of the Philippine tarsier: cryptic genetic variation restructures conservation priorities for an island archipelago primate.

Establishment of conservation priorities for primates is a particular concern in the island archipelagos of Southeast Asia, where rates of habitat destruction are among the highest in the world. Conservation programs require knowledge of taxonomic diversity to ensure success. The Philippine tarsier is a flagship species that promotes environmental awareness and a thriving ecotourism economy in the Philippines. However, assessment of its conservation status has been impeded by taxonomic uncertainty, a paucity of field studies, and a lack of vouchered specimens and genetic samples available for study in biodiversity repositories. Consequently, conservation priorities are unclear. In this study we use mitochondrial and nuclear DNA to empirically infer geographic partitioning of genetic variation and to identify evolutionarily distinct lineages for conservation action. The distribution of Philippine tarsier genetic diversity is neither congruent with expectations based on biogeographical patterns documented in other Philippine vertebrates, nor does it agree with the most recent Philippine tarsier taxonomic arrangement. We identify three principal evolutionary lineages that do not correspond to the currently recognized subspecies, highlight the discovery of a novel cryptic and range-restricted subcenter of genetic variation in an unanticipated part of the archipelago, and identify additional geographically structured genetic variation that should be the focus of future studies and conservation action. Conservation of this flagship species necessitates establishment of protected areas and targeted conservation programs within the range of each genetically distinct variant of the Philippine tarsier.

Reduced representation genome sequencing suggests low diversity on the sex chromosomes of tonkean macaque monkeys.

In species with separate sexes, social systems can differ in the relative variances of male versus female reproductive success. Papionin monkeys (macaques, mangabeys, mandrills, drills, baboons, and geladas) exhibit hallmarks of a high variance in male reproductive success, including a female-biased adult sex ratio and prominent sexual dimorphism. To explore the potential genomic consequences of such sex differences, we used a reduced representation genome sequencing approach to quantifying polymorphism at sites on autosomes and sex chromosomes of the tonkean macaque (Macaca tonkeana), a species endemic to the Indonesian island of Sulawesi. The ratio of nucleotide diversity of the X chromosome to that of the autosomes was less than the value (0.75) expected with a 1:1 sex ratio and no sex differences in the variance in reproductive success. However, the significance of this difference was dependent on which outgroup was used to standardize diversity levels. Using a new model that includes the effects of varying population size, sex differences in mutation rate between the autosomes and X chromosome, and GC-biased gene conversion (gBGC) or selection on GC content, we found that the maximum-likelihood estimate of the ratio of effective population size of the X chromosome to that of the autosomes was 0.68, which did not differ significantly from 0.75. We also found evidence for 1) a higher level of purifying selection on genic than nongenic regions, 2) gBGC or natural selection favoring increased GC content, 3) a dynamic demography characterized by population growth and contraction, 4) a higher mutation rate in males than females, and 5) a very low polymorphism level on the Y chromosome. These findings shed light on the population genomic consequences of sex differences in the variance in reproductive success, which appear to be modest in the tonkean macaque; they also suggest the occurrence of hitchhiking on the Y chromosome.