Resolving tricky nodes in the tree of life through amino acid recoding.

Genomic data allowed a detailed resolution of the Tree of Life, but "tricky nodes" such as the root of the animals remain unresolved. Genome-scale datasets are heterogeneous as genes and species are exposed to different pressures, and this can negatively impacts phylogenetic accuracy. We use simulated genomic-scale datasets and show that recoding amino acid data improves accuracy when the model does not account for the compositional heterogeneity of the amino acid alignment. We apply our findings to three datasets addressing the root of the animal tree, where the debate centers on whether sponges (Porifera) or comb jellies (Ctenophora) represent the sister of all other animals. We show that results from empirical data follow predictions from simulations and suggest that, at the least in phylogenies inferred from amino acid sequences, a placement of the ctenophores as sister to all the other animals is best explained as a tree reconstruction artifact.

Was There a Cambrian Explosion on Land? The Case of Arthropod Terrestrialization.

Arthropods, the most diverse form of macroscopic life in the history of the Earth, originated in the sea. Since the early Cambrian, at least ~518 million years ago, these animals have dominated the oceans of the world. By the Silurian-Devonian, the fossil record attests to arthropods becoming the first animals to colonize land, However, a growing body of molecular dating and palaeontological evidence suggests that the three major terrestrial arthropod groups (myriapods, hexapods, and arachnids), as well as vascular plants, may have invaded land as early as the Cambrian-Ordovician. These dates precede the oldest fossil evidence of those groups and suggest an unrecorded continental "Cambrian explosion" a hundred million years prior to the formation of early complex terrestrial ecosystems in the Silurian-Devonian. We review the palaeontological, phylogenomic, and molecular clock evidence pertaining to the proposed Cambrian terrestrialization of the arthropods. We argue that despite the challenges posed by incomplete preservation and the scarcity of early Palaeozoic terrestrial deposits, the discrepancy between molecular clock estimates and the fossil record is narrower than is often claimed. We discuss strategies for closing the gap between molecular clock estimates and fossil data in the evolution of early ecosystems on land.

Biochemical Evolution of a Potent Target of Mosquito Larvicide, 3-Hydroxykynurenine Transaminase.

A specific mosquito enzyme, 3-hydroxykynurenine transaminase (HKT), is involved in the processing of toxic metabolic intermediates of the tryptophan metabolic pathway. The HKT enzymatic product, xanthurenic acid, is required for Plasmodium spp. development in the mosquito vectors. Therefore, an inhibitor of HKT may not only be a mosquitocide but also a malaria-transmission blocker. In this work, we present a study investigating the evolution of HKT, which is a lineage-specific duplication of an alanine glyoxylate aminotransferases (AGT) in mosquitoes. Synteny analyses, together with the phylogenetic history of the AGT family, suggests that HKT and the mosquito AGTs are paralogous that were formed via a duplication event in their common ancestor. Furthermore, 41 amino acid sites with significant evidence of positive selection were identified, which could be responsible for biochemical and functional evolution and the stability of conformational stabilization. To get a deeper understanding of the evolution of ligands' capacity and the ligand-binding mechanism of HKT, the sequence and the 3D homology model of the common ancestor of HKT and AGT in mosquitoes, ancestral mosquito AGT (AncMosqAGT), were inferred and built. The homology model along with 3-hydroxykynurenine, kynurenine, and alanine were used in docking experiments to predict the binding capacity and ligand-binding mode of the new substrates related to toxic metabolites detoxification. Our study provides evidence for the dramatic biochemical evolution of the key detoxifying enzyme and provides potential sites that could hinder the detoxification function, which may be used in mosquito larvicide and design.

A Practical Guide to Design and Assess a Phylogenomic Study.

Over the last decade, molecular systematics has undergone a change of paradigm as high-throughput sequencing now makes it possible to reconstruct evolutionary relationships using genome-scale datasets. The advent of "big data" molecular phylogenetics provided a battery of new tools for biologists but simultaneously brought new methodological challenges. The increase in analytical complexity comes at the price of highly specific training in computational biology and molecular phylogenetics, resulting very often in a polarized accumulation of knowledge (technical on one side and biological on the other). Interpreting the robustness of genome-scale phylogenetic studies is not straightforward, particularly as new methodological developments have consistently shown that the general belief of "more genes, more robustness" often does not apply, and because there is a range of systematic errors that plague phylogenomic investigations. This is particularly problematic because phylogenomic studies are highly heterogeneous in their methodology, and best practices are often not clearly defined. The main aim of this article is to present what I consider as the ten most important points to take into consideration when planning a well-thought-out phylogenomic study and while evaluating the quality of published papers. The goal is to provide a practical step-by-step guide that can be easily followed by nonexperts and phylogenomic novices in order to assess the technical robustness of phylogenomic studies or improve the experimental design of a project.

De novo transcriptome sequencing of the northern fowl mite, Ornithonyssus sylviarum, shed light on parasitiform poultry mites evolution and its chemoreceptor repertoires.

The northern fowl mite (NFM), Ornithonyssus sylviarum, and the poultry red mite (PRM), Dermanyssus gallinae, are the most serious pests of poultry, both of which have an expanding global prevalence. Research on NFM has been constrained by a lack of genomic and transcriptomic data. Here, we report and analyze the first global transcriptome data across all mite live stages and sexes. A total of 28,999 unigenes were assembled, of which 19,750 (68.10%) were annotated using seven functional databases. The biological function of these unigenes was classified using the GO, KOG, and KEGG databases. To gain insight into the chemosensory receptor-based system of parasitiform mites, we furthermore assessed the gene repertoire of gustatory receptors (GRs) and ionotropic receptors (IRs), both of which encode putative ligand-gated ion channel proteins. While these receptors are well characterized in insect model species, our understanding of chemosensory detection in mites and ticks is in its infancy. To address this paucity of data, we identified 9 IR/iGluRs and 2 GRs genes by analyzing transcriptome data in the NFM, while 9 GRs and 41 IR/iGluRs genes were annotated in the PRM genome. Taken together, the transcriptomic and genomic annotation of these two species provide a valuable reference for studies of parasitiform mites and also help to understand how chemosensory gene family expansion/contraction events may have been reshaped by an obligate parasitic lifestyle compared with their free-living closest relatives. Future studies should include additional species to validate this observation and functional characterization of the identified proteins as a step forward in identifying tools for controlling these poultry pests.

The evolution of insect biodiversity.

Insects comprise over half of all described animal species. Together with the Protura (coneheads), Collembola (springtails) and Diplura (two-pronged bristletails), insects form the Hexapoda, a terrestrial arthropod lineage characterised by possessing six legs. Exponential growth of genome-scale data for the hexapods has substantially altered our understanding of the origin and evolution of insect biodiversity. Phylogenomics has provided a new framework for reconstructing insect evolutionary history, resolving their position among the arthropods and some long-standing internal controversies such as the placement of the termites, twisted-winged insects, lice and fleas. However, despite the greatly increased size of phylogenomic datasets, contentious relationships among key insect clades remain unresolved. Further advances in insect phylogeny cannot rely on increased depth and breadth of genome and taxon sequencing. Improved modelling of the substitution process is fundamental to countering tree-reconstruction artefacts, while gene content, modelling of duplications and deletions, and comparative morphology all provide complementary lines of evidence to test hypotheses emerging from the analysis of sequence data. Finally, the integration of molecular and morphological data is key to the incorporation of fossil species within insect phylogeny. The emerging integrated framework of insect evolution will help explain the origins of insect megadiversity in terms of the evolution of their body plan, species diversity and ecology. Future studies of insect phylogeny should build upon an experimental, hypothesis-driven approach where the robustness of hypotheses generated is tested against increasingly realistic evolutionary models as well as complementary sources of phylogenetic evidence.

Arachnid monophyly: Morphological, palaeontological and molecular support for a single terrestrialization within Chelicerata.

The majority of extant arachnids are terrestrial, but other chelicerates are generally aquatic, including horseshoe crabs, sea spiders, and the extinct eurypterids. It is necessary to determine whether arachnids are exclusively descended from a single common ancestor (monophyly), because only that relationship is compatible with one land colonisation in chelicerate evolutionary history. Some studies have cast doubt on arachnid monophyly and recast the origins of their terrestrialization. These include some phylogenomic analyses placing horseshoe crabs within Arachnida, and from aquatic Palaeozoic stem-group scorpions. Here, we evaluate the possibility of arachnid monophyly by considering morphology, fossils and molecules holistically. We argue arachnid monophyly obviates the need to posit reacquisition/retention of aquatic characters such as gnathobasic feeding and book gills without trabeculae from terrestrial ancestors in horseshoe crabs, and that the scorpion total-group contains few aquatic taxa. We built a matrix composed of 200 slowly-evolving genes and re-analysed two published molecular datasets. We retrieved arachnid monophyly where other studies did not - highlighting the difficulty of resolving chelicerate relationships from current molecular data. As such, we consider arachnid monophyly the best-supported hypothesis. Finally, we inferred that arachnids terrestrialized during the Cambrian-Ordovician using the slow-evolving molecular matrix, in agreement with recent analyses.

A Cambrian-Ordovician Terrestrialization of Arachnids.

Understanding the temporal context of terrestrialization in chelicerates depends on whether terrestrial groups, the traditional Arachnida, have a single origin and whether or not horseshoe crabs are primitively or secondarily marine. Molecular dating on a phylogenomic tree that recovers arachnid monophyly, constrained by 27 rigorously vetted fossil calibrations, estimates that Arachnida originated during the Cambrian or Ordovician. After the common ancestor colonized the land, the main lineages appear to have rapidly radiated in the Cambrian-Ordovician boundary interval, coinciding with high rates of molecular evolution. The highest rates of arachnid diversification are detected between the Permian and Early Cretaceous. A pattern of ancient divergence estimates for terrestrial arthropod groups in the Cambrian while the oldest fossils are Silurian (seen in both myriapods and arachnids) is mirrored in the molecular and fossil records of land plants. We suggest the discrepancy between molecular and fossil evidence for terrestrialization is likely driven by the extreme sparseness of terrestrial sediments in the rock record before the late Silurian.

Author Correction: Increasing species sampling in chelicerate genomic-scale datasets provides support for monophyly of Acari and Arachnida.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Pancrustacean Evolution Illuminated by Taxon-Rich Genomic-Scale Data Sets with an Expanded Remipede Sampling.

The relationships of crustaceans and hexapods (Pancrustacea) have been much discussed and partially elucidated following the emergence of phylogenomic data sets. However, major uncertainties still remain regarding the position of iconic taxa such as Branchiopoda, Copepoda, Remipedia, and Cephalocarida, and the sister group relationship of hexapods. We assembled the most taxon-rich phylogenomic pancrustacean data set to date and analyzed it using a variety of methodological approaches. We prioritized low levels of missing data and found that some clades were consistently recovered independently of the analytical approach used. These include, for example, Oligostraca and Altocrustacea. Substantial support was also found for Allotriocarida, with Remipedia as the sister of Hexapoda (i.e., Labiocarida), and Branchiopoda as the sister of Labiocarida, a clade that we name Athalassocarida (="nonmarine shrimps"). Within Allotriocarida, Cephalocarida was found as the sister of Athalassocarida. Finally, moderate support was found for Hexanauplia (Copepoda as sister to Thecostraca) in alliance with Malacostraca. Mapping key crustacean tagmosis patterns and developmental characters across the revised phylogeny suggests that the ancestral pancrustacean was relatively short-bodied, with extreme body elongation and anamorphic development emerging later in pancrustacean evolution.

Increasing species sampling in chelicerate genomic-scale datasets provides support for monophyly of Acari and Arachnida.

Chelicerates are a diverse group of arthropods, represented by such forms as predatory spiders and scorpions, parasitic ticks, humic detritivores, and marine sea spiders (pycnogonids) and horseshoe crabs. Conflicting phylogenetic relationships have been proposed for chelicerates based on both morphological and molecular data, the latter usually not recovering arachnids as a clade and instead finding horseshoe crabs nested inside terrestrial Arachnida. Here, using genomic-scale datasets and analyses optimised for countering systematic error, we find strong support for monophyletic Acari (ticks and mites), which when considered as a single group represent the most biodiverse chelicerate lineage. In addition, our analysis recovers marine forms (sea spiders and horseshoe crabs) as the successive sister groups of a monophyletic lineage of terrestrial arachnids, suggesting a single colonisation of land within Chelicerata and the absence of wholly secondarily marine arachnid orders.

Homeobox Gene Duplication and Divergence in Arachnids.

Homeobox genes are key toolkit genes that regulate the development of metazoans and changes in their regulation and copy number have contributed to the evolution of phenotypic diversity. We recently identified a whole genome duplication (WGD) event that occurred in an ancestor of spiders and scorpions (Arachnopulmonata), and that many homeobox genes, including two Hox clusters, appear to have been retained in arachnopulmonates. To better understand the consequences of this ancient WGD and the evolution of arachnid homeobox genes, we have characterized and compared the homeobox repertoires in a range of arachnids. We found that many families and clusters of these genes are duplicated in all studied arachnopulmonates (Parasteatoda tepidariorum, Pholcus phalangioides, Centruroides sculpturatus, and Mesobuthus martensii) compared with nonarachnopulmonate arachnids (Phalangium opilio, Neobisium carcinoides, Hesperochernes sp., and Ixodes scapularis). To assess divergence in the roles of homeobox ohnologs, we analyzed the expression of P. tepidariorum homeobox genes during embryogenesis and found pervasive changes in the level and timing of their expression. Furthermore, we compared the spatial expression of a subset of P. tepidariorum ohnologs with their single copy orthologs in P. opilio embryos. We found evidence for likely subfunctionlization and neofunctionalization of these genes in the spider. Overall our results show a high level of retention of homeobox genes in spiders and scorpions post-WGD, which is likely to have made a major contribution to their developmental evolution and diversification through pervasive subfunctionlization and neofunctionalization, and paralleling the outcomes of WGD in vertebrates.

Soft-Bodied Fossils Are Not Simply Rotten Carcasses - Toward a Holistic Understanding of Exceptional Fossil Preservation: Exceptional Fossil Preservation Is Complex and Involves the Interplay of Numerous Biological and Geological Processes.

Exceptionally preserved fossils are the product of complex interplays of biological and geological processes including burial, autolysis and microbial decay, authigenic mineralization, diagenesis, metamorphism, and finally weathering and exhumation. Determining which tissues are preserved and how biases affect their preservation pathways is important for interpreting fossils in phylogenetic, ecological, and evolutionary frameworks. Although laboratory decay experiments reveal important aspects of fossilization, applying the results directly to the interpretation of exceptionally preserved fossils may overlook the impact of other key processes that remove or preserve morphological information. Investigations of fossils preserving non-biomineralized tissues suggest that certain structures that are decay resistant (e.g., the notochord) are rarely preserved (even where carbonaceous components survive), and decay-prone structures (e.g., nervous systems) can fossilize, albeit rarely. As we review here, decay resistance is an imperfect indicator of fossilization potential, and a suite of biological and geological processes account for the features preserved in exceptional fossils.

RelTime Rates Collapse to a Strict Clock When Estimating the Timeline of Animal Diversification.

Establishing an accurate timescale for the history of life is crucial to understand evolutionary processes. For this purpose, relaxed molecular clock models implemented in a Bayesian MCMC framework are generally used. However, these methods are time consuming. RelTime, a non-Bayesian method implementing a fast, ad hoc, algorithm for relative dating, was developed to overcome the computational inefficiencies of Bayesian software. RelTime was recently used to investigate the timing of origin of animals, yielding results consistent with early strict clock studies from the 1980s and 1990s, estimating metazoans to have a Mesoproterozoic origin-over a billion years ago. RelTime results are unexpected and disagree with the largest majority of modern, relaxed, Bayesian molecular clock analyses, which suggest animals originated in the Tonian-Cryogenian (less that 850 million years ago). Here, we demonstrate that RelTime-inferred divergence times for the origin of animals are spurious, a consequence of the inability of RelTime to relax the clock along the internal branches of the animal phylogeny. RelTime-inferred divergence times are comparable to strict-clock estimates because they are essentially inferred under a strict clock. Our results warn us of the danger of using ad hoc algorithms making implicit assumptions about rate changes along a tree. Our study roundly rejects a Mesoproterozoic origin of animals; metazoans emerged in the Tonian-Cryogenian, and diversified in the Ediacaran, in the immediate prelude to the routine fossilization of animals in the Cambrian associated with the emergence of readily preserved skeletons.

Uncertain-tree: discriminating among competing approaches to the phylogenetic analysis of phenotype data.

Morphological data provide the only means of classifying the majority of life's history, but the choice between competing phylogenetic methods for the analysis of morphology is unclear. Traditionally, parsimony methods have been favoured but recent studies have shown that these approaches are less accurate than the Bayesian implementation of the Mk model. Here we expand on these findings in several ways: we assess the impact of tree shape and maximum-likelihood estimation using the Mk model, as well as analysing data composed of both binary and multistate characters. We find that all methods struggle to correctly resolve deep clades within asymmetric trees, and when analysing small character matrices. The Bayesian Mk model is the most accurate method for estimating topology, but with lower resolution than other methods. Equal weights parsimony is more accurate than implied weights parsimony, and maximum-likelihood estimation using the Mk model is the least accurate method. We conclude that the Bayesian implementation of the Mk model should be the default method for phylogenetic estimation from phenotype datasets, and we explore the implications of our simulations in reanalysing several empirical morphological character matrices. A consequence of our finding is that high levels of resolution or the ability to classify species or groups with much confidence should not be expected when using small datasets. It is now necessary to depart from the traditional parsimony paradigms of constructing character matrices, towards datasets constructed explicitly for Bayesian methods.

A molecular palaeobiological exploration of arthropod terrestrialization.

Understanding animal terrestrialization, the process through which animals colonized the land, is crucial to clarify extant biodiversity and biological adaptation. Arthropoda (insects, spiders, centipedes and their allies) represent the largest majority of terrestrial biodiversity. Here we implemented a molecular palaeobiological approach, merging molecular and fossil evidence, to elucidate the deepest history of the terrestrial arthropods. We focused on the three independent, Palaeozoic arthropod terrestrialization events (those of Myriapoda, Hexapoda and Arachnida) and showed that a marine route to the colonization of land is the most likely scenario. Molecular clock analyses confirmed an origin for the three terrestrial lineages bracketed between the Cambrian and the Silurian. While molecular divergence times for Arachnida are consistent with the fossil record, Myriapoda are inferred to have colonized land earlier, substantially predating trace or body fossil evidence. An estimated origin of myriapods by the Early Cambrian precedes the appearance of embryophytes and perhaps even terrestrial fungi, raising the possibility that terrestrialization had independent origins in crown-group myriapod lineages, consistent with morphological arguments for convergence in tracheal systems.This article is part of the themed issue 'Dating species divergences using rocks and clocks'.