Cretaceous amber inclusions illuminate the evolutionary origin of tardigrades.

Tardigrades are a diverse phylum of microscopic invertebrates widely known for their extreme survival capabilities. Molecular clocks suggest that tardigrades diverged from other panarthropods before the Cambrian, but their fossil record is extremely sparse. Only the fossil tardigrades Milnesium swolenskyi (Late Cretaceous) and Paradoryphoribius chronocaribbeus (Miocene) have resolved taxonomic positions, restricting the availability of calibration points for estimating for the origin of this phylum. Here, we revise two crown-group tardigrades from Canadian Cretaceous-aged amber using confocal fluorescence microscopy, revealing critical morphological characters that resolve their taxonomic positions. Formal morphological redescription of Beorn leggi reveals that it features Hypsibius-type claws. We also describe Aerobius dactylus gen. et sp. nov. based on its unique combination of claw characters. Phylogenetic analyses indicate that Beo. leggi and Aer. dactylus belong to the eutardigrade superfamily Hypsibioidea, adding a critical fossil calibration point to investigate tardigrade origins. Our molecular clock estimates suggest an early Paleozoic diversification of crown-group Tardigrada and highlight the importance of Beo. leggi as a calibration point that directly impacts estimates of shallow nodes. Our results suggest that independent terrestrialization of eutardigrades and heterotardigrades occurred around the end-Carboniferous and Lower Jurassic, respectively. These estimates also provide minimum ages for convergent acquisition of cryptobiosis.

Pangenomes at the limits of evolution.

Evolutionary pathways can be random or deterministic. In a recent article, Beavan et al. investigate this balance by applying machine learning models to microbial pangenomes. The presence of almost one-third of genes can be reliably inferred, indicating a surprising amount of predictable evolution.

Convergent adaptation of true crabs (Decapoda: Brachyura) to a gradient of terrestrial environments.

For much of terrestrial biodiversity, the evolutionary pathways of adaptation from marine ancestors are poorly understood, and have usually been viewed as a binary trait. True crabs, the decapod crustacean infraorder Brachyura, comprise over 7,600 species representing a striking diversity of morphology and ecology, including repeated adaptation to non-marine habitats. Here, we reconstruct the evolutionary history of Brachyura using new and published sequences of 10 genes for 344 tips spanning 88 of 109 brachyuran families. Using 36 newly vetted fossil calibrations, we infer that brachyurans most likely diverged in the Triassic, with family-level splits in the late Cretaceous and early Paleogene. By contrast, the root age is underestimated with automated sampling of 328 fossil occurrences explicitly incorporated into the tree prior, suggesting such models are a poor fit under heterogeneous fossil preservation. We apply recently defined trait-by-environment associations to classify a gradient of transitions from marine to terrestrial lifestyles. We estimate that crabs left the marine environment at least seven and up to 17 times convergently, and returned to the sea from non-marine environments at least twice. Although the most highly terrestrial- and many freshwater-adapted crabs are concentrated in Thoracotremata, Bayesian threshold models of ancestral state reconstruction fail to identify shifts to higher terrestrial grades due to the degree of underlying change required. Lineages throughout our tree inhabit intertidal and marginal marine environments, corroborating the inference that the early stages of terrestrial adaptation have a lower threshold to evolve. Our framework and extensive new fossil and natural history datasets will enable future comparisons of non-marine adaptation at the morphological and molecular level. Crabs provide an important window into the early processes of adaptation to novel environments, and different degrees of evolutionary constraint that might help predict these pathways.

Major Revisions in Pancrustacean Phylogeny and Evidence of Sensitivity to Taxon Sampling.

The clade Pancrustacea, comprising crustaceans and hexapods, is the most diverse group of animals on earth, containing over 80% of animal species and half of animal biomass. It has been the subject of several recent phylogenomic analyses, yet relationships within Pancrustacea show a notable lack of stability. Here, the phylogeny is estimated with expanded taxon sampling, particularly of malacostracans. We show small changes in taxon sampling have large impacts on phylogenetic estimation. By analyzing identical orthologs between two slightly different taxon sets, we show that the differences in the resulting topologies are due primarily to the effects of taxon sampling on the phylogenetic reconstruction method. We compare trees resulting from our phylogenomic analyses with those from the literature to explore the large tree space of pancrustacean phylogenetic hypotheses and find that statistical topology tests reject the previously published trees in favor of the maximum likelihood trees produced here. Our results reject several clades including Caridoida, Eucarida, Multicrustacea, Vericrustacea, and Syncarida. Notably, we find Copepoda nested within Allotriocarida with high support and recover a novel relationship between decapods, euphausiids, and syncarids that we refer to as the Syneucarida. With denser taxon sampling, we find Stomatopoda sister to this latter clade, which we collectively name Stomatocarida, dividing Malacostraca into three clades: Leptostraca, Peracarida, and Stomatocarida. A new Bayesian divergence time estimation is conducted using 13 vetted fossils. We review our results in the context of other pancrustacean phylogenetic hypotheses and highlight 15 key taxa to sample in future studies.

Three-dimensional morphology of the biramous appendages in Isoxys from the early Cambrian of South China, and its implications for early euarthropod evolution.

Early euarthropod evolution involved a major transition from lobopodian-like taxa to organisms featuring a segmented, well-sclerotized trunk (arthrodization) and limbs (arthropodization). However, the precise origin of a completely arthrodized trunk and arthropodized ventral biramous appendages remain controversial, as well as the early onset of anterior-posterior limb differentiation in stem-group euarthropods. New fossil material and micro-computed tomography inform the detailed morphology of the arthropodized biramous appendages in the carapace-bearing euarthropod Isoxys curvirostratus from the early Cambrian Chengjiang biota. In addition to well-developed grasping frontal appendages, I. curvirostratus possesses two batches of morphologically and functionally distinct biramous limbs. The first batch consists of four pairs of short cephalic appendages with robust endites with a feeding function, whereas the second batch has more elongate trunk appendages for locomotion. Critically, our new material shows that the trunk of I. curvirostratus was not arthrodized. The results of our phylogenetic analyses recover isoxyids as some of the earliest branching sclerotized euarthropods, and strengthens the hypothesis that arthropodized biramous appendages evolved before full body arthrodization.

Ordovician opabiniid-like animals and the role of the proboscis in euarthropod head evolution.

A crucial step in the evolution of Euarthropoda (chelicerates, myriapods, pancrustaceans) was the transition between fossil groups that possessed frontal appendages innervated by the first segment of the brain (protocerebrum), and living groups with a protocerebral labrum and paired appendages innervated by the second brain segment (deutocerebrum). Appendage homologies between the groups are controversial. Here we describe two specimens of opabiniid-like euarthropods, each bearing an anterior proboscis (a fused protocerebral appendage), from the Middle Ordovician Castle Bank Biota, Wales, UK. Phylogenetic analyses support a paraphyletic grade of stem-group euarthropods with fused protocerebral appendages and a posterior-facing mouth, as in the iconic Cambrian panarthropod Opabinia. These results suggest that the labrum may have reduced from an already-fused proboscis, rather than a pair of arthropodized appendages. If some shared features between the Castle Bank specimens and radiodonts are considered convergent rather than homologous, phylogenetic analyses retrieve them as opabiniids, substantially extending the geographic and temporal range of Opabiniidae.

New opabiniid diversifies the weirdest wonders of the euarthropod stem group.

Once considered 'weird wonders' of the Cambrian, the emblematic Burgess Shale animals Anomalocaris and Opabinia are now recognized as lower stem-group euarthropods and have provided crucial data for constraining the polarity of key morphological characters in the group. Anomalocaris and its relatives (radiodonts) had worldwide distribution and survived until at least the Devonian. However, despite intense study, Opabinia remains the only formally described opabiniid to date. Here we reinterpret a fossil from the Wheeler Formation of Utah as a new opabiniid, Utaurora comosa nov. gen. et sp. By visualizing the sample of phylogenetic topologies in treespace, our results fortify support for the position of U. comosa beyond the nodal support traditionally applied. Our phylogenetic evidence expands opabiniids to multiple Cambrian stages. Our results underscore the power of treespace visualization for resolving imperfectly preserved fossils and expanding the known diversity and spatio-temporal ranges within the euarthropod lower stem group.

Evolution: Oh, my Cambrian nerves.

Developmental gene expression suggests a cryptic subdivision of the anterior brain in euarthropods. A new study illustrates delicate details of the nervous system from exceptionally preserved 500-million-year-old Chinese fossils, supporting the bipartite origin of the anterior brain among Cambrian representatives.

How to become a crab: Phenotypic constraints on a recurring body plan.

A fundamental question in biology is whether phenotypes can be predicted by ecological or genomic rules. At least five cases of convergent evolution of the crab-like body plan (with a wide and flattened shape, and a bent abdomen) are known in decapod crustaceans, and have, for over 140 years, been known as "carcinization." The repeated loss of this body plan has been identified as "decarcinization." In reviewing the field, we offer phylogenetic strategies to include poorly known groups, and direct evidence from fossils, that will resolve the history of crab evolution and the degree of phenotypic variation within crabs. Proposed ecological advantages of the crab body are summarized into a hypothesis of phenotypic integration suggesting correlated evolution of the carapace shape and abdomen. Our premise provides fertile ground for future studies of the genomic and developmental basis, and the predictability, of the crab-like body form.

The appendicular morphology of Sinoburius lunaris and the evolution of the artiopodan clade Xandarellida (Euarthropoda, early Cambrian) from South China.

BACKGROUND: Artiopodan euarthropods represent common and abundant faunal components in sites with exceptional preservation during the Cambrian. The Chengjiang biota in South China contains numerous taxa that are exclusively known from this deposit, and thus offer a unique perspective on euarthropod diversity during the early Cambrian. One such endemic taxon is the non-trilobite artiopodan Sinoburius lunaris, which has been known for approximately three decades, but few details of its anatomy are well understood due to its rarity within the Chengjiang, as well as technical limitations for the study of these fossils. Furthermore, the available material does not provide clear information on the ventral organization of this animal, obscuring our understanding of phylogenetically significant details such as the appendages. RESULTS: We employed X-ray computed tomography to study the non-biomineralized morphology of Sinoburius lunaris. Due to the replacement of the delicate anatomy with pyrite typical of Chengjiang fossils, computed tomography reveals substantial details of the ventral anatomy of Sinoburius lunaris, and allow us to observe in detail the three-dimensionally preserved appendicular organization of this taxon for the first time. The dorsal exoskeleton consists of a crescent-shaped head shield with well-developed genal spines, a thorax with seven freely articulating tergites, and a fused pygidium with lateral and median spines. The head bears a pair of ventral stalked eyes that are accommodated by dorsal exoskeletal bulges, and an oval elongate ventral hypostome. The appendicular organization of the head is unique among Artiopoda. The deutocerebral antennae are reduced, consisting of only five podomeres, and bear an antennal scale on the second podomere that most likely represents an exite rather than a true ramus. The head includes four post-antennal biramous limb pairs. The first two biramous appendages are differentiated from the rest. The first appendage pair consists of a greatly reduced endopod coupled with a greatly elongated exopod with a potentially sensorial function. The second appendage pair carries a more conventionally sized endopod, but also has an enlarged exopod. The remaining biramous appendages are homonomous in their construction, but decrease in size towards the posterior end of the body. They consist of a basipodite with ridge-like crescentic endites, an endopod with seven podomeres and a terminal claw, and a lamellae-bearing exopod with a slender shaft. Contrary to previous reports, we confirm the presence of segmental mismatch in Sinoburius lunaris, expressed as diplotergites in the thorax. Maximum parsimony and Bayesian phylogenetic analyses support the monophyly of Xandarellida within Artiopoda, and illuminate the internal relationships within this enigmatic clade. Our results allow us to propose a transformation series explaining the origin of archetypical xandarellid characters, such as the evolution of eye slits in Xandarella spectaculum and Phytophilaspis pergamena as derivates from the anterolateral notches in the head shield observed in Cindarella eucalla and Luohuilinella species. In this context, Sinoburius lunaris is found to feature several derived characters within the group, such as the secondary loss of eye slits and a high degree of appendicular tagmosis. Contrary to previous findings, our analyses strongly support close affinities between Sinoburius lunaris, Xandarella spectaculum and Phytophilaspis pergamena, although the precise relationships between these taxa are sensitive to different methodologies. CONCLUSIONS: The revised morphology of Sinoburius lunaris, made possible through the use of computed tomography to resolve details of its three-dimensionally preserved appendicular anatomy, contributes towards an improved understanding of the morphology of this taxon and the evolution of Xandarellida more broadly. Our results indicate that Sinoburius lunaris possesses an unprecedented degree of appendicular tagmosis otherwise unknown within Artiopoda, with the implication that this iconic group of Palaeozoic euarthropods likely had a more complex ecology and functional morphology than previously considered. The application of computer tomographic techniques to the study of Chengjiang euarthropods holds exceptional promise for understanding the morphological diversity of these organisms, and also better reconstructing their phylogenetic relationships and evolutionary history.

A phylogenomic framework, evolutionary timeline and genomic resources for comparative studies of decapod crustaceans.

Comprising over 15 000 living species, decapods (crabs, shrimp and lobsters) are the most instantly recognizable crustaceans, representing a considerable global food source. Although decapod systematics have received much study, limitations of morphological and Sanger sequence data have yet to produce a consensus for higher-level relationships. Here, we introduce a new anchored hybrid enrichment kit for decapod phylogenetics designed from genomic and transcriptomic sequences that we used to capture new high-throughput sequence data from 94 species, including 58 of 179 extant decapod families, and 11 of 12 major lineages. The enrichment kit yields 410 loci (greater than 86 000 bp) conserved across all lineages of Decapoda, more clade-specific molecular data than any prior study. Phylogenomic analyses recover a robust decapod tree of life strongly supporting the monophyly of all infraorders, and monophyly of each of the reptant, 'lobster' and 'crab' groups, with some results supporting pleocyemate monophyly. We show that crown decapods diverged in the Late Ordovician and most crown lineages diverged in the Triassic-Jurassic, highlighting a cryptic Palaeozoic history, and post-extinction diversification. New insights into decapod relationships provide a phylogenomic window into morphology and behaviour, and a basis to rapidly and cheaply expand sampling in this economically and ecologically significant invertebrate clade.

Paleozoic diversification of terrestrial chitin-degrading bacterial lineages.

BACKGROUND: Establishing the divergence times of groups of organisms is a major goal of evolutionary biology. This is especially challenging for microbial lineages due to the near-absence of preserved physical evidence (diagnostic body fossils or geochemical biomarkers). Horizontal gene transfer (HGT) can serve as a temporal scaffold between microbial groups and other fossil-calibrated clades, potentially improving these estimates. Specifically, HGT to or from organisms with fossil-calibrated age estimates can propagate these constraints to additional groups that lack fossils. While HGT is common between lineages, only a small subset of HGT events are potentially informative for dating microbial groups. RESULTS: Constrained by published fossil-calibrated studies of fungal evolution, molecular clock analyses show that multiple clades of Bacteria likely acquired chitinase homologs via HGT during the very late Neoproterozoic into the early Paleozoic. These results also show that, following these HGT events, recipient terrestrial bacterial clades likely diversified ~ 300-500 million years ago, consistent with established timescales of arthropod and plant terrestrialization. CONCLUSIONS: We conclude that these age estimates are broadly consistent with the dispersal of chitinase genes throughout the microbial world in direct response to the evolution and ecological expansion of detrital-chitin producing groups. The convergence of multiple lines of evidence demonstrates the utility of HGT-based dating methods in microbial evolution. The pattern of inheritance of chitinase genes in multiple terrestrial bacterial lineages via HGT processes suggests that these genes, and possibly other genes encoding substrate-specific enzymes, can serve as a "standard candle" for dating microbial lineages across the Tree of Life.

Three-Dimensionally Preserved Appendages in an Early Cambrian Stem-Group Pancrustacean.

Pancrustaceans boast impressive diversity, abundance, and ecological impact in the biosphere throughout the Phanerozoic [1]. Molecular clock estimates suggest an early Cambrian divergence for pancrustaceans [2, 3]. Despite the wealth of Palaeozoic exceptional fossiliferous deposits [4-7], the early evolution of Pancrustacea remains elusive given the difficulty of recognizing synapomorphies between Cambrian forms and extant representatives. Although early studies suggested crustacean affinities for Cambrian bivalved euarthropods [8-11], this view has fallen out of favor by recent reappraisals of their morphology [12-16]. The best evidence for total-group pancrustaceans comes from Cambrian microfossils preserved as three-dimensional phosphatic replicates in Orsten-type assemblages [4, 17-19] or as "small carbonaceous fossils" (SCFs) [20, 21]. Although these taphonomic windows capture minute morphology enabling detailed comparisons with extant representatives, these microfossils are limited to larval stages (Orsten) or recalcitrant fragmentary remains (SCFs) restricting their phylogenetic precision [5, 12, 19, 20, 22, 23]. We employed X-ray computed tomography [24] to reveal the three-dimensionally appendage morphology of the Chengjiang bivalved euarthropod Ercaicunia multinodosa [25] from the early Cambrian of China. E. multinodosa possesses characters uniquely shared with extant crustaceans, including differentiated tritocerebral antennae and epipodite-bearing biramous trunk appendages. Similarities between E. multinodosa with clypecaridids [9], waptiids [16] and hymenocarines [11, 14] suggest that these euarthropods may also possess similarly differentiated appendages, but these details are obstructed by the limits of preservation of compacted macrofossils. E. multinodosa illuminates the early evolution of pancrustacean appendage differentiation and represents the oldest unequivocal crown-group mandibulate known from complete macrofossils [22].

Horizontal gene transfer constrains the timing of methanogen evolution.

Microbial methanogenesis may have been a major component of Earth's carbon cycle during the Archaean eon, generating a methane greenhouse that increased global temperatures enough for a liquid hydrosphere, despite the Sun's lower luminosity at the time. Evaluation of potential solutions to the 'faint young Sun' hypothesis by determining the age of microbial methanogenesis has been limited by ambiguous geochemical evidence and the absence of a diagnostic fossil record. To overcome these challenges, we use a temporal constraint: a horizontal gene transfer event from within archaeal methanogens to the ancestor of Cyanobacteria, one of the few microbial clades with recognized crown-group fossils. Results of molecular clock analyses calibrated by this horizontal-gene-transfer-propagated constraint show methanogens diverging within Euryarchaeota no later than 3.51 billion years ago, with methanogenesis itself probably evolving earlier. This timing provides independent support for scenarios wherein microbial methane production was important in maintaining temperatures on the early Earth.

Metamorphosis Is Ancestral for Crown Euarthropods, and Evolved in the Cambrian or Earlier.

Macroevolutionary developmental biology employs fossilized ontogenetic data and phylogenetic comparative methods to probe the evolution of development at ancient nodes. Despite the prevalence of ecologically differentiated larval forms in marine invertebrates, it has been frequently presumed that the ancestors of arthropods were direct developers, and that metamorphosis may not have evolved until the Ordovician or later. Using fossils and new dated phylogenies, I infer that metamorphosis was likely ancestral for crown arthropods, contradicting this assumption. Based on a published morphological dataset encompassing 217 exceptionally preserved fossil and 96 extant taxa, fossils were directly incorporated into both the topology and age estimates, as in "tip dating" analyses. Using data from post-embryonic fossils representing 25 species throughout stem and crown arthropod lineages (as well as most of the 96 extant taxa), characters for metamorphosis were assigned based on inferred ecological changes in development (e.g., changes in habitat and adaptive landscape). Under all phylogenetic hypotheses, metamorphosis was supported as most likely ancestral to both ecdysozoans and euarthropods. Care must be taken to account for potential drastic post-embryonic morphological changes in evolutionary analyses. Many stem group euarthrpods may have had ecologically differentiated larval stages that did not preserve in the fossil record. Moreover, a complex life cycle and planktonic ecology may have evolved in the Ediacaran or earlier, and may have typified the pre-Cambrian explosion "wormworld" prior to the origin of crown group euarthropods.

Testing the phylogenetic position of Cambrian pancrustacean larval fossils by coding ontogenetic stages.

The study of ontogeny as an integral part of understanding the pattern of evolution dates back over 200 years, but only recently have ontogenetic data been explicitly incorporated into phylogenetic analyses. Pancrustaceans undergo radical ontogenetic changes. The spectacular upper Cambrian "Orsten" fauna preserves phosphatized fossil larvae, including putative crown-group pancrustaceans with amazingly complete developmental sequences. The putative presence and nature of adult stages remains a source of debate, causing spurious placements in a traditional morphological analysis. We introduce a new coding method where each semaphoront (discrete larval or adult stage) is considered an operational taxonomic unit. This avoids a priori assumptions of heterochrony. Characters and their states are defined to identify changes in morphology throughout ontogeny. Phylogenetic analyses of semaphoronts produced possible relationships of each Orsten fossil to the crown-group clade expected from morphology shared with extant larvae. Bredocaris is a member of the stem lineage of Thecostraca or (Thecostraca + Copepoda), and Yicaris and Rehbachiella are probably members of the stem lineage of Cephalocarida. These placements rely directly on comparisons between extant and fossil larval character states. The position of Phosphatocopina remains unresolved. This method may have broader applications to other phylogenetic problems which may rely on ontogenetically variable homology statements.

Next-generation phenomics for the Tree of Life.

The phenotype represents a critical interface between the genome and the environment in which organisms live and evolve. Phenotypic characters also are a rich source of biodiversity data for tree building, and they enable scientists to reconstruct the evolutionary history of organisms, including most fossil taxa, for which genetic data are unavailable. Therefore, phenotypic data are necessary for building a comprehensive Tree of Life. In contrast to recent advances in molecular sequencing, which has become faster and cheaper through recent technological advances, phenotypic data collection remains often prohibitively slow and expensive. The next-generation phenomics project is a collaborative, multidisciplinary effort to leverage advances in image analysis, crowdsourcing, and natural language processing to develop and implement novel approaches for discovering and scoring the phenome, the collection of phentotypic characters for a species. This research represents a new approach to data collection that has the potential to transform phylogenetics research and to enable rapid advances in constructing the Tree of Life. Our goal is to assemble large phenomic datasets built using new methods and to provide the public and scientific community with tools for phenomic data assembly that will enable rapid and automated study of phenotypes across the Tree of Life.

Phylotranscriptomics to bring the understudied into the fold: monophyletic ostracoda, fossil placement, and pancrustacean phylogeny.

An ambitious, yet fundamental goal for comparative biology is to understand the evolutionary relationships for all of life. However, many important taxonomic groups have remained recalcitrant to inclusion into broader scale studies. Here, we focus on collection of 9 new 454 transcriptome data sets from Ostracoda, an ancient and diverse group with a dense fossil record, which is often undersampled in broader studies. We combine the new transcriptomes with a new morphological matrix (including fossils) and existing expressed sequence tag, mitochondrial genome, nuclear genome, and ribosomal DNA data. Our analyses lead to new insights into ostracod and pancrustacean phylogeny. We obtained support for three epic pancrustacean clades that likely originated in the Cambrian: Oligostraca (Ostracoda, Mystacocarida, Branchiura, and Pentastomida); Multicrustacea (Copepoda, Malacostraca, and Thecostraca); and a clade we refer to as Allotriocarida (Hexapoda, Remipedia, Cephalocarida, and Branchiopoda). Within the Oligostraca clade, our results support the unresolved question of ostracod monophyly. Within Multicrustacea, we find support for Thecostraca plus Copepoda, for which we suggest the name Hexanauplia. Within Allotriocarida, some analyses support the hypothesis that Remipedia is the sister taxon to Hexapoda, but others support Branchiopoda + Cephalocarida as the sister group of hexapods. In multiple different analyses, we see better support for equivocal nodes using slow-evolving genes or when excluding distant outgroups, highlighting the increased importance of conditional data combination in this age of abundant, often anonymous data. However, when we analyze the same set of species and ignore rate of gene evolution, we find higher support when including all data, more in line with a "total evidence" philosophy. By concatenating molecular and morphological data, we place pancrustacean fossils in the phylogeny, which can be used for studies of divergence times in Pancrustacea, Arthropoda, or Metazoa. Our results and new data will allow for attributes of Ostracoda, such as its amazing fossil record and diverse biology, to be leveraged in broader scale comparative studies. Further, we illustrate how adding extensive next-generation sequence data from understudied groups can yield important new phylogenetic insights into long-standing questions, especially when carefully analyzed in combination with other data.