Single-cell RNA sequencing of mid-to-late stage spider embryos: new insights into spider development.

BACKGROUND: The common house spider Parasteatoda tepidariorum represents an emerging new model organism of arthropod evolutionary and developmental (EvoDevo) studies. Recent technical advances have resulted in the first single-cell sequencing (SCS) data on this species allowing deeper insights to be gained into its early development, but mid-to-late stage embryos were not included in these pioneering studies. RESULTS: Therefore, we performed SCS on mid-to-late stage embryos of Parasteatoda and characterized resulting cell clusters by means of in-silico analysis (comparison of key markers of each cluster with previously published information on these genes). In-silico prediction of the nature of each cluster was then tested/verified by means of additional in-situ hybridization experiments with additional markers of each cluster. CONCLUSIONS: Our data show that SCS data reliably group cells with similar genetic fingerprints into more or less distinct clusters, and thus allows identification of developing cell types on a broader level, such as the distinction of ectodermal, mesodermal and endodermal cell lineages, as well as the identification of distinct developing tissues such as subtypes of nervous tissue cells, the developing heart, or the ventral sulcus (VS). In comparison with recent other SCS studies on the same species, our data represent later developmental stages, and thus provide insights into different stages of developing cell types and tissues such as differentiating neurons and the VS that are only present at these later stages.

New insights into mesoderm and endoderm development, and the nature of the onychophoran blastopore.

BACKGROUND: Early during onychophoran development and prior to the formation of the germ band, a posterior tissue thickening forms the posterior pit. Anterior to this thickening forms a groove, the embryonic slit, that marks the anterior-posterior orientation of the developing embryo. This slit is by some authors considered the blastopore, and thus the origin of the endoderm, while others argue that the posterior pit represents the blastopore. This controversy is of evolutionary significance because if the slit represents the blastopore, then this would support the amphistomy hypothesis that suggests that a slit-like blastopore in the bilaterian ancestor evolved into protostomy and deuterostomy. RESULTS: In this paper, we summarize our current knowledge about endoderm and mesoderm development in onychophorans and provide additional data on early endoderm- and mesoderm-determining marker genes such as Blimp, Mox, and the T-box genes. CONCLUSION: We come to the conclusion that the endoderm of onychophorans forms prior to the development of the embryonic slit, and thus that the slit is not the primary origin of the endoderm. It is thus unlikely that the embryonic slit represents the blastopore. We suggest instead that the posterior pit indeed represents the lips of the blastopore, and that the embryonic slit (and surrounding tissue) represents a morphologically superficial archenteron-like structure. We conclude further that both endoderm and mesoderm development are under control of conserved gene regulatory networks, and that many of the features found in arthropods including the model Drosophila melanogaster are likely derived.

Two Notorious Nodes: a Critical Examination of Relaxed Molecular Clock Age Estimates of the Bilaterian Animals and Placental Mammals.

The popularity of relaxed clock Bayesian inference of clade origin timings has generated several recent publications with focal results considerably older than the fossils of the clades in question. Here we critically examine two such clades: the animals (with focus on the bilaterians); and the mammals (with focus on the placentals). Each example displays a set of characteristic pathologies which, although much commented on, are rarely corrected for. We conclude that in neither case does the molecular clock analysis provide any evidence for an origin of the clade deeper than what is suggested by the fossil record. In addition, both these clades have other features (including, in the case of the placental mammals, proximity to a large mass extinction) that allow us to generate precise expectations of the timings of their origins. Thus, in these instances the fossil record can provide a powerful test of molecular clock methodology, and why it goes astray; and we have every reason to think these problems are general.

Comment on "The lower Cambrian lobopodian Cardiodictyon resolves the origin of euarthropod brains".

Strausfeld et al. (Report, 24 Nov 2022, p. 905) claim that Cambrian fossilized nervous tissue supports the interpretation that the ancestral panarthropod brain was tripartite and unsegmented. We argue that this conclusion is unsupported, and developmental data from living onychophorans contradict it.

Evolutionary contingency in lingulid brachiopods across mass extinctions.

Morphology usually serves as an effective proxy for functional ecology,1,2,3,4,5 and evaluating morphological, anatomical, and ecological changes permits a deeper understanding of the nature of diversification and macroevolution.5,6,7,8,9,10,11,12 Lingulid (order Lingulida) brachiopods are both diverse and abundant during the early Palaeozoic but decrease in diversity over time, with only a few genera of linguloids and discinoids present in modern marine ecosystems, resulting in them frequently being referred to as "living fossils."13,14,15 The dynamics that drove this decline remain uncertain, and it has not been determined if there is an associated decline in morphological and ecological diversity. Here, we apply geometric morphometrics to reconstruct global morphospace occupation for lingulid brachiopods through the Phanerozoic, with results showing that maximum morphospace occupation was reached by the Early Ordovician. At this time of peak diversity, linguloids with a sub-rectangular shell shape already possessed several evolutionary features, such as the rearrangement of mantle canals and reduction of the pseudointerarea, common to all modern infaunal forms. The end Ordovician mass extinction has a differential effect on linguloids, disproportionally wiping out those forms with a rounded shell shape, while forms with sub-rectangular shells survived both the end Ordovician and the Permian-Triassic mass extinctions, leaving a fauna predominantly composed of infaunal forms. For discinoids, both morphospace occupation and epibenthic life strategies remain consistent through the Phanerozoic. Morphospace occupation over time, when considered using anatomical and ecological analyses, suggests that the limited morphological and ecological diversity of modern lingulid brachiopods reflects evolutionary contingency rather than deterministic processes.

Expression of netrin and its receptors uncoordinated-5 and frazzled in arthropods and onychophorans suggests conserved and diverged functions in neuronal pathfinding and synaptogenesis.

BACKGROUND: Development of the nervous system and the correct connection of nerve cells require coordinated axonal pathfinding through an extracellular matrix. Outgrowing axons exhibit directional growth toward or away from external guidance cues such as Netrin. Guidance cues can be detected by growth cones that are located at the end of growing axons through membrane-bound receptors such as Uncoordianted-5 and Frazzled. Binding of Netrin causes reformation of the cytoskeleton and growth of the axon toward (or away from) the source of Netrin production. RESULTS: Here, we investigate the embryonic mRNA expression patterns of netrin genes and their potential receptors, uncoordinated-5 and frazzled in arthropod species that cover all main branches of Arthropoda, that is, Pancrustacea, Myriapoda, and Chelicerata. We also studied the expression patterns in a closely related outgroup species, the onychophoran Euperipatoides kanangrensis, and provide data on expression profiles of these genes in larval tissues of the fly Drosophila melanogaster including the brain and the imaginal disks. CONCLUSION: Our data reveal conserved and diverged aspects of neuronal guidance in Drosophila with respect to the other investigated species and suggest a conserved function in nervous system patterning of the developing appendages.

Reassessing a cryptic history of early trilobite evolution.

Trilobites are an iconic Paleozoic group of biomineralizing marine euarthropods that appear abruptly in the fossil record (c. 521 million years ago) during the Cambrian 'explosion'. This sudden appearance has proven controversial ever since Darwin puzzled over the lack of pre-trilobitic fossils in the Origin of Species, and it has generally been assumed that trilobites must have an unobserved cryptic evolutionary history reaching back into the Precambrian. Here we review the assumptions behind this model, and suggest that a cryptic history creates significant difficulties, including the invocation of rampant convergent evolution of biomineralized structures and the abandonment of the synapomorphies uniting the clade. We show that a vicariance explanation for early Cambrian trilobite palaeobiogeographic patterns is inconsistent with factors controlling extant marine invertebrate distributions, including the increasingly-recognized importance of long-distance dispersal. We suggest that survivorship bias may explain the initial rapid diversification of trilobites, and conclude that the group's appearance at c. 521 Ma closely reflects their evolutionary origins.

A comprehensive study of arthropod and onychophoran Fox gene expression patterns.

Fox genes represent an evolutionary old class of transcription factor encoding genes that evolved in the last common ancestor of fungi and animals. They represent key-components of multiple gene regulatory networks (GRNs) that are essential for embryonic development. Most of our knowledge about the function of Fox genes comes from vertebrate research, and for arthropods the only comprehensive gene expression analysis is that of the fly Drosophila melanogaster. For other arthropods, only selected Fox genes have been investigated. In this study, we provide the first comprehensive gene expression analysis of arthropod Fox genes including representative species of all main groups of arthropods, Pancrustacea, Myriapoda and Chelicerata. We also provide the first comprehensive analysis of Fox gene expression in an onychophoran species. Our data show that many of the Fox genes likely retained their function during panarthropod evolution highlighting their importance in development. Comparison with published data from other groups of animals shows that this high degree of evolutionary conservation often dates back beyond the last common ancestor of Panarthropoda.

The evolution of biramous appendages revealed by a carapace-bearing Cambrian arthropod.

Biramous appendages are a common feature among modern marine arthropods that evolved deep in arthropod phylogeny. The branched appendage of Cambrian arthropods has long been considered as the ancient biramous limb, sparking numerous investigations on its origin and evolution. Here, we report a new arthropod, Erratus sperare gen. et sp. nov., from the Lower Cambrian (Stage 3, 520 Ma) Chengjiang biota of Yunnan, China, with unique trunk appendages formed of lateral anomalocaridid-type flaps and ventral subconical endopods. These appendages represent an intermediate stage of biramous limb evolution, i.e. from 'two pairs of flap appendages' in radiodonts to 'flap + endopod' in Erratus, to 'exopod + endopod' in the rest of carapace-bearing arthropods that populate the basal region of the upper-stem lineage arthropods (deuteropods). The new species occupies a phylogenetic position at the first node closer to deuteropods than to radiodonts, and therefore pinpoints the earliest occurrence of the endopod within Deuteropoda. The primitive endopod is weakly sclerotized, and has unspecialized segments without endites or claw. The findings might support previous claims that the outer branch of the biramous limb of fossil marine arthropods, such as trilobites, is not a true exopod, but is instead a modified exite. This article is part of the theme issue 'The impact of Chinese palaeontology on evolutionary research'.

Morphospace.

In his famous (if uncharacteristic) burst of lyricism at the end of the Origin Darwin described biodiversity as "endless forms most beautiful and wonderful". It is easy to agree with him when one considers red-lipped batfish or pelagic holothurians. But are they endless, or are there limitations to the variety of forms - and if there are, where do they come from? Can morphological evolution be described by Brownian motion of a gas, slowly diffusing to fill up all the space of possible forms, or does it operate within a certain set of constraints? And if there are constraints, where do they come from? The concept of morphospace is an attempt to map out the products of evolution within a quantitative framework to try to shed light on these questions.

Molecular evidence for a single origin of ultrafiltration-based excretory organs.

Excretion is an essential physiological process, carried out by all living organisms, regardless of their size or complexity.1-3 Both protostomes (e.g., flies and flatworms) and deuterostomes (e.g., humans and sea urchins) possess specialized excretory organs serving that purpose. Those organs exhibit an astonishing diversity, ranging from units composed of just few distinct cells (e.g., protonephridia) to complex structures, built by millions of cells of multiple types with divergent morphology and function (e.g., vertebrate kidneys).4,5 Although some molecular similarities between the development of kidneys of vertebrates and the regeneration of the protonephridia of flatworms have been reported,6,7 the molecular underpinnings of the development of excretory organs have never been systematically studied in a comparative context.4 Here, we show that a set of transcription factors (eya, six1/2, pou3, sall, lhx1/5, and osr) and structural proteins (nephrin, kirre, and zo1) is expressed in the excretory organs of a phoronid, brachiopod, annelid, onychophoran, priapulid, and hemichordate that represent major protostome lineages and non-vertebrate deuterostomes. We demonstrate that the molecular similarity observed in the vertebrate kidney and flatworm protonephridia6,7 is also seen in the developing excretory organs of those animals. Our results show that all types of ultrafiltration-based excretory organs are patterned by a conserved set of developmental genes, an observation that supports their homology. We propose that the last common ancestor of protostomes and deuterostomes already possessed an ultrafiltration-based organ that later gave rise to the vast diversity of extant excretory organs, including both proto- and metanephridia.

Panarthropod tiptop/teashirt and spalt orthologs and their potential role as "trunk"-selector genes.

BACKGROUND: In the vinegar fly Drosophila melanogaster, the homeodomain containing transcription factor Teashirt (Tsh) appears to specify trunk identity in concert with the function of the Hox genes. While in Drosophila there is a second gene closely related to tsh, called tiptop (tio), in other arthropods species only one copy exists (called tio/tsh). The expression of tsh and tio/tsh, respectively, is surprisingly similar among arthropods suggesting that its function as trunk selector gene may be conserved. Other research, for example on the beetle Tribolium castaneum, questions even conservation of Tsh function among insects. The zinc-finger transcription factor Spalt (Sal) is involved in the regulation of Drosophila tsh, but this regulatory interaction does not appear to be conserved in Tribolium either. Whether the function and interaction of tsh and sal as potential trunk-specifiers, however, is conserved is still unclear because comparative studies on sal expression (except for Tribolium) are lacking, and functional data are (if at all existing) restricted to Insecta. RESULTS: Here, we provide additional data on arthropod tsh expression, show the first data on onychophoran tio/tsh expression, and provide a comprehensive investigation on sal expression patterns in arthropods and an onychophoran. CONCLUSIONS: Our data support the idea that tio/tsh genes are involved in the development of "trunk" segments by regulating limb development. Our data suggest further that the function of Sal is indeed unlikely to be conserved in trunk vs head development like in Drosophila, but early expression of sal is in line with a potential homeotic function, at least in Arthropoda.

The origin and evolution of the euarthropod labrum.

A widely (although not universally) accepted model of arthropod head evolution postulates that the labrum, a structure seen in almost all living euarthropods, evolved from an anterior pair of appendages homologous to the frontal appendages of onychophorans. However, the implications of this model for the interpretation of fossil arthropods have not been fully integrated into reconstructions of the euarthropod stem group, which remains in a state of some disorder. Here I review the evidence for the nature and evolution of the labrum from living taxa, and reconsider how fossils should be interpreted in the light of this. Identification of the segmental identity of head appendage in fossil arthropods remains problematic, and often rests ultimately on unproven assertions. New evidence from the Cambrian stem-group euarthropod Parapeytoia is presented to suggest that an originally protocerebral appendage persisted well up into the upper stem-group of the euarthropods, which prompts a re-evaluation of widely-accepted segmental homologies and the interpretation of fossil central nervous systems. Only a protocerebral brain was implicitly present in a large part of the euarthropod stem group, and the deutocerebrum must have been a relatively late addition.

Oscillating waves of Fox, Cyclin and CDK gene expression indicate unique spatiotemporal control of cell cycling during nervous system development in onychophorans.

Forkhead box (Fox) genes code for a class of transcription factors with many different fundamental functions in animal development including cell cycle control. Other important factors of cell cycle control are Cyclins and Cyclin-dependent kinases (CDKs). Here we report on the oscillating expression of three Fox genes, FoxM, FoxN14 (jumeaux) and FoxN23 (Checkpoint suppressor like-1), Cyclins and CDKs in an onychophoran, a representative of a relatively small group of animals that are closely related to the arthropods. Expression of these genes is in the form of several waves that start as dot-like domains in the center of each segment and then transform into concentric rings that run towards the periphery of the segments. This oscillating gene expression, however, occurs exclusively along the anterior-posterior body axis in the tissue ventral to the base of the appendages, a region where the central nervous system and the enigmatic ventral and preventral organs of the onychophoran develop. We suggest that the oscillating gene expression and the resulting waves of expression we report are likely correlated with cell cycle control during the development of the onychophoran nervous system. This intriguing patterning appears to be unique for onychophorans as it is not found in any of the arthropods we also investigated in this study, and is likely correlated with the slow embryonic development of onychophorans compared to arthropods.

Animal Phylogeny: Resolving the Slugfest of Ctenophores, Sponges and Acoels?

Animal phylogeny has always been controversial, but a new study brings some much-needed order for two infamous wandering groups, the ctenophores and the Xenacoelomorphs. The study introduces an innovative approach to dissect systematic errors in the underlying methodology of molecular phylogenies.

Impacts of speciation and extinction measured by an evolutionary decay clock.

The hypothesis that destructive mass extinctions enable creative evolutionary radiations (creative destruction) is central to classic concepts of macroevolution1,2. However, the relative impacts of extinction and radiation on the co-occurrence of species have not been directly quantitatively compared across the Phanerozoic eon. Here we apply machine learning to generate a spatial embedding (multidimensional ordination) of the temporal co-occurrence structure of the Phanerozoic fossil record, covering 1,273,254 occurrences in the Paleobiology Database for 171,231 embedded species. This facilitates the simultaneous comparison of macroevolutionary disruptions, using measures independent of secular diversity trends. Among the 5% most significant periods of disruption, we identify the 'big five' mass extinction events2, seven additional mass extinctions, two combined mass extinction-radiation events and 15 mass radiations. In contrast to narratives that emphasize post-extinction radiations1,3, we find that the proportionally most comparable mass radiations and extinctions (such as the Cambrian explosion and the end-Permian mass extinction) are typically decoupled in time, refuting any direct causal relationship between them. Moreover, in addition to extinctions4, evolutionary radiations themselves cause evolutionary decay (modelled co-occurrence probability and shared fraction of species between times approaching zero), a concept that we describe as destructive creation. A direct test of the time to over-threshold macroevolutionary decay4 (shared fraction of species between two times ≤ 0.1), counted by the decay clock, reveals saw-toothed fluctuations around a Phanerozoic mean of 18.6 million years. As the Quaternary period began at a below-average decay-clock time of 11 million years, modern extinctions further increase life's decay-clock debt.

Survival and selection biases in early animal evolution and a source of systematic overestimation in molecular clocks.

Important evolutionary events such as the Cambrian Explosion have inspired many attempts at explanation: why do they happen when they do? What shapes them, and why do they eventually come to an end? However, much less attention has been paid to the idea of a 'null hypothesis'-that certain features of such diversifications arise simply through their statistical structure. Such statistical features also appear to influence our perception of the timing of these events. Here, we show in particular that study of unusually large clades leads to systematic overestimates of clade ages from some types of molecular clocks, and that the size of this effect may be enough to account for the puzzling mismatches seen between these molecular clocks and the fossil record. Our analysis of the fossil record of the late Ediacaran to Cambrian suggests that it is likely to be recording a true evolutionary radiation of the bilaterians at this time, and that explanations involving various sorts of cryptic origins for the bilaterians do not seem to be necessary.

Evolution: Mapping Out Early Echinoderms.

The rich early fossil record of the echinoderms reveals surprisingly dynamic patterns of body plan evolution and suggests that currently popular theories about how the major features of the animal originated and were maintained are unlikely to be correct.

Expression of the zinc finger transcription factor Sp6-9 in the velvet worm Euperipatoides kanangrensis suggests a conserved role in appendage development in Panarthropoda.

The Sp-family genes encode important transcription factors in animal development. Here we investigate the embryonic expression patterns of the complete set of Sp-genes in the velvet worm Euperipatoides kanangrensis (Onychophora), with a special focus on the Sp6-9 ortholog. In arthropods, Sp6-9, the ortholog of the Drosophila melanogaster D-Sp1 gene plays a conserved role in appendage development. Our data show that the expression of Sp6-9 during the development of the velvet worm is conserved, suggesting that the key function of the Sp6-9 gene dates back to at least the last common ancestor of arthropods and onychophorans and thus likely the last common ancestor of Panarthropoda.