Echinoids are key components of modern marine ecosystems. Despite a remarkable fossil record, the emergence of their crown group is documented by few specimens of unclear affinities, rendering their early history uncertain. The origin of sand dollars, one of its most distinctive clades, is also unclear due to an unstable phylogenetic context. We employ 18 novel genomes and transcriptomes to build a phylogenomic dataset with a near-complete sampling of major lineages. With it, we revise the phylogeny and divergence times of echinoids, and place their history within the broader context of echinoderm evolution. We also introduce the concept of a chronospace - a multidimensional representation of node ages - and use it to explore methodological decisions involved in time calibrating phylogenies. We find the choice of clock model to have the strongest impact on divergence times, while the use of site-heterogeneous models and alternative node prior distributions show minimal effects. The choice of loci has an intermediate impact, affecting mostly deep Paleozoic nodes, for which clock-like genes recover dates more congruent with fossil evidence. Our results reveal that crown group echinoids originated in the Permian and diversified rapidly in the Triassic, despite the relative lack of fossil evidence for this early diversification. We also clarify the relationships between sand dollars and their close relatives and confidently date their origins to the Cretaceous, implying ghost ranges spanning approximately 50 million years, a remarkable discrepancy with their rich fossil record.

Animal mitogenomes are typically devoid of introns. Here, we report the largest number of mitochondrial introns ever recorded from bilaterian animals. Mitochondrial introns were identified for the first time from the phylum Bryozoa. They were found in four species from three families (Order Cheilostomatida). A total of eight introns were found in the complete mitogenome of Exechonella vieirai, and five, 17 and 18 introns were found in the partial mitogenomes of Parantropora penelope, Discoporella cookae and Cupuladria biporosa, respectively. Intron-encoded protein domains reverse transcriptase and intron maturase (RVT-IM) were identified in all species. Introns in E. vieirai and P. penelope had conserved Group II intron ribozyme domains V and VI. Conserved domains were lacking from introns in D. cookae and C. biporosa, preventing their further categorization. Putative origins of metazoan introns were explored in a phylogenetic context, using an up-to-date alignment of mitochondrial RVT-IM domains. Results confirmed previous findings of multiple origins of annelid, placozoan and sponge RVT-IM domains and provided evidence for common intron donor sources across metazoan phyla. Our results corroborate growing evidence that some metazoans with regenerative abilities (i.e. placozoans, sponges, annelids and bryozoans) are susceptible to intron integration, most likely via horizontal gene transfer.

Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Materials and methods Appendix 1 Appendix 2 Appendix 3 Data availability References Decision letter Author response Article and author information Metrics Abstract Echinoids are key components of modern marine ecosystems. Despite a remarkable fossil record, the emergence of their crown group is documented by few specimens of unclear affinities, rendering their early history uncertain. The origin of sand dollars, one of its most distinctive clades, is also unclear due to an unstable phylogenetic context. We employ 18 novel genomes and transcriptomes to build a phylogenomic dataset with a near-complete sampling of major lineages. With it, we revise the phylogeny and divergence times of echinoids, and place their history within the broader context of echinoderm evolution. We also introduce the concept of a chronospace – a multidimensional representation of node ages – and use it to explore methodological decisions involved in time calibrating phylogenies. We find the choice of clock model to have the strongest impact on divergence times, while the use of site-heterogeneous models and alternative node prior distributions show minimal effects. The choice of loci has an intermediate impact, affecting mostly deep Paleozoic nodes, for which clock-like genes recover dates more congruent with fossil evidence. Our results reveal that crown group echinoids originated in the Permian and diversified rapidly in the Triassic, despite the relative lack of fossil evidence for this early diversification. We also clarify the relationships between sand dollars and their close relatives and confidently date their origins to the Cretaceous, implying ghost ranges spanning approximately 50 million years, a remarkable discrepancy with their rich fossil record. Editor's evaluation The study by Mongiardino Koch et al., presents new phylogenomic and molecular clock analyses of echinoids. The study uses state of the art phylogenetic approaches and includes 18 newly sequenced genomes and transcriptomes, which are used to estimate the tree topology and divergence times of major groups of echinoids. The molecular clock-estimated times of origin of particular echinoid lineages predate the lineages' appearance on the fossil record by tens of millions of years, prompting re-evaluation of the early evolution of echinoid diversity. https://doi.org/10.7554/eLife.72460.sa0 Decision letter Reviews on Sciety eLife's review process Introduction The fossil record represents the best source of primary data for constraining the origins of major lineages across the tree of life. However, the fossil record is not perfect, and even for groups with an excellent fossilization potential, constraining their age of origin can be difficult (Smith and Peterson, 2002; Donoghue and Benton, 2007). Furthermore, as many traditional hypotheses of relationships have been revised in light of large-scale molecular datasets, the affinities of fossil lineages and their bearings on inferred times of divergence have also required a reassessment. An exemplary case of this is Echinoidea, a clade comprising sea urchins, heart urchins, sand dollars, and allies, for which phylogenomic trees have questioned the timing of previously well-constrained nodes (Mongiardino Koch et al., 2018; Mongiardino Koch and Thompson, 2021d). Echinoids are easily recognized by their spine-covered skeletons or tests, composed of numerous tightly interlocking plates. Slightly over 1000 living species have been described to date (Kroh and Mooi, 2020), a diversity that populates every marine benthic environment from intertidal to abyssal depths (Schultz, 2015). Echinoids are usually subdivided into two morpho-functional groups with similar species-level diversities: ‘regular’ sea urchins, a paraphyletic assemblage of hemispherical, epibenthic consumers protected by large spines; and irregulars (Irregularia), a clade of predominantly infaunal and bilaterally symmetrical forms covered by small and specialized spines. In today’s oceans, regular echinoids act as ecosystem engineers in biodiverse coastal communities such as coral reefs (Edmunds and Carpenter, 2001) and kelp forests (Harrold and Pearse, 1987), where they are often the main consumers. They are first well known in the fossil record on either side of the Permian-Triassic (P-T) mass extinction event when many species occupied reef environments similar to those inhabited today by their descendants (Zonneveld et al., 2016; Thompson et al., 2017b). This extinction event was originally thought to have radically impacted the macroevolutionary history of the clade, decimating the echinoid stem group and leading to the radiation of crown group taxa from a single surviving lineage (Kier, 1977b; Twitchett and Oji, 2005). However, it is now widely accepted that the origin of crown group Echinoidea (i.e., the divergence between its two main lineages, Cidaroidea and Euechinoidea) occurred in the Late Permian, as supported by molecular estimates of divergence (Smith et al., 2006; Thompson et al., 2017a), as well as the occurrence of Permian fossils with morphologies typical of modern cidaroids (Smith and Hollingworth, 1990; Thompson et al., 2015). However, a recent total-evidence study recovered many taxa previously classified as crown group members along the echinoid stem, while also suggesting that up to three crown group lineages survived the P-T mass extinction (Mongiardino Koch and Thompson, 2021d). This result increases the discrepancy between molecular estimates and the fossil record and renders uncertain the early evolutionary history of crown group echinoids. Constraining the timing of origin of this clade relative to the P-T mass extinction (Mongiardino Koch et al., 2018; Mongiardino Koch and Thompson, 2021d) is further complicated by the poor preservation potential of stem group echinoids, and the difficulty assigning available disarticulated remains from the Late Paleozoic and Early Triassic to specific clades (Kier, 1977b; Twitchett and Oji, 2005; Smith, 2007; Kroh and Smith, 2010; Thompson et al., 2018; Thompson et al., 2019). Compared to the morphological conservatism of regular sea urchins, the evolutionary history of the relatively younger Irregularia was characterized by dramatic levels of morphological and ecological innovation (Kier, 1982; Saucède et al., 2006; Barras, 2008; Hopkins and Smith, 2015). Within the diversity of irregulars, sand dollars are the most easily recognized (Figure 1). The clade includes greatly flattened forms that live in high-energy sandy environments where they feed using a unique mechanism for selecting and transporting organic particles to the mouth, where these are crushed using well-developed jaws (Mooi, 1990a; Nebelsick, 2020). Sand dollars (Scutelloida) were long thought to be most closely related to sea biscuits (Clypeasteroida) given a wealth of shared morphological characters (Mooi, 1990a; Kroh and Smith, 2010). The extraordinary fossil record of both sand dollars and sea biscuits suggested their last common ancestor originated in the early Cenozoic from among an assemblage known as ‘cassiduloids’ (Mooi, 1990a; Saucède et al., 2006), a once diverse group that is today represented by three depauperate lineages: cassidulids (and close relatives), echinolampadids, and apatopygids (Smith, 2016; Kroh and Smith, 2010). These taxa not only lack the defining features of both scutelloids and clypeasteroids but have experienced little morphological change since their origin deep in the Mesozoic (Kier, 1962; Smith, 2016; Hopkins and Smith, 2015; Souto et al., 2019). However, early molecular phylogenies supported both cassidulids and echinolampadids as close relatives of sand dollars (e.g., Littlewood and Smith, 1995; Smith et al., 2006), a topology initially disregarded for its conflicts with both morphological and paleontological evidence, but later confirmed using phylogenomic approaches (Mongiardino Koch et al., 2018). While many of the traits shared by sand dollars and sea biscuits have since been suggested to represent a mix of convergences and ancestral synapomorphies secondarily lost by some ‘cassiduloids’ (Mongiardino Koch et al., 2018; Mongiardino Koch and Thompson, 2021d), the strong discrepancy between molecular topologies and the fossil record remains unexplained. Central to this discussion is the position of apatopygids, a clade so far unsampled in molecular studies. Apatopygids have a fossil record stretching more than 100 million years and likely have phylogenetic affinities with even older extinct lineages (Kier, 1962; Kroh and Smith, 2010; Souto et al., 2019; Mongiardino Koch and Thompson, 2021d). Although current molecular topologies already imply ghost ranges for scutelloids and clypeasteroids that necessarily extend beyond the Cretaceous-Paleogene (K-Pg) boundary, the phylogenetic position of apatopygids could impose even earlier ages on these lineages (Figure 1). Constraining these divergences is necessary to understand the timing of origin of the sand dollars, one of the most specialized lineages of echinoids (Mooi, 1990a; Smith, 2016; Hopkins and Smith, 2015; Nebelsick, 2020). Resolving some phylogenetic relationships within scutelloids has also been complicated by their recurrent miniaturization and associated loss of morphological features (Figure 1; Mooi, 1990a; Mooi, 1990b; Mongiardino Koch, 2021a). Figure 1 Download asset Open asset Neognathostomate diversity and phylogenetic relationships. (A) Fellaster zelandiae, North Island, New Zealand (Clypeasteroida). (B) Large specimen: Peronella japonica, Ryukyu Islands, Japan; Small specimen: Echinocyamus crispus, Maricaban Island, Philippines (Laganina: Scutelloida). (C) Large specimen: Leodia sexiesperforata, Long Key, Florida; Small specimen: Sinaechinocyamus mai, Taiwan (Scutellina: Scutelloida). (D) Rhyncholampas pacificus, Isla Isabela, Galápagos Islands (Cassidulidae). (E) Conolampas sigsbei, Bimini, Bahamas (Echinolampadidae). (F)Apatopygus recens, Australia (Apatopygidae). (G) Hypotheses of relationships among neognathostomates. Top: Morphology supports a clade of Clypeasteroida + Scutelloida originating after the Cretaceous-Paleogene (K-Pg) boundary, subtended by a paraphyletic assemblage of extant (red) and extinct (green) ‘cassiduloids’ (Kroh and Smith, 2010). Bottom: A recent total-evidence study split cassiduloid diversity into a clade of extant lineages closely related to scutelloids, and an unrelated clade of extinct forms (Nucleolitoida; Mongiardino Koch and Thompson, 2021d). Divergence times are much older and conflict with fossil evidence. Cassidulids and apatopygids lacked molecular data in this analysis. Scale bars = 10 mm. Echinoidea constitutes a model clade in developmental biology and genomics. As these fields embrace a more comparative approach (Thompson et al., 2017a; Dunn et al., 2018; Smith et al., 2020), robust and time-calibrated phylogenies are expected to play an increasingly important role. Likewise, the extraordinary fossil record of echinoids and the ease with which echinoid fossils can be incorporated in phylogenetic analyses make them an ideal system to explore macroevolutionary dynamics using phylogenetic comparative methods (Mongiardino Koch, 2021a; Mongiardino Koch and Thompson, 2021d). In this study, we build upon available molecular resources with 18 novel genome-scale datasets and build the largest molecular matrix for echinoids yet compiled. Our expanded phylogenomic dataset extends sampling to 16 of the 17 currently recognized echinoid orders – plus the unassigned apatopygids (Kroh, 2020) – and is the first to bracket the extant diversity of both sand dollars and sea biscuits and include members of all three lineages of living ‘cassiduloids’ (cassidulids, echinolampadids, and apatopygids). We also incorporate a diverse sample of outgroups, providing access to the deepest nodes within the crown groups of all other echinoderm classes (holothuroids, asteroids, ophiuroids, and crinoids). With it, we reconstruct the phylogenetic relationships and divergence times of the major lineages of living echinoids and place their diversification within the broader context of echinoderm evolution. Results Phylogeny of Echinoidea Analyses relied on a 70% occupancy supermatrix composed of 1346 loci (327,695 amino acid sites), and including 54 echinoid terminals plus 12 outgroups. Inference was performed under multiple concatenation and coalescent-aware methodologies, as well as relying on maximum likelihood and Bayesian implementations of site-homogeneous and site-heterogeneous models, as these approaches are known to differ in their susceptibility to model violations (Lartillot et al., 2007; Kainer and Lanfear, 2015; Jiang et al., 2020; see Materials and methods for further details). Phylogenetic relationships supported by the full dataset were remarkably stable, with all nodes but one being identically resolved and fully supported across all methods (Figure 2A). While recovering a topology similar to those of previous molecular studies (Littlewood and Smith, 1995; Smith et al., 2006; Thompson et al., 2017a; Mongiardino Koch et al., 2018; Lin et al., 2020; Mongiardino Koch and Thompson, 2021d), this analysis is the first to sample and confidently place micropygoids and aspidodiadematoids within Aulodonta, as well as resolve the relationships among all major clades of Neognathostomata (scutelloids, clypeasteroids and the three lineages of extant ‘cassiduloids’). Our results show that Apatopygus recens is not related to the remaining ‘cassiduloids’ but is instead the sister clade to all other sampled neognathostomates. The strong support for this placement, as well as for a clade of cassidulids and echinolampadids (Cassiduloida sensu stricto) as the sister group to sand dollars, provides a basis for an otherwise elusive phylogenetic classification of neognathostomates. Our topology also confirms that Sinaechinocyamus mai, a miniaturized species once considered a plesiomorphic member of Scutelloida based on the reduction or loss of diagnostic features (Figure 1), is in fact a derived paedomorphic lineage closely related to Scaphechinus mirabilis (Mooi, 1990b). Figure 2 Download asset Open asset Phylogenetic relationships among major clades of Echinoidea. (A) Favored topology, as obtained using the full supermatrix and a best-fit partitioning scheme in IQ-TREE (Nguyen et al., 2015). With the exception of a single contentious node within Echinacea (marked with a yellow star), all methods supported the same pattern of relationships, and assigned maximum support values to all nodes. Numbers below major clades correspond to the current numbers of described living species (obtained from Kroh and Mooi, 2020). (B) Likelihood-mapping analysis showing the proportion of quartets supporting different resolutions within Echinacea. While the majority of quartets support the topology depicted in A (shown in red), a relatively large number support an alternative resolution that has been recovered in morphological analyses (shown in blue; Kroh and Smith, 2010). (C) Difference in likelihood score (delta likelihood) for the two resolutions of Echinacea most strongly supported in the likelihood-mapping analysis. Genes were sorted based on their inferred phylogenetic usefulness (Mongiardino Koch, 2021b), and gene-wise delta scores were averaged for datasets composed of multiples of 20 loci. Support for a clade of Salenioida + (Camarodonta + Stomopneustoida), as depicted in A, is seen as positive delta scores and is predominantly concentrated among the most phylogenetically useful loci. This signal is attenuated in larger datasets that contain less reliable genes, eventually favoring an alternative resolution (as seen by negative scores for the largest datasets). Only the 584 loci containing data for the three main lineages of Echinacea were considered. The line corresponds to a second-degree polynomial regression. (D) Resolution and bootstrap scores (see color scale) of the topology within Echinacea found using datasets of different sizes and alternative methods of inference. Salenioida is another major lineage sampled here for the first time, and whose exact position among regular echinoids proved difficult to resolve. While some methods supported salenioids as the sister group to a clade of camarodonts, stomopneustoids, and arbacioids (a topology previously supported by morphology; Kroh and Smith, 2010), others recovered a closer relationship of salenioids to Camarodonta + Stomopneustoida, with arbacioids sister to them all (as shown in Figure 2A). As revealed using likelihood mapping, these results do not stem from a lack of phylogenetic signal, but rather from the presence of strong and conflicting evidence in the dataset regarding the position of salenioids (Figure 2B). However, a careful dissection of these signals shows that loci with high phylogenetic usefulness (as defined by Mongiardino Koch, 2021b; Mongiardino Koch and Thompson, 2021d; see Materials and methods) favor the topology shown in Figure 2A, with the morphological hypothesis becoming dominant only after incorporating less reliable loci (Figure 2C). In line with these results, moderate levels of gene subsampling (down to 500 loci) targeting the most phylogenetically useful loci unambiguously support the placement of arbacioids as sister to the remaining taxa, regardless of the chosen method of inference (Figure 2D). More extreme subsampling (down to 100 loci) again results in disagreement among methods. This possibly stems from the increasing effect of stochastic errors in smaller datasets, as less than half of the sampled loci in these reduced datasets contain data for all branches of this quartet (see Figure 2C). This result shows the importance of ensuring that datasets (especially subsampled ones) retain appropriate levels of occupancy for clades bracketing contentious nodes (Dell’Ampio, 2014). Despite these disagreements, several lines of evidence favor the topology shown in Figure 2A, including the results of likelihood mapping, and the increased support for this resolution among the most phylogenetically useful loci and when using more complex methods of reconstruction, such as partitioned and site-heterogeneous models, which always favor this topology regardless of dataset size (Figure 2D). Sensitivity of node ages While alternative methods of inference had minor effects on phylogenetic relationships, they did impact the reconstruction of branch lengths (Figure 3). Site-heterogeneous models (such as CAT + GTR + G) returned longer branch lengths overall, but also uncovered a larger degree of molecular change among echinoderm classes. Branches connecting these clades were stretched to a much larger extent than those within the ingroup, a phenomenon that might affect the inference of node ages. We tested this hypothesis by exploring the sensitivity of divergence times to the use of alternative models of molecular evolution (site-homogeneous vs. site-heterogeneous), as well as different clocks (autocorrelated vs. uncorrelated), prior node distributions (Cauchy vs. uniform), and gene sampling strategies (using five different approaches; see Materials and methods). All combinations of these factors were explored, resulting in 40 different time calibration settings that were run using Bayesian approaches under a constrained tree topology (shown in Figure 2A). While the nodes connecting some outgroup taxa were among those most sensitive to these methodological decisions, large effects were also seen among nodes relating to the origin and diversification of the echinoid clades Cidaroidea, Aulodonta, and Neognathostomata. All of these nodes varied in age by more than 35 Myr – and up to 115 Myr – among the consensus topologies of different analyses (Figure 4). Figure 3 Download asset Open asset Estimated branch lengths across different models of molecular evolution. Different site-homogeneous models (left) infer similar levels of divergence, and the choice between them induces little distortion in the general tree structure. Site-heterogeneous models on the other hand not only infer a larger degree of divergence between terminals relative to site-homogeneous ones (center and right), but they also distort the tree (i.e., impose a non-isometric stretching), with branch lengths connecting outgroup taxa expanding much more than those within the ingroup clade. Figure 4 with 1 supplement see all Download asset Open asset The 10 most sensitive node dates are found within Cidaroidea, Aulodonta, Neognathostomata, and among outgroup nodes. For each, the range shown spans the interval between the minimum and maximum ages found among the consensus topologies of the 80 time-calibrated runs performed. In order to isolate and visualize the impact of each of these factors on divergence time estimation, chronograms were represented in a multidimensional space of node dates, with each axis representing the age of a given node. We term this type of graph a chronospace given its similarities to the treespaces commonly used to explore topological differences among phylogenetic trees (Hillis et al., 2005). Each observation (chronogram) was classified as obtained under a specific clock, model of molecular evolution, node prior distribution, and gene sampling strategy, and the major effects of each of these choices were extracted with the use of between-group principal component analyses (bgPCAs). The single dimension of chronospace maximizing the distinctiveness of chronograms obtained under different clocks explained 53.4% of the total variance in node ages across all analyses (Figure 5). In contrast, the choice of different loci, models of molecular evolution, and prior distributions on node ages showed much lesser effects, explaining 10.7%, 3.9%, and 0.4% of the total variance, respectively (Figure 5 and Figure 5—figure supplement 1). Even though most of these decisions affected a similar set of sensitive nodes (those mentioned above, as well as some relationships within Atelostomata), the choice of clock model modified the ages of 17 of these by more than 20 Myr (Figure 5—figure supplement 2). This degree of change was induced on only four nodes by selecting alternative loci, and was not induced on any node by enforcing different models of evolution or node age priors (Figure 5—figure supplements 3–5). Regarding gene choice, the ages most different to those obtained under random loci selection were found when using the most clock-like genes (Figure 5C). Figure 5 with 5 supplements see all Download asset Open asset Sensitivity of divergence time estimation to methodological decisions. Between-group principal component analysis (bgPCA) was used to retrieve axes that separate chronograms based on the clock model (A), model of molecular evolution (B), and gene sampling strategy (C) employed. In the latter case, only the first two out of four bgPCA dimensions are shown. The inset shows the centroid for each loci sampling strategy, and the width of the lines connecting them are scaled to the inverse of the Euclidean distances that separates them (as a visual summary of overall similarity). The proportions of total variance explained are shown on the axis labels. The impact of the clock model is such that a bimodal distribution of chronograms can be seen even when bgPCA are built to discriminate based on other factors (as in C). Echinoid (and echinoderm) divergence times Even when the age of crown Echinodermata was constrained to postdate the appearance of stereom (the characteristic skeletal microstructure of echinoderms) in the Early Cambrian (Bottjer et al., 2006; Zamora et al., 2013), only analyses using the most clock-like loci recovered ages concordant with this (i.e., median ages younger than the calibration enforced; Figure 5—figure supplement 3). Instead, most consensus trees favored markedly older ages for the clade, in some cases even predating the origin of the Ediacaran biota (Pu et al., 2016; Figure 4—figure supplement 1). Despite the relative sensitivity of many of the earliest nodes to methodological choices (Figure 4 and Figure 4—figure supplement 1), the split between Crinoidea and all other echinoderms (Eleutherozoa) is always inferred to have predated the end of the Cambrian (youngest median age = 492.1 Ma), and the divergence among the other major lineages (classes) of extant echinoderms are constrained to have happened between the Late Cambrian and Middle Ordovician (Figure 4—figure supplement 1). Our results also recover an early origin of crown group Holothuroidea (sea cucumbers; range of median ages = 350.4–384.2 Ma), well before the crown groups of other extant echinoderm classes. These dates markedly postdate the first records of holothuroid calcareous rings in the fossil record (Reich, 2015; Miller et al., 2017), and imply that this trait does not define the holothuroid crown group but instead evolved from an echinoid-like jaw-apparatus along its stem (Rahman et al., 2019). The other noteworthy disagreement between our results and those of previous studies (Rouse et al., 2013) involves dating crown group Crinoidea to times that precede the P-T mass extinction (range of median ages = 268.0–329.7 Ma, although highest posterior density intervals are always wide and include Triassic ages). Across all of the analyses performed, the echinoid crown group is found to have originated somewhere between the Pennsylvanian and Cisuralian, with 30.2% posterior probability falling within the late Carboniferous and 69.1% within the early Permian (Figure 6 and Figure 6—figure supplement 1). An origin of the clade postdating the P-T mass extinction is never recovered, even when such ages are common under the joint prior (Figure 6—figure supplement 2). While the posterior distribution of ages for Euechinoidea spans both sides of the P-T boundary, the remaining earliest splits within the echinoid tree are constrained to have occurred during the Triassic, including the origins of Aulodonta, Carinacea, Echinacea, and Irregularia (Figure 6 and Figure 4—figure supplement 1). Many echinoid orders are also inferred to have diverged from their respective sister clades during this period, including aspidodiadematoids, pedinoids, echinothurioids, arbacioids, and salenioids. Lineage-through-time plots confirm that diversification proceeded rapidly throughout the Triassic (Figure 6B). Despite the topological reorganization of Neognathostomata, the clade is dated to a relatively narrow time interval in the Late to Middle Jurassic (range of median ages = 169.48–180.93 Ma), in agreement with recent estimates (Mongiardino Koch and Thompson, 2021d). Within this clade, the origins of both scutelloids and clypeasteroids confidently predate the K-Pg mass extinction (posterior probability of origination before the boundary = 1.00 and 0.97, respectively), despite younger ages being allowed by the joint prior (Figure 6—figure supplement 2). Figure 6 with 2 supplements see all Download asset Open asset Divergence times among major clades of Echinoidea and other echinoderms. (A) Consensus chronogram of the two PhyloBayes (Lartillot et al., 2013) runs using clock-like genes under a CAT + GTR + G model of evolution, an autocorrelated log-normal (LN) clock, and Cauchy prior distributions. Node ages correspond to median values, and bars show the 95% highest posterior density intervals. (B) Lineage-through-time plot, showing the rapid divergence of higher-level clades following the P-T mass extinction (shown with dashed lines, along with the Cretaceous-Paleogene [K-Pg] boundary). Each line corresponds to an individual consensus topology from among the 80 time-calibrated runs performed. (C) Posterior distributions of the ages of selected nodes (identified in A with numbers). The effects introduced by the use of different models of molecular evolution and node age prior distributions are not shown, as they represent the least important factors (see Figure 5); the posterior distributions obtained under different settings of these were merged for every combination of targeted loci and clock prior. Tick marks = 10 Myr. Discussion The echinoid tree of life In agreement with previous phylogenomic studies (Mongiardino Koch et al., 2018; Mongiardino Koch and Thompson, 2021d), echinoid diversity can be subdivided into five major clades (Figure 2A). Cidaroids form the sister group to all other crown group echinoids (Euechinoidea). Some aspects of the relationships among sampled cidaroids are consistent with previous molecular (Brosseau et al., 2012) and morphological studies (Kroh and Smith, 2010), including an initial split between Histocidaris and the remaining taxa, representing the two main branches of extant cidaroids (Kroh, 2020; Kroh and Mooi, 2020). Others, such as the nested position of Prionocidaris baculosa within the genus Eucidaris, not only implies paraphyly of this genus but also suggests the need for a taxonomic reorganization of the family Cidaridae. Within euechinoids, the monophyly of Aulodonta is supported for the first time with sampling of all of its major groups. The subdivision of these into a clade that includes diadematoids plus micropygoids (which we propose should retain the name Diadematacea), sister to a clade including echinothurioids and pedinoids (Echinothuriacea sensu Mongiardino Koch et al., 2018) is strongly reminiscent of some early classifications (e.g., Durham and Melville, 1957). Our expanded phylogenomic sampling also confirms an aulodont affinity for aspidodiadematoids (Kroh, 2020; M

1 Department of Earth & Planetary Sciences, Yale University, New Haven CT, USA. 2 Scripps Institution of 9 Oceanography, University of California San Diego, La Jolla CA, USA. 3 Department of Earth Sciences, 10 Natural History Museum, Cromwell Road, SW7 5BD London, UK. 4 University College London Center for 11 Life’s Origins and Evolution, London, UK. 5 Department of Invertebrate Zoology and Geology, California 12 Academy of Sciences, San Francisco CA, USA. 6 Bader International Study Centre, Queen's University, 13 Herstmonceux Castle, East Sussex, UK. 7 Departamento de Bioquímica y Biología Molecular, Facultad de 14 Ciencias Biológicas, Universidad de Concepción, Concepción, Chile. 8 School of Zoology, Faculty of Life 15 Sciences, Tel Aviv University, Tel Aviv, Israel. 9 Steinhardt Museum of Natural History, Tel-Aviv, Israel. 10 16 Department of Geology and Palaeontology, Natural History Museum Vienna, Vienna, Austria 17

The sea urchins Echinothrix calamaris and Echinothrix diadema have sympatric distributions throughout the Indo-Pacific. Diverse colour variation is reported in both species. To reconstruct the phylogeny of the genus and assess gene flow across the Indo-Pacific we sequenced mitochondrial 16S rDNA, ATPase-6, and ATPase-8, and nuclear 28S rDNA and the Calpain-7 intron. Our analyses revealed that E. diadema formed a single trans-Indo-Pacific clade, but E. calamaris contained three discrete clades. One clade was endemic to the Red Sea and the Gulf of Oman. A second clade occurred from Malaysia in the West to Moorea in the East. A third clade of E. calamaris was distributed across the entire Indo-Pacific biogeographic region. A fossil calibrated phylogeny revealed that the ancestor of E. diadema diverged from the ancestor of E. calamaris ~ 16.8 million years ago (Ma), and that the ancestor of the trans-Indo-Pacific clade and Red Sea and Gulf of Oman clade split from the western and central Pacific clade ~ 9.8 Ma. Time since divergence and genetic distances suggested species level differentiation among clades of E. calamaris. Colour variation was extensive in E. calamaris, but not clade or locality specific. There was little colour polymorphism in E. diadema.

The pan-tropical sea urchin Tripneustes gratilla is an ecologically and economically important shallow water algal grazer. The aquaculture of T. gratilla has spurred growing interest in the population biology of the species, and by extension the generation of more molecular resources. To this purpose, de novo transcriptomes of T. gratilla were generated for two adults, a male and a female, as well as for a cohort of approximately 1000 plutei larvae. Gene expression profiles of three adult tissue samples were quantified and compared. These samples were of gonadal tissue, the neural ring, and pooled tube feet and pedicellariae. Levels of shared and different gene expression between sexes, as well as across functional categories of interest, including the immune system, toxins, genes involved in fertilization, and sensory genes are highlighted. Differences in expression of isoforms between the sexes and Sex determining Region Y-related High Mobility Group box groups is observed. Additionally an expansion of the tumor suppressor DMBT1 is observed in T. gratilla when compared to the annotated genome of the sea urchin Strongylocentrotus purpuratus. The draft transcriptome of T. gratilla is presented here in order to facilitate more genomic level analysis of emerging model sea urchin systems.

The pan-tropical sea urchin Tripneustes gratilla is an ecologically and economically important shallow water algal grazer. The aquaculture of T. gratilla has spurred growing interest in the population biology of the species, and by extension the generation of more molecular resources. To this purpose, de novo transcriptomes of T. gratilla were generated for two adults, a male and a female, as well as for a cohort of approximately 1,000 plutei larvae. Gene expression profiles of three adult tissue samples were quantified and compared. These samples were of gonadal tissue, the neural ring, and pooled tube feet and pedicellariae. Levels of shared and different gene expression between sexes, as well as across functional categories of interest, including the immune system, toxins, genes involved in fertilization, and sensory genes are highlighted. Differences in expression of Sex determining Region Y-related High Mobility Group box groups and general isoform expression between the sexes is observed. Additionally an expansion of the tumor suppressor DMBT1 was observed in T. gratilla when compared to the annotated genome of the sea urchin Strongylocentrotus purpuratus. The draft transcriptome of T. gratilla is presented here in order to facilitate more genomic level analysis of de-novo sea urchin systems.

The purpose of this application, under Article 23.9.3 of the Code, is to conserve the widely used generic name StenonasterLambert, 1922 (Echinoidea, Stenonasteridae). The name Stenonaster is threatened by its senior objective synonym Stenocorys Lambert, 1917, that was erroneously considered a junior homonym of StenocorisBurmeister, 1839 (Arthropoda, Hemiptera, Alydidae) and StenocorisRambur, 1839 (= ParomiusFieber, 1861; Arthropoda, Hemiptera, Rhyparochromidae). Strict application of the Code would result in unnecessary confusion since the name Stenonaster has been explicitly used in the geological and palaeontological literature for the last 100 years whereas its senior objective synonym Stenocorys has not been used since it was (erroneously) replaced by Stenonaster. Stenonaster is the type-genus of the family Stenonasteridae, an enigmatic, but important missing link between the two major groups of atelostomate echinoids: Holasteroida and Spatangoida. Reverting the genus name to its senior synonym would necessitate changing the name of this family, which would lead to nomenclatural instability and confusion in the literature. We therefore request a reversal of precedence under the Commission's plenary power, with Stenocorys and Stenocoridae, the senior objective synonyms being suppressed and Stenonaster and Stenonasteridae given precedence.

BACKGROUND:Echinoidea is a clade of marine animals including sea urchins, heart urchins, sand dollars and sea biscuits. Found in benthic habitats across all latitudes, echinoids are key components of marine communities such as coral reefs and kelp forests. A little over 1000 species inhabit the oceans today, a diversity that traces its roots back at least to the Permian. Although much effort has been devoted to elucidating the echinoid tree of life using a variety of morphological data, molecular attempts have relied on only a handful of genes. Both of these approaches have had limited success at resolving the deepest nodes of the tree, and their disagreement over the positions of a number of clades remains unresolved.RESULTS:We performed de novo sequencing and assembly of 17 transcriptomes to complement available genomic resources of sea urchins and produce the first phylogenomic analysis of the clade. Multiple methods of probabilistic inference recovered identical topologies, with virtually all nodes showing maximum support. In contrast, the coalescent-based method ASTRAL-II resolved one node differently, a result apparently driven by gene tree error induced by evolutionary rate heterogeneity. Regardless of the method employed, our phylogenetic structure deviates from the currently accepted classification of echinoids, with neither Acroechinoidea (all euechinoids except echinothurioids), nor Clypeasteroida (sand dollars and sea biscuits) being monophyletic as currently defined. We show that phylogenetic signal for novel resolutions of these lineages is strong and distributed throughout the genome, and fail to recover systematic biases as drivers of our results.CONCLUSIONS:Our investigation substantially augments the molecular resources available for sea urchins, providing the first transcriptomes for many of its main lineages. Using this expanded genomic dataset, we resolve the position of several clades in agreement with early molecular analyses but in disagreement with morphological data. Our efforts settle multiple phylogenetic uncertainties, including the position of the enigmatic deep-sea echinothurioids and the identity of the sister clade to sand dollars. We offer a detailed assessment of evolutionary scenarios that could reconcile our findings with morphological evidence, opening up new lines of research into the development and evolutionary history of this ancient clade.

Vicariant events have been widely used to calibrate rates of molecular evolution, the completion of the Central American Isthmus more extensively than any other. Recent studies have claimed that rather than the generally accepted date of ~3 million years ago (Ma), the Isthmus was effectively complete by the middle Miocene, 13 Ma. We present a fossil calibrated phylogeny of the new world sand dollar genus Encope, based on one nuclear and four mitochondrial genes, calibrated with fossils at multiple nodes. Present day distributions of Encope are likely the result of multiple range contractions and extinction events. Most species are now endemic to a single region, but one widely distributed species in each ocean is composed of morphotypes previously described as separate species. The most recent separation between eastern Pacific and Caribbean extant clades occurred at 4.90 Ma, indicating that the Isthmus of Panama allowed genetic exchange until the Pliocene. The rate of evolution of mitochondrial genes in Encope has been ten times slower than in the closely related genera Mellita and Lanthonia. This large difference in rates suggests that splits between eastern Pacific and Caribbean biota, dated on the assumption of a “universal” mitochondrial DNA clock are not valid.

The formation of the Isthmus of Panama stands as one of the greatest natural events of the Cenozoic, driving profound biotic transformations on land and in the oceans. Some recent studies suggest that the Isthmus formed many millions of years earlier than the widely recognized age of approximately 3 million years ago (Ma), a result that if true would revolutionize our understanding of environmental, ecological, and evolutionary change across the Americas. To bring clarity to the question of when the Isthmus of Panama formed, we provide an exhaustive review and reanalysis of geological, paleontological, and molecular records. These independent lines of evidence converge upon a cohesive narrative of gradually emerging land and constricting seaways, with formation of the Isthmus of Panama sensu stricto around 2.8 Ma. The evidence used to support an older isthmus is inconclusive, and we caution against the uncritical acceptance of an isthmus before the Pliocene.

Lanthonia gen. nov. Coppard 2016 is a genus of clypeasteroid sand dollar whose members inhabit shallow, sandy waters from Mexico (including the Gulf of California) to Colombia in the tropical and subtropical eastern Pacific. Lanthonia includes Lanthonia longifissa (Michelin, 1858) and Lanthonia grantii (Mortensen, 1948), with L. longifissa hereby designated as the type species. Both L. longifissa and L. grantii were previously placed in the genus Mellita (L. Agassiz, 1841). However, levels of genetic divergence between a lineage containing L. longifissa and L. grantii and a lineage containing all other species of Mellita, including the type species M. quinquiesperforata (Leske, 1778), indicate genus level differentiation. The systematic interpretation of this group also supports the designation of this new genus as it allows the tree topology to be recovered from the nomenclature and clarifies the historical biogeography of these clades. This has resulted in members of both lineages today being sympatric in the eastern Pacific. Members of Lanthonia are morphologically differentiated from the type species of Mellita and all Pacific Mellita in having very narrow ambulacral regions between the food grooves and the ambulacral lunules on the oral surface, these being very broad in both M. quinquiesperforata and M. notabilis. The dentation of the bidentate pedicellariae also differentiate these genera, with small peripheral teeth present along the edge of the blade in species of Lanthonia and one to three enlarged intersecting teeth present distally in all species of Mellita.

Many free-spawning marine invertebrates, such as sea urchins, lack any courtship or assortative mating behavior. Mate recognition in such cases occur at the gametic level, and molecules present on the sperm and egg are major determinants of species-specific fertilization. These molecules must also coevolve in relation to each other in order to preserve functional integrity. When sea urchins release their gametes in seawater, diffusible molecules from the egg, termed sperm-activating peptides, activate and attract the sperm to swim toward the egg, initiating a series of interactions between the gametes. Although the compositions and diversity of such sperm-activating peptides have been characterized in a variety of sea urchins, little is known about the evolution of their genes. Here we characterize the genes encoding the sperm-activating peptide of the egg (speract) and its receptor on the sperm, and examine their evolutionary dynamics in the sea urchin genus Diadema, in the interest of determining whether they are involved in reproductive isolation between the species. We found evidence of purifying selection on several codon sites in both molecules and of selectively neutral evolution in others. The diffusible speract peptide that activates sperm is invariant across species, indicating that Diadema egg peptides do not discriminate between con- and hetero-specific sperm at this stage of the process. Speract and its receptor do not contribute to reproductive isolation in Diadema.

Sand dollars of the genus Mellita are members of the sandy shallow-water fauna. The genus ranges in tropical and subtropical regions on the two coasts of the Americas. To reconstruct the phylogeography of the genus we sequenced parts of the mitochondrial cytochrome oxidase I and of 16S rRNA as well as part of the nuclear 28S rRNA gene from a total of 185 specimens of all ten described morphospecies from 31 localities. Our analyses revealed the presence of eleven species, including six cryptic species. Sequences of five morphospecies do not constitute monophyletic molecular units and thus probably represent ecophenotypic variants. The fossil-calibrated phylogeny showed that the ancestor of Mellita diverged into a Pacific lineage and an Atlantic+Pacific lineage close to the Miocene/Pliocene boundary. Atlantic M. tenuis, M. quinquiesperforata and two undescribed species of Mellita have non-overlapping distributions. Pacific Mellita consist of two highly divergent lineages that became established at different times, resulting in sympatric M. longifissa and M. notabilis. Judged by modern day ranges, not all divergence in this genus conforms to an allopatric speciation model. Only the separation of M. quinquiesperforata from M. notabilis is clearly due to vicariance as the result of the completion of the Isthmus of Panama. The molecular phylogeny calibrated on fossil evidence estimated this event as having occurred ~3 Ma, thus providing evidence that, contrary to a recent proposal, the central American Isthmus was not completed until this date.

Diadenia africanum sp. nov. Rodríguez et al. 2013 occurs in the eastern Atlantic Ocean at depths of 1-80 meters off Ma- deira Islands, Salvage Islands, Canary Islands, Cape Verde Islands, Sâo Tome Islands and at the continental coast off Sen- egal and Ghana. This species was previously considered an eastern Atlantic population of D. antillarum. Genetic distances between the holotype of D. africanum and the neotype of D. antillarun herein designated, measured 3.34% in Cytochrome oxidase I, 3.80% in ATPase-8 and 2.31% in ATPase-6. Such divergence is similar to that already highlighted between other accepted species of Diadena. Morphometric analysis of test, spine and pedicellarial characters also separated D. africanum from D. antillartn and reveals that this new species is morphologically similar to D. antillarum ascensionis from the mid Atlantic. The tridentate pedicellariae, which have been shown to have diagnostic characters which discriminate among species of Diadema, occur as both broad and narrow valved forms in D. antillarumn from the western Atlantic. In D. africanum the tridentate pedicellariae occur only as a single form which is characterized by moderately broad and curved valves, with an expanded distal gripping region. This form of tridentate pedicellaria is very similar to that of D. antillarum ascensionis from the central Atlantic, with only slight variations in valve serration and valve curvature differ- entiating the two forms.

Coppard, S.E., Kroh, A. and Smith, A.B. (2010). The evolution of pedicellariae in echinoids: an arms race against pests and parasites. —Acta Zoologica (Stockholm) 00:1–24. Sea urchins (Echinoidea) have evolved a diverse array of jawed appendages termed pedicellariae to deter pests and predators. Pedicellarial structure and function are reviewed and their distribution mapped in 75 extant genera. Using a phylogeny of echinoids at family level constructed from 353 skeletal characters scored across 162 extant and fossil taxa, the evolution of pedicellarial form and function is reconstructed. For much of the Palaeozoic echinoids possessed a very restricted pedicellarial armament. By the early Mesozoic a diverse array of pedicellarial types had become established, implying that the threat from predators and pests markedly increased at this time. Since the Triassic, echinoids have continued to improve their defensive capability by evolving more effective venom delivery in globiferous pedicellariae, developing spatulate-tips and curved blades for a more efficient grab in tridentate pedicellariae, and stouter, more robust valves with a stronger bite in ophicephalous pedicellariae to disable and remove ectoparasites. However, pedicellarial types are shown to be particularly prone to subsequent secondary loss, especially among infaunal echinoids, and thus have higher homoplasy levels than other phylogenetically useful skeletal structures.

Information on biodiversity and community structure is vital for monitoring the effects of climate change and other anthropogenic impacts. Benthic ecosystems of 5 sites off Viti Levu (Fiji), comprising 50 stations were sampled quantitatively revealing 13 128 individuals of 230 species at a mean density of 273.5 ind. m(-2). Common taxa included polychaetes (89 species), crustaceans (84 species), molluscs (50 species) and echinoderms (7 species). No species occurred in all 50 stations; the maximum distribution range was 45 stations occupied by the polychaete Aglaophamus sp. A total of 81 species (35.2 %) were restricted to single sites ('uniques'), highlighting spot endemism. Species richness and rarefaction curves provided high estimates of diversity. Multivariate analyses incorporating biological abundances and environmental factors showed 3 distinct clusters among sites characterising differences in benthic community structure. Strongest determinants of faunal distribution were depth, distance from reef and river, and sand content. The presence of heterogeneous faunal assemblages suggests the interplay of these factors at each site. Fauna in Nadi Bay (Shannon-Weiner diversity index H': 3.26), Suva Harbour (H': 3.19) and Laucala Bay Lagoon (I-F: 3.06) had high diversity indicative of biologically accommodated communities. Rewa River Estuary (H': 2.42) and Nukubuco Reef drop-off (H': 2,48) had low diversities, typical of habitats subjected to fluctuating environmental conditions. Benthic community structure in the lagoons around Viti Levu was rich and diverse. Biodiversity was greater than previously recorded from the Great Astrolabe Reef, Fiji (207 to 211 species) and Australia's Great Barrier Reef (154 species), but lower than in New Caledonia (311 species) and Tahiti (315 species).