Monday, 1 June 2020

Tropical Storm Amanda kills at least eighteen in Central America.

At least eighteen people have died and several more are missing after Tropical Storm Amanda made landfall on the Pacific Coast of El Salvador on Sunday 31 May 2020, bringing with it over 500 mm of rain in twenty four hours. The majority of the fatalities occurred in El Salvador, where fifteen people are known to have died, including a family of three who died when their home was swept away by a landslide in San Juan Opico. Landslides are a common problem after severe weather events, as excess pore water pressure can overcome cohesion in soil and sediments, allowing them to flow like liquids. Approximately 90% of all landslides are caused by heavy rainfall. Another two people died in Guatemala, including a a nine-year-old-boy swept away by a flash flood and another person killed in a house collapse, and one person has lost their life in Honduras, which has also suffered a series of landslips and flood events.

Damage caused by Tropical Storm Amanda in San Salvador, the capital city of El Salvador, on 31 May 2020. Jose Cabezas/Reuters.

Tropical storms are caused by solar energy heating the air above the oceans, which causes the air to rise leading to an inrush of air. If this happens over a large enough area the inrushing air will start to circulate, as the rotation of the Earth causes the winds closer to the equator to move eastwards compared to those further away (the Coriolis Effect). This leads to tropical storms rotating clockwise in the southern hemisphere and anticlockwise in the northern hemisphere. These storms tend to grow in strength as they move across the ocean and lose it as they pass over land (this is not completely true: many tropical storms peter out without reaching land due to wider atmospheric patterns), since the land tends to absorb solar energy while the sea reflects it.

A landslide caused by Tropical Storm Amanda in El Salvador on 31 May 2020. Yuri Cortez/AFP.

Despite the obvious danger of winds of this speed, which can physically blow people, and other large objects, away as well as damaging buildings and uprooting trees, the real danger from these storms comes from the flooding they bring. Each drop millibar drop in air-pressure leads to an approximate 1 cm rise in sea level, with big tropical storms capable of causing a storm surge of several meters. This is always accompanied by heavy rainfall, since warm air over the ocean leads to evaporation of sea water, which is then carried with the storm. These combined often lead to catastrophic flooding in areas hit by tropical storms. 

Floodwaters on the River Los Esclavos in Guatemala on 31 May 2020. Moises Castillo/AP.

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Residents evacuated from their homes after landslide on the Isle of Sheppey.

Around 20 households have been evacuated following a landslide on the north coast of the Isle of Sheppey, on the north coast of Kent in southeast England, on Monday 1 June 2020. The event, a slump on an exposure of London Clay in the cliffs, is about fifteen metres wide and about two metres deep, and partially undermined one house, with the clear potential to extend further, leading the Kent Fire and Rescue Service to order the evacuation of twenty homes, with a further thirteen warned that they could be asked to leave if the slip spread further.

Aerial view of a landslide that partially undermined a house on the north coast of the Isle of Sheppey on Monday 1 June 2020. Kent Fire and Rescue Service.

Photographs of the landslide show excessive water pooled within the slumped material, this is fairly typical of landslides, as excess pore water pressure can overcome cohesion in soil and sediments, allowing them to flow like liquids. Approximately 90% of all landslides are caused by heavy rainfall. However, the north Kent coastal area has been suffering a prolonged bout of dry weather, making such an explanation for the event unlikely. Reports in the local press state that cracks were first seen on the cliffs on Friday 29 May, probably a result of dry cracking of the clay, and that locals had responded by pouring water onto the affected area in the hope of improving the cliffs cohesion, something which had reportedly worked in the past, but which seems highly likely to have triggered the landslip.

Wider angle view of the 1 June 2020 Sheppey landslip. SWNS.

The north coast of the Isle of Sheppey is popular with fossil collectors due to the large number of Eocene fossils it produces. The London Clay outcrops directly on the coast here, and as this is a poorly consolidated, soft sediment, it is easily eroded by the action of the sea, revealing large numbers of highly fossiliferous phosphate nodules, noted for the high quality plant macrofossils (particularly Mangrove Plants) and marine invertebrates (particularly articulated Decapod Crustaceans) that they produce. Unfortunately the same sediment makes conditions at the site extremely treacherous, as the clay readily absorbs large volumes of water, turning into a highly sticky mud that can cause cliff failures in the area, exposing new fossil material, but which can also  trap the unwary, as the weight of a human footfall is sufficient to squeeze the water out of the mud beneath the foot, creating a vacuum that prevents the foot from being lifted again.

A fossil Shark's tooth from the London Clay on the north coast of the Isle of Sheppey. UK Fossils.

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Searching for the origin of biomineralisation in Animals.

In modern ecosystems, Animal skeletons are hugely diverse in terms of morphology, ecology, function, and mineralogy, and are found across all the major divisions of Metazoa (Animals). Latest Ediacaran and early Cambrian rocks also preserve a staggering diversity of Animal skeletons, largely in the form of small shelly fossils. Although many remain enigmatic, they are increasingly being recognised as representatives of modern skeleton-building Animal groups. They also provide evidence of a huge ecological shift across the Ediacaran–Cambrian transition. In fact, the origin of biomineralisation is an integral component of the complex interaction of factors responsible for the explosion in Animal diversity. Developmental genetics, following the identification of deep homology in regulatory genes, provides an alternative, independent, record for unravelling early Animal evolution. Regulatory genes associated with a range of Bilaterian-specific developmental programs have homologues in Sponges and Cnidarians, and it seems that the vast majority of regulatory genes are not novel to specific Eumetazoan groups, but were pre-adapted in their unicellular ancestors. The discovery of this developmental ‘toolkit’ has generated hypotheses that can, in part, explain the rapid diversification of the Metazoa during the Cambrian. The establishment of this conserved toolkit is thought to have been followed by successive ‘canalisation’ since the Cambrian, that precluded the evolution of novel high-level morphological traits after the evolution of the majority of Animal phyla. First-appearance data from fossil evidence of Animal phyla and classes, coupled with molecular clock divergence dates, indicate that (with the exception of groups with very low preservation potential) all major phyla originate in the Ediacaran–Cambrian transition, and all Bilaterian classes are in place by the end of the Cambrian, with virtually no innovation at higher taxonomic levels in the subsequent 500 million years. One of the key innovations during the Cambrian Explosion is the widespread use of mineralised skeletons, and there is a growing body of evidence (reviewed herein) that a preadapted ‘biomineralization toolkit’ played a crucial role. This hypothesis makes a number of predictions of the fossil record of the earliest skeletons: (i) mineralised tissues should appear independently in different biomineralising lineages; (ii) biomineralised taxa should be pre-dated by soft-bodied representatives; (iii) the first skeletal tissues should show greater disparity than their descendants, being subject to looser biological control prior to canalisation. These predictions are here tested by compiling what is known of the earliest fossil examples of each of the major groups of biomineralising Animals, alongside evidence from the skeletogenesis of their extant counterparts.

In a paper published in the journal Biological Reviews on 23 May 2020, Duncan Murdock of the Oxford University Museum of Natural History, presents a review of the origin of biomineralisation across all Animal groups, combined with a time-calibrated consensus Animal phylogeny in order to better understand the divergence time betwen animal groups and origin of biomineralisation within the different groups.

The earliest evidence for widespread and relatively largescale (i.e. over 1 mm) mineralised skeletons with possible Animal affinity is around 550 million years old, in the terminal Ediacaran. This suite of largely tubular organisms dominate the fossil record of biomineralised organisms up until midway through the Fortunian, around 536 million years ago. The affinity of these (and the majority of) organisms of Ediacaran age remains controversial. However, when interpreted as metazoans, they are generally regarded as Cnidarian- or Poriferan-grade animals, although an alternative interpretation of some Cloudinomorph fossils has recently been proposed. The fossils of this period exhibit a range of mineralogies in their skeletons. Sinotubulites is testament to aragonitic biomineralisation in a complex organism in the terminal Ediacaran. Cloudina-type fossils may represent the earliest example of cnidarian biomineralisation, in the form of stacked cones composed of high-magnesium calcite. Many of these Ediacaran macrobiota exhibit microgranular or fibrous microstructures with a non-hierarchical organisation, suggestive of biomineralisation mechanisms under loose control. Further to this, a number of entirely organic contemporary counterparts to Ediacaran skeletal fossils have been described, including for Cloudina and Sinotubulites. These are reconstructed as representing the independent acquisition of biomineralised tissue utilising common calcification processes and pre-existing organic scaffolds.

Microstructural diversity in the first biomineralising animals. (A) The ‘tube-in-tube’ structure of Sinotubulites in longitudinal section exemplified at point ‘a’, arrow at ‘b’ indicates point of fracture. (B) Transverse sections through several Cloudina showing ‘funnel-in-funnel’ structure and calcite joining neighbouring individuals indicated by arrows. (C) Tripartite organisation of the skeleton of Namacalathus, columnar microlamellar inflections indicated by arrow. (D) Anabarites with close-up of apical end showing fibrous microtexture, at the point indicated by the arrow. Scale bars: (A) 800 μm; (B) 10 mm; (C) 100 μm; (D) 500 μm. Murdock (2020).

Anabaritids have their origin in the terminal Ediacaran, and are one of the most abundant components of the earliest small shelly faunas. These triradially symmetrical tubes made up of growth lamellae consisting of bundles of fibres re interpreted to have been originally aragonitic. Their affinity remains enigmatic, but they are usually regarded as closely related to Cnidarians. Their fibrous aragonitic microstructure is consistent with a pattern of early loose control over biomineralisation, but without a better constrained phylogenetic hypothesis it is difficult to establish a pattern. Similarly, first appearing through the earliest three stages of the Cambrian, the aragonitic Coleoloida and phosphatic Hyolithemintida and Byroniida, are tubular problematica with possible Cnidarian affinity. These groups would increase the diversity of modes of biomineralisation in the early evolution of the Cnidarian skeleton. The earliest putative Anthozoans (Corals) are a suite of fossils known as ‘Coralomorphs’ including the Khasaktiidae (high-magnesium calcite),the Hydroconozoa (low-magnesium calcite) and the Tabulaconida, with both aragonitic and high-magnesium calcite. The ‘Coralomorphs’ are not a natural group, but a grade of taxa best regarded as closely related to Corals. Medusozoans (‘Jellyfish’) are in most cases entirely soft-bodied, however, the phosphatic Conulariids are generally regarded as an extinct group of Scyphozoans, with possible origins in the latest Ediacaran.

Recent evidence supports a Poriferan affinity for the Ediacaran skeletal macrofossil Namapoikia, with a likely originally aragonitic skeleton formed by the calcification of a pre-existing organic framework comparable to the extant calcifying Demosponge Vaceletia, although it has also been interpreted as a calcifying microbe. If the sponge-affinity of Namapoikia is supported, it may represent the earliest evidence of co-option of a biocalcification toolkit inherited from the last common ancestor of metazoans for building skeletons.

Alternative hypothesis for the origin of the Poriferan skeleton (A), (D) with examples of early Sponge skeletons (B), (C), (E), (F). (A) A single origin of bimineralic spicules (exemplified by those of Lenica) with subsequent reduction and modification in each of the extant Sponge groups. (D) Alternatively, there were multiple origins of calcareous and siliceous spicules, supported by the high diversity of spicule types in Cambrian Stages 2 and 3. This is reflected by a range of taxa with disparate skeletal forms near the base of the Porifera, encompassed by the dark ellipse. The relationships between extinct Sponge groups, and other taxa with proposed Sponge affinities is unclear; dashed lines reflect this uncertainty. (B) Eiffelia globosa, a likely stem-Calcarean. (C) Different modes of preservation in spicules of Lenica: external mould (em), pyrite steinkern (ps), removed external laminae (l) and aluminosilicate replacement (c). (E) The putative Ediacaran Sponge Namapoikia rietoogensis. (F) Cross section of the demineralised spicule of Vasispongia delicata showing inner core. Scale bars: (B) 2 mm; (C) 5 mm; (E) 50 mm; (F) 5 μm. Murdock (2020).

Stem, and possibly crown (an fossil thought to represent an organism more closely related to a living group than to any other living group, but not deecended from any the last common ancestor of all living members of that group is considered to be part of the 'stem group'; a fossil thought to bedecended from the last common ancestor of all living members of the group is part of the 'crown group'), representative of three of the extant Sponge classes are also found in the Cambrian, but the pattern of acquisition of Sponge skeletons is far from clear. Essentially the problem can be couched as either favouring convergence of spiculation in different lineages or plesiomorphy of the Poriferan skeleton. The fossil record is not decisive on either competing hypothesis, but there is palaeontological evidence for homology of the siliceous spicules of Demosponges and Hexactinellids. Furthermore, some early Sponge fossils possess a mosaic of characters seen in different extant groups, such as bimineralic sclerites of Lenica that may represent the primitive sponge skeleton; originally siliceous spicules with Hexactinellid-type morphology and Calcarean-type organic sheaths; and Eiffelia from the Burgess Shale that exhibits Calcarean and Hexactinellid-like symmetries and organic sheaths. Some authors argue these lines of evidence support bimineralic biomineralisation in the last common ancestor of modern Sponges, implying loss of silica in the Calcarea, and loss of calcite in the other classes. However, another analysis suggests a more parsimonious solution is convergent evolution of sheaths and Hexactine-like spicules, and that the early Sponge skeleton exhibited high initial disparity perhaps followed by increasing constraint and canalization, entirely consistent with the biomineralisation toolkit hypothesis. The recently described Vasispongia delicata, interpreted as a stem-Silicean or stem-Hexactinellid sponge, possessed spicules with an organic axial filament surrounded by a weakly silicified layer. In the preferred scenario for Sponge character evolution presented by Murdock, the spicules of Vasispongia delicata represent another example of early independent evolution of biomineralisation under weak biological control.

The enigmatic sac-like Chancelloriids (part of the ‘Coeloscleritophora’) are problematic because although their overall morphology, inferred mode of life, and growth are very Sponge-like, their sclerite microstructure is closely comparable to that of Halkieriids, Sachitids and Siphogonuchitids which have a Molluscan affinity. It has been argued that commonalities of microstructure are indicative of homology of the sclerites of Coeloscleritophorans; in order to reconcile this with their proposed affinities, their sclerites must be plesiomorphic for Eumetazoa, and Coeloscleritophora must be paraphyletic. Alternatively, the common features of the skeletons of Coeloscleritophorans are independently acquired but perhaps by co-option of the same molecular processes under similar selective pressures. This would predict the existence of a Chancelloriid with an entirely organic precursor skeleton. Intriguingly, such a ‘naked’ Chancelloriid was recently described, although its affinities are as yet not entirely resolved.

Two studies have described the molecular toolkit for biocalcification of the Demosponges Astrosclera willeyana and Vaceletia sp., finding deeply conserved genes with a key role in biomineralisation, which, in combination with the distribution of α-carbonic anhydrases across Metazoa, suggests a single ancestral α-carbonic anhydrase common to all Metazoans that was subsequently duplicated and diversified in separate lineages. Furthermore, an even more complex repertoire of α-carbonic anhydrasess has been found in two Calcarean Sponges, and reconstruct eight α-carbonic anhydrases in the last common ancestor of Porifera, with different and independent histories of duplication and loss in each Sponge lineage. This is further elaborated by the acquisition of spicule-type-specific genes in individual lineages, such as the Calcareous Sponge Sycon ciliatum, demonstrating the independent evolution of a skeleton in each lineage.

This evidence for a ‘biomineralization toolkit’ in the first Animals to biomineralise provides a mechanism, but what was the control on the timing of this evolutionary shift? One proposal is that initial calcification of skeletons by Animals in the terminal Ediacaran was a non-selective response triggered by environmental change, but by the early Cambrian increasing predation pressure drove an escalating defensive response, and tighter developmental control. This is supported by a shift into clastic environments and the decoupling of skeletal mineralogy from ambient seawater chemistry seen through the early Cambrian. Thus, a combination of pre-adaptation in the genomes of soft-bodied ancestors of biomineralising taxa, a changing environment and ecological escalation can explain the role of the skeleton in the Ediacaran–Cambrian transition.

Lophotrochozoan skeletons are hugely diverse, numerous and disparate in both extant taxa and throughout the Phanerozoic. Relationships between Spiralian phyla, i.e. Protostomes not included within Ecdysozoa, are not well resolved. Within Spiralia, mineralised skeletons are restricted to the Lophotrochozoa (sensu stricto), namely Bryozoans (or Ectoprocts), Brachiopods, Molluscs and Annelids; calcareous structures are also known from Nemerteans and Flatworms. The most robustly supported hypotheses for the relative relationships of these biomineralising phyla resolve Bryozoa as sister to a clade consisting of Annelids, Brachiopods and Molluscs (along with non-biomineralising Nemerteans and Phoronids), the Trochozoa, and is the phylogenetic framework used by Murdock. Nevertheless, a number of alternative hypotheses exist for the interrelationships of the Lophotrochozoan phyla, with different resulting implications for the evolution of mineralised skeletons. Although rarely recovered bymolecular phylogenetics, traditionally Bryozoans were included in a clade with Brachiopods, the Lophophorata, based on the presence of a horseshoe-shaped tentacular feeding apparatus. This could imply a common ancestry of Bryozoan skeletons and those of Brachiopods. Although they share several ultrastructural features, there are none to the exclusion of Molluscs. Furthermore, there is no evidence for such a common ancestor in the fossil record, despite several fossils of putative stem-Brachiopods. The relative relationships between Annelids, Molluscs and Brachiopods are not well resolved, but studies that place Molluscs as sister to Brachiopods (plus Phoronids) would be consistent with a single origin of external mineralised ‘shells’ within Trochozoa (and secondary loss in Phoronids). However, this topology is increasingly poorly supported, with a sister relationship between Annelids and Brachiopods recovered by a greater number of analyses. Furthermore, evidence from transcriptomic and proteomic studies comparing Brachiopod and Mollusc shell biomineralisation is revealing significant differences in the genetic machinery involved. A sister-group relationship between Annelids and Brachiopods presents an intriguing hypothesis for the homology of chaetae across both phyla. A chaetae-like structure is exactly what would be expected of the organic precursor of a biomineralised skeleton. Furthermore, there are structural similarities between Brachiopod chaetae and aculiferan Mollusc sclerites.

Summary of hypotheses for the origin of Lophotrochozoan skeletons. (A) A common organic skeleton underwent mineralisation in independent lineages numerous times during the Cambrian, with several subsequent instances of loss. In Brachiopods, calcitic skeletons likely derived from the organo-phosphatic skeleton in different groups of small shelly fossils. The fossil evidence is currently unable to distinguish between a single origin of an organophosphatic skeleton for Brachiopods and Phoronids with subsequent loss or change of mineral system in a number of groups or multiple origins. An ancestral chaetae-like structure may be the precursor organic skeleton for a putative clade containing Annelids and Brachiopods, all Trochozoa, or all biomineralising Lophotrochozoans. (B) The calcitic skeleton of the putative Aculiferan Mollusc Ocruranus showing lamello-fibrillar shell microstructure, example of complete valve inset. (C) Diversity of microstructure in the Tommotiids, phosphatic stem-Brachiopods. Virtual cross sections with renderings of entire valves inset: Eccentrotheca (top), Lapworthella (right), Micrina (bottom). Scale bars: (B) 100 μm; (C) 100 μm (top), 150 μm (right), 100 μm (bottom). Murdock (2020).

There have been a number of fossil candidates for the common ancestor of some, or all, of the Lophotrochozoan phyla that bear mineralised skeletons. The enigmatic Ediacaran organism Namacalathus exhibits features indicative of a possible Lophotrochozoan affinity. These include asexual budding in a Bilateral pattern and a skeleton with an organic-rich inner layer and a regular foliated ultrastructure with columnar microlamellar inflections, reminiscent of some Brachiopod and Bryozoan skeletons. The common features suggest a similarity of mechanism for biomineralization between Namacalathus and Lophophorate phyla, independently utilising a common genetic toolkit, given that the weight of evidence indicates that Brachiopod and Bryozoan skeletons are independently derived. Nevertheless, the evidence for a Lophophorate affinity of Namacalathus has been contested and has to remain equivocal given the uncertainty regarding the relationships between the extant taxa.

Aside from Namacalathus, perhaps the oldest fossil skeletons proposed to have Lophotrochozoan (sensu lato) affinity are those of the Protoconodont Protohertzina (Protoconodonts are no longer thought to be relate to true Conodonts), appearing in the Fortunian (the first stage of the Cambrian, roughly 541-529 million years ago). Similarities in morphology, apparatus arrangement and internal structure, suggest Protoconodonts represent the grasping spines of stem-Chaetognaths. The Chaetognatha has proved to be one of the most problematic Bilaterian phyla to resolve in the tree of life, but current consensus suggests they are Protostomes more closely allied with Spiralians than Ecdysozoans. Grasping spines of extant Chaetognaths are composed of chitin, whereas Protoconodont elements are formed of primary calcium phosphate. Without better resolution of the relationships between Chaetognaths and other biomineralising groups it is difficult to explore this further, but the current evidence is consistent with Protoconodonts representing an independent origin of biomineralisation that was either lost in subsequent lineages or was restricted to a nowextinct clade on the Chaetognath stem. 

Also among the small shelly fossils, along with rare examples preserving unmineralised and/or articulated skeletons, are the ‘Coeloscleritophorans’. Although now not regarded as a natural group, largely through the discovery of exceptionally preserved fossils, various members of the ‘Coeloscleritophora’ remain pertinent to Murdock's discussion. Sclerite-bearing Animals known from Cambrian lagerstätten, Halkieria, Wiwaxia, and Orthrozanclus, along with similar disarticulated sclerites (e.g. Siphogonuchitids), have been interpreted to have a close affinity to Molluscs, Brachiopods and Annelids. The most widely supported consensus supports a Molluscan affinity, with Wiwaxia recovered as a stem-Mollusc, suggesting a lack of a mineralised skeleton at the origin of crown-Mollusca, and support an independent Terreneuvian origin of biomineralisation in Conchiferans and Aculiferans, including Halkieriids and Siphogonuchitids as early biomineralized stem-Aculiferans. Alternatively, a Brachiopod affinity for Halkieriids would shift an independent origin of the Aculiferan skeleton to the ‘palaeoloricates’ of the Furongian, but has no direct bearing on the presence or absence of a mineralised skeleton in the last common ancestor of crown-Molluscs. Similarly, alternative affinities of Wiwaxia as a stem-Annelid or sister to a clade consisting of Annelida plus Mollusca are all consistent with an unmineralised last common Trochozoan ancestor, and supportive of common ancestry of nonmineralised skeletal elements, such as sclerites and setae, being derived from serially repeated elements of an unmineralised scleritome.

Evolution of the conchiferan skeleton. (A) The first conchiferan molluscs exhibit various loosely constrained lamello-fibrillar fabrics. From an ancestral organic scaffold, different Mollusc groups first evolved this inner lamello-fibrillar mineralised layer in the Cambrian. These give rise in different groups, sometimes in different minerals, to laths and foliated textures, and ultimately nacre later in the lower Palaeozoic. Crucially, this represents a general trend in increasing control over biomineralisation in several lineages evolving in parallel. Uncertainty around the phylogenetic relationships of the extant classes, and affinities of some fossil taxa, leaves some ambiguity over the origin of the first lamello-fibrillar tissues. A recent analysis suggests a sister relationship for Gastropoda and Scaphopoda and a later origin of the clade (the starred lineage), in which case the fossil taxa represented would be stem to this clade with a single common origin of a complex shell microstructure. (B) Prismatic microstructure of Pojetaia and (C) lamello-fibrillar fabrics of Aldanella; examples of entire shells inset. Scale bars 100 μm. Murdock (2020).

Biomineralization is not a common feature of modern Annelids, being restricted to a few groups with much more recent origins. However, throughout much of the Palaeozoic, an extinct group of Annelids, the Machaeridians, bore a skeleton composed of calcitic shell plates that may represent another independent calcification of similar structures in a Lophotrochozoan clade. Similarly mineralised tubes of Vestimentiferan worms first occur much later in the Palaeozoic, with superficial similarities with various Ediacaran and Cambrian tubular fossils. An Annelid affinity for some Cloudinomorph fossils has also been supported recently by putative soft tissues interpreted as guts. However, given these Cloudinomorph fossils and Vestimentiferan tubes have complex, different taphonomic histories and are peculiar to restricted environments, a close affinity is not well supported. Nevertheless, should an Annelid affinity for Cloudinomorphs be verified, and given the known distribution of Annelid skeletons, they would best be interpreted as an independent origin of biomineralisation in the Annelid stem. 

Some Nemerteans have been observed to bear calcified stylets. They appear to be formed of amorphous calcium phosphate, likely via calcification of a pre-existing organic scaffold. The mechanisms of calcification in Nemerteans is not well understood, nor is the evolution of these structures. The position of Nemerteans as sister to Annelids has been recovered in some molecular analyses, but not widely, and has few morphological characters to support it. Nemertean stylets, therefore, may also represent an independent evolution of biomineralisation from a chaetae-like organic structure. However, without better resolution of the phylogenetic affinities of Nemerteans and/or a fossil record of mineralised \Nemertean stylets, this remains equivocal.

Despite their skeletons being independently acquired and having an entirely soft-bodied common ancestor, Molluscs, Brachiopods and, to a lesser extent Bryozoans, share some common skeletal features, including shell pores, an organic-rich shell, a secretory mantle, a periostracum, a complex shell differentiated into layers, and similar shell microstructures. Comparison of Hox gene clusters in Brachiopods, Molluscs and Annelids supports the homology of chaetae across the Lophotrochozoa, and the homology of the chitin network associated with the shell fields of Brachiopods and Molluscs. However, different Hox genes are deployed in the shell fields of Brachiopods and Molluscs, suggesting they do not share an ancestral role in specification of the shell-forming epithelium. Expression patterns of engrailed in larvae of Molluscs and Brachiopods show it is involved in shell formation in both phyla, but there are no conserved non-coding sequences and a comparison of gene synteny shows significant differences in the organisation of their engrailed genes, suggesting independent co-option of engrailed for shell formation (rather than by common ancestry) in Brachiopods and Molluscs, at least.

Based on the distribution of skeletons in Molluscs and Brachiopods, and the identification of commonalities in chitin synthases and bone morphogenetic protein signalling, researchers have suggested three models for the evolution of common Molluscan and Brachiopod skeletons. Two models propose a common ancestor with a mineralised skeleton (either phosphatic or calcareous) with subsequent loss or modification in different lineages. Given the evidence for multiple origins of skeletal tissues in Molluscs, and the absence of such an ancestor in the fossil record, Murdock rejects both these models. The third model proposes a common ancestor from which both phyla inherited a chitinous scaffold, mineralised with different phases in different lineages. This is consistent with all the evidence reviewed by Murdock, and is consequently supported.

Although the deep relationships of the extant Mollusc groups are not fully resolved, and the position of the numerous fossil groups with proposed Molluscan affinity is equally equivocal, Molluscs are arguably the oldest Bilaterian Animals with a skeletal fossil record (with the possible exception of Chaetognaths). A division between those Molluscs that bear a conch or shell (Bivalves, Gastropods, Scaphopods, Cephalopods and Monoplacophorans), the Conchifera, and those that do not (Chitons and Aplacophorans), the Aculifera, is well supported. The origin of Conchifera and the divergence and diversification of its constituent classes is estimated to have taken place across a very short window of time in the very latest Ediacaran to earliest Cambrian, roughly coincident with the first appearance of their fossils. This, along with the diverse derived body plans of extant conchiferans, has made establishing their relationships challenging. Monoplacophora are generally regarded as sister to a clade containing the four other Conchiferan classes, but virtually all other possible topologies have been suggested. The fossil record is also ambiguous, with representatives of all five Conchiferan groups present by the end of the Fortunian, including stem and potentially crown taxa. They exhibit a range of mineralogies, including aragonite, high- and low-magnesium calcite and unknown or mixed calcareous forms. Among the earliest of these are the enigmatic ‘Helcionelliforms’ many of which cannot be confidently placed within any one group. Many Helcionelliforms, along with taxa for which there is better resolution of their affinity, exhibit various lamello-fibrillar (Canopoconus, stem-Monoplacophora; Aldanella stem-Gastropoda) and crossed-bladed aragonitic (Anabarella, Watsonella, stem-Bivalvia) microstructures indicative of loose control over biomineralisation. These gave rise in different groups, sometimes in different minerals, to laths and foliated textures: foliated aragonite in the stem-Bivalves Fordilla and Pojetaia and the paragastropod Yuwenia; a unique cross foliated aragonitic microstructure in the putative stem-Gastropod Pelagiella; and calcitic semi-nacre in the probable stem-Scaphopod Mellopegma. Ultimately nacre evolved independently in each Conchiferan group later in the lower Palaeozoic. The fossil evidence suggests that from an ancestral organic scaffold, different Mollusc groups first evolved an inner lamello-fibrillar mineralised layer in the Terreneuvian (541-521 million years ago), then foliated and prismatic layers, and ultimately multiple independent origins of nacre in different lineages, with a combination of crossed lamellar microstructures and nacre being the dominant fabrics from the Ordovician. Crucially, this represents a general trend in increasing control over biomineralisation in several lineages evolving in parallel. This hypothesis is compatible with the competing resolutions of their interrelationships and with the evidence from divergence time estimates which place the diversification of the Conchiferan classes shortly after the origin of crown-Conchifera. The fossil record suggests either entirely soft-bodied ancestors to each of the Conchiferan classes, or an inherited inner lamello-fibrillar calcified layer, independently elaborated in each lineage, perhaps represented by some Helcionelliform fossil taxa. However, a recent study recovered a sister relationship of Gastropoda and Scaphopoda and estimated a maximum divergence time of this clade to around the base of Cambrian Stage 3 (429.25–525.494 million years ago). This would exclude older fossils as stem-Gastropods (e.g. the earliest Paragastropods, Pelagiellids and Aldanellids) or stem-Scaphopods (e.g. Mellopegma), and implies the assembly of a complex multi-layered shell structure in the common ancestors of Gastropods and Scaphopods. More work needs to be done on the affinity of these fossils, and on biomineralisation in Scaphopods, to test this hypothesis.

The Aculiferan Molluscs today consist of the Polyplacophora (Chitons) which possess a shell composed of eight aragonitic valves, and the so-called Aplacophoran ‘Worms’ (the Aplacophora, which comprises the  Caudeofovates and Solenogastres) that lack a mineralised skeleton entirely. The fossil record, however, reveals that Aplacophoran ancestors bore similar (calcareous, possibly aragonitic) skeletons consisting of imbricated valves to those of Chitons. The ‘Palaeoloricates’ originated in the Furongian, and are reconstructed as a paraphyletic assemblage of stem-Aplacophorans. The Polyplacophora have their immediate origins in the ‘Multiplacophorans’ of the Ordovician. However, the Polyplacophoran skeleton may be present in the Fortunian; the small shelly fossils Ocruranus and Eohalobia are reconstructed as part of a multi-plated scleritome of a stem-Polyplacophoran. They possess a lamello-fibrillar fabric, similar to other Cambrian Molluscs among the small shelly fossils, and were likely originally aragonitic. The Paracarinachitidae and Cambroclavida are also Lophotrochozoans with a possible stem-Polyplacophoran affinity, although their scleritome reconstruction remains enigmatic. They bear a skeleton with an original aragonitic mineralogy and similar fibrous ultrastructures. Whether or not the last common ancestor of all Aculiferans had a mineralised skeleton, or if biomineralisation was independently derived in Paleoloricates and Polyplacophorans, remains unclear. However, the Siphogonuchitids and Halkieriids have been proposed as stem-Aculiferans and possess mineralised (aragonitic) sclerites with some unique microstructural features consisting of an outer organic layer and inner layer comprised of aragonitic fibres. Although the affinity of Siphogonuchitids and Halkieriids is still under debate, if they do represent the plesiomorphic Aculiferan skeleton, they may reveal an initially less well-constrained mode of shell secretion, followed by a lamello-fibrillar fabric evident in Ocruranus/Eohalobia, and ultimately the condition seen in modern Polyplacophorans.

There is a great deal of evidence for the biomineralisation toolkit in modern Molluscan skeletons. One group of researchers compared the skeleton-building gene sets of the Abalone (Haliotis asinina) and the Pearl Oyster (Pinctada maxima), two Molluscs that share a common ancestor in deep time, along with those expressed in embryonic skeletogenesis of the Purple Sea Urchin (Strongylocentrotus purpuratus). They found very few commonalities in the transcriptome of the two Molluscs, suggesting they independently evolved nacre since they diverged in the latest Pre-Cambrian. Critically, many of the genes shared by the Abalone and the Pearl Oyster are also common to the Sea Urchin. This suggests that genes present in the (entirely soft) common ancestor of all Bilaterians were exapted into a role of building skeletons. The independent origin of genes encoding mantle secretory proteins in Gastropods and Bivalves was further supported by subsequent studies and the common role of repetitive low-complexity domains in Molluscan biomineralisation is now well established use comparative transcriptomics to confirm different origins of biomineralisation-related genes in Molluscs, Brachiopods and Vertebrates. Most of these genes can be found in both the Brachiopod Lingula and Humans, suggesting that they have general functions other than shell formation. The 30 genes shared by all selected genomes are mainly involved in cellular and metabolic processes and with other diverse functions not limited to biomineralisation, suggesting that these genes may have been co-opted independently in each Mollusc lineage. There is also new evidence from whole-genome and transcriptomic data for a much more recent co-option of the biomineralisation toolkit in Molluscs. The Scaly-foot Snail (Chrysomallon squamiferum) bears a complex skeleton of shells and plates reminiscent of certain Cambrian fossil taxa, but evolved in the Cenozoic. It was found that the transcription factors in the scale-secreting mantle differ to those of the shell-secreting mantle and are common not only to relatively distantly related Molluscs (e.g. in Aculiferan mantle) but also to a wider sample of Lophotrochozoans (e.g. in Brachiopod shells). This implies an ancient origin for these transcription factors as part of a biomineralisation toolkit. Finally, the biomineralisation toolkit hypothesis predicts a pre-existing organic skeleton, for which there is good evidence in Molluscs, chitin being a basic component of the nacre matrix.

Brachiopods comprise three clades, Linguliformea, Craniformea, and Rhynchonelliformea, which likely diverged prior to the advent of mineralisation in the Brachiopod skeleton. If we examine the fossil record of Brachiopod skeletons, there is growing evidence that each of the major Brachiopod clades, along with their likely sister group the Phoronids, can trace their origins to phosphatic microfossils in Cambrian Stages 2 and 3. The fossil group most closely associated with the origin of Linguliform Brachiopods are the Tommotiids. Although the validity of some of the characters used to ally Tommotiids with Brachiopods has been challenged, and their precise relationships remain unresolved, the microstructural evidence for this affinity is robust. The phosphatic microfossils Salanygolina and Fengzuella have been proposed as stem-Rhychonelliform and stem-Craniiform Brachiopods, respectively, based on clear similarities in morphology and microstructure or shared characters. Given the predominance of low-magnesium calcite in these groups, this suggests the calcareous Brachiopods independently acquired calcite skeletons. Furthermore, given the lack of evidence for a stem-Brachiopod with a skeleton, even phosphatic biomineralisation could be independent in each group. The Linguliform Brachiopod Eoobolus, known from Cambrian Series 2 (521-509 million years ago), with a bimineralic shell structure incorporating calcium carbonate and calcium phosphate, gives an indication of how this transition may have occurred. Nevertheless, more work needs to be done to establish the relationships between the extant clades and their fossil relatives. Furthermore, the taxa inferred to occupy these stem positions often exhibit similar microtextures within an organic-rich shell plus some considerable variation within and between individual sclerites. This is suggestive of loose control over biomineralisation.

Phoronids are the closest living relatives of Brachiopods, and have even been suggested as the sister group to Linguliforms (i.e. within a paraphyletic ‘Brachiopoda’ or ‘Brachiozoa’) although the weight of molecular evidence supports Brachiopod monophyly. The phoronid Phoronis possesses far fewer chitin synthase genes, known to play a key role in biomineralisation, than the Brachiopod Lingula, including all of those with close orthologues in Molluscs, therefore, attribute this to loss of these genes in the Phoronid lineage. This is consistent with the hypothesis that crown-Phoronids derive from a biomineralising ancestor and with the reconstruction of Eccentrotheca as a stem-Phoronid. The suite of shell-matrix proteins in the organophosphatic Lingula anatina (Linguliformea) are almost entirely different from those of the calcitic Magellania venosa (Rhynchonelliformea), with only five in common; which are all also found in the non-mineralizing Phoronis australis (Phoronida) and in other Metazoans with functions not related to biomineralisation. This is suggestive that, lineage-specific gene expansions, acquisition of novel genes and redeployment of extra-cellular matrix genes are involved in the evolution of Lophophorate biomineralisation. This supports multiple independent origins of mineralised skeletons in Brachiopods, as in Molluscs.

Biomineralisation is not as widespread in extant Ecdysozoans as in the other major divisions of the Bilaterians, the calcified exoskeleton of several groups of Crustaceans being a notable exception. Crustacean skeletons are also well represented in the fossil record, with stem-Crustaceans bearing low-magnesium calcite skeletons known from Miaolingian (509 to 497 million years ago) and Furongian ‘Orsten’-type deposits. However, these are pre-dated by phosphatic groups (Bradoriids and Phosphatocopids) appearing in Stages 2 and 3, respectively, that have been suggested to have Crustacean affinity. Further adding to the diversity of Arthropod mineralised skeletons in the Cambrian are other phosphatic extinct taxa such as the Aglaspidids and Phytophilaspis that may be more closely allied to Chelicerates. The prevalence of both calcite and apatite as Crustacean biominerals is not restricted to the Cambrian, with dual mineralisation documented in a range of Crustacean mandibles.

Perhaps the best known of all extinct Arthropod groups, however, are the Trilobites. Trilobite body fossils are first known from the beginning of Cambrian Series 2; their skeletons predominantly composed of low-magnesium calcite, but with some phosphatic examples, appearing relatively abruptly with high diversity, disparity, and established provincialism. A recent study has estimated that the true origin of trilobites pre-dates their fossil record, roughly coincident with the first Rusophycus-like trace fossils in the Fortunian, to which Trilobites are often attributed as likely trace-makers. This Terreneuvian Trilobite gap could be indicative of a lack of biomineralisation in the earliest Trilobites, with an independent and synchronous origin of calcareous skeletons across multiple Trilobite lineages. Trilobites also provide evidence for canalisation of skeletal characters, a study of polymorphism in Palaeozoic Trilobites shows a significant post- Cambrian reduction in morphological variability. There is, therefore, a consistent pattern in the fossil record of several Arthropod groups independently deriving calcareous and phosphatic biomineralisation.

Examples of biomineralization in Cambrian Ecdysozoans and Deuterostomes. (A) Partial cephalon of one of the earliest Trilobites, Profallotaspis. (B) Dorsal view and cross section of mineralised cuticular plates of the Palaeoscolecid Hadimopanella. (C) Isolated Cambrian Series 2 Echinoderm sclerite with characteristic stereom. (D) Virtual cross section of the early Euconodont Proconodontus. Scale bars: (A) 2.5 mm; (B) 5 mm; (C) 200 μm; (D) 100 μm. Murdock (2020).

In addition to Arthropods, a host of other phosphatic microfossils believed to have Ecdysozoan affinity appear in Cambrian Stages 2 and 3, some of which are also known from articulated or exceptionally preserved specimens. A number of Lobopodian taxa bear phosphatic plates or spines. The lack of consensus over the affinity of the representatives in this grade of Panarthropod taxa makes it difficult to assess the distribution of skeletal characters, nevertheless multiple independent origins (and subsequent losses) of phosphatic mineralisation in stem-Onychophorans (e.g. Microdictyon and Collinsium), and stem-Tardigrades (e.g. Onychodictyon ferox) is supported by recent phylogenetic reconstructions. The Palaeoscolecids are Cycloneuralian Worms bearing small phosphatic plates proposed as stem-Priapulids, but which have been allied to other groups such as Nematomorphs. As with Lobopodians, and given the distribution of soft-bodied-sister-groups, biomineralising Palaeoscolecids most likely represent another example of an independent foray into hard skeletons in the early diversification of Animals.

In Deuterostomes, the phosphatic Vertebrate skeleton and the calcitic Echinoderm skeleton (intercalated by the soft-bodied Cephalochordates, Tunicates and Hemichordates) share cellular and molecular processes in their early skeletogenesis, yet the protein-coding genes essential to downstream biomineralisation processes were acquired largely independently in both lineages. These observations support the hypothesis that there may have been a set of pre-adapted genes with elements that were independently utilised many times in the evolution of the skeleton, a ‘biomineralisation toolkit’. The Vertebrate endoskeleton is also highly likely to have evolved via the calcification of an organic scaffold. This hypothesis has been strengthened by the presence of a collagenous skeleton in the Cephalochordate Branchiostoma floridae. The generally accepted model for the origin of the Vertebrate skeleton is that initial Vertebratetissue mineralisation utilised collagen as a scaffold and specific protein acidic and cysteine rich gene as a critical mediator of mineral crystallisation. Differentiation into different fibrillar collagens and a greater diversity of skeletal tissues followed a series of whole-genome or large segmental duplications, resulting in multiple independent origins of skeletal tissues. 

The fossil evidence broadly supports this, with independent modifications to the plesiomorphic skeleton in every major skeletonising Vertebrate lineage and multiple times in different lineages. The oldest Vertebrates with a mineralized skeleton are the Conodonts (including ‘Paraconodonts’ and ‘Euconodonts’); these otherwise entirely soft-bodied Jawless Fish possessed a set of phosphatic tooth-like elements, with an abundant and widespread fossil record. Paraconodonts, with a dentin-like skeleton, are first known from the Guzhangian (500.5-497 million years ago) followed by Euconodonts after the evolution of the Eonodont crown, an enamel-like tissue unique to Euconodonts. The first Conodont skeletal tissues likely evolved via the calicification of cyclostome-like organic mouthparts and are largely organic, with hard parts apparently under relatively loose biological control. Subsequently we see the evolution of increasing basal body complexity, and ultimately the origin of the Euconodont crown.

In parallel with vertebrates, Echinoderms are a major biomineralising phylum of Deuterostomes. Echinoderms have a near-synchronous global origin around 525 million years ago, with a good Cambrian and Ordovician record of both articulated and isolated skeletal elements. While the five modern Echinoderm body plans are present in the early Ordovician, the earliest (Cambrian) Echinoderms belong to a number of strange groups, with a greater plasticity of construction. The diagnostic structure of stereom, the calcium carbonate mineral that comprises the Echinoderm skeleton, is common to all five modern classes of Echinoderm, along with fossil representatives. However, the larval Echinoderm skeleton is not so widespread, being either lost in Asteroids and much reduced in Holothurians or independently evolved in Echinoids and Ophiuroids. Developmental evidence suggests that a common set of transcription factors are involved in adult skeletogenesis in all Echinoderms, and some used in the developing larval skeleton evolved at the base of the phylum. Clade-specific parts of the regulatory network may have evolved in Echinoids and Ophiuroids during the independent evolution of the larval skeleton. However, more recent evidence supports the loss of the Asteroid larval skeleton rather than independent origins in other groups. The rare aragonitic epidermal ossicles of Hemichordates also appear to be independently derived from other Deuterostome skeletons, with several unique biomineralization genes in the Enteropneust Saccoglossus kowalevskii, while still sharing carbonic anhydrase involved in biomineralisation with the Sea Urchin Strongylocentrotus purpuratus. Furthermore, the Tunicates (phylogenetically intermediate between Echinoderms and Vertebrates) share the same ancient gene regulatory networks, but do not produce widespread biominerals, with the calcareous spicules seen in extant Tunicates not known prior to the Upper Triassic, providing further support for independent co-option for biomineralisation in different Deuterostome clades.

Murdock compiled records of the first fossil appearances of extant biomineralising groups, at (predominantly) class level, for all Animal phyla. These are combined with example taxa from the Ediacaran–Ordovician fossil record that are interpreted as their closest extinct sister taxa, plus fossil taxa placed as sister to more inclusive clades, and selected additional, often enigmatic, taxa to cover the range of biomineralising Animals through this interval. These fossil ranges, along with the mineralogy of the skeletons they possessed, are mapped onto a consensus Animal phylogeny, then time-calibrated. Along with the extant and fossil ranges, minimum node age estimates were applied to 17 nodes representing high-level crown clades, except for crown-Brachiozoa, where the minimum node age was used. These were chosen to reflect a conservative but realistic minimum age for the nodes for which the fossil ranges are uninformative. No minimum node age was used for Porifera, owing to the lack of consensus around the age and relationships within and immediately outside this clade. These data were plotted against the International Commission on Stratigraphy timescale using the paleotree package in R, employing a minimum branch length of 0.5 million years.

The distribution of different mineral systems across biomineralising Animals (particularly if the fossil record is included) favours multiple independent origins of skeletal tissues with successive waves of first appearances of biomineralising groups throughout the latest Ediacaran and Cambrian. The timing of origin of biomineralising classes and, to a large extent, phyla is coincident with their first appearance in the fossil record (with fossils pre-dating the minimum estimates in some cases), while the minimum dates for the more inclusive clades pre-date the fossil record of Animal skeletons. This is precisely the pattern to be expected if biomineralised skeletons, and the increased preservation potential they afford, post-date the divergence of skeletonized clades, supporting the hypothesis that the common ancestor of deeply divergent biomineralising clades was unmineralized. Fossils representing aragonitic, calcitic (high- and low-magnesium), organophosphatic and siliceous taxa are all present by the end of Cambrian Stage 2, and with a range of interpreted ecologies. This rapid diversification of known skeletal fossils, as well as the fact that nonmineralising groups are intercalated with carbonate-,phosphate- and silica-mineralisers, does not support a single origin of the animal skeleton followed by modifications and radiations of mineral types, but instead also points to multiple independent origins of biomineralisation from descendants of a last common Metazoan ancestor that was entirely soft-bodied. Furthermore, this pattern of multiple origins of skeletal tissues extends within individual phyla (and in some cases, classes). For example: the first Cambrian Molluscs, Cnidarians and Sponges show a greater degree of diversity of mineral system than their descendent clades, and calcitic skeletons likely evolved multiple times within Brachiopods and Arthropods as well as diverse phosphatic skeletons that are otherwise underrepresented in modern assemblages.

The phylogenetic and temporal distribution of the first Animal skeletons. First appearance data for Animal phyla and classes, plotted on a consensus phylogeny, with some clades collapsed to reflect disagreement in the literature, plotted against the International Commission on Stratigraphy timescale. The bars represent stratigraphic ranges in stage level (or local stages for the Terreneuvian and Cambrian Series 2) time bins for the first appearances of their skeletons, colour-coded by their main mineralogy. Murdock (2020).

From the compiled data, Murdock subsampled to retain only the extant lineages, their fossil ranges, and minimum node estimates. Fossils were excluded owing to the biased nature of the fossil sampling method, i.e. choosing representative taxa from a restricted temporal window. The tree was time-calibrated, to assign branch lengths, and, along with the dominant mineralogy of the extant biomineralising representatives of the clade, was used to estimate the ancestral state of each node. This was achieved using the rerooting Method in the R package phytools, using an equal rates model. This maximum likelihood method estimates the marginal likelihood of each state of each character (in this case, biomineralisation) for each internal node in the tree. An equal rates model, essentially an Mk model, assumes an equal probability of changing between all pairs of states, across all branches in the tree. By using the character states at the tips, along with the branch lengths over which those states may change, the conditional probability of observing each character state, given the data, is first estimated for the root node. The tree is then progressively rerooted to estimate the marginal likelihoods for all internal nodes. The sum of the likelihoods for all possible states must equal one, thus the marginal likelihoods for each node are displayed as a pie chart. This methodology was chosen as it makes the fewest possible assumptions of character evolution, and can accommodate uncertainty of tree topology by randomly resolving polytomies with branches of zero length then matching nodes to derive the original topology.

Ancestral state reconstruction of composition of Animal skeletons. Nodes are colour-coded based on the marginal probability for each state at that node, estimated under a Mk model, represented by a pie chart. Colour-coding and topology are as above. Given the deep divergence times of these lineages, branch length is estimated from the fossil record of Animal skeletons. With the possible exception of the Porifera, the common ancestor of all phyla, and the higher taxa to which they belong, is reconstructed as entirely soft-bodied. Murdock (2020).

This analysis strongly supports soft-bodied common ancestors to virtually all extant biomineralising phyla and classes, which is consistent with the fossil and molecular evidence reviewed by Murdock. The exception are the Sponges, which are reconstructed as likely ancestrally sharing a siliceous skeleton. This is supported by some fossil evidence, however, uncertainties in the results shed doubt on this conclusion. The branch lengths in the tree are estimated based on the fossil data and minimum limits of posterior divergence times. Due to uncertainty around the deep interrelationships of the Poriferan classes, uncertainty of the relationship between Sponges and the rest of the Metazoa, and a lack of a robust fossil calibration, the limits for this node are wide with a minimum divergence in the Carboniferous. Therefore, the node age is derived from the oldest crown Sponge fossil included, which almost certainly represents a considerable underestimation of the true origin of the Poriferan crown. With more fossils resolved as stem taxa to the crown-poriferan classes, and/or less uncertainty in the divergence time estimates, there would be much longer branch length between the origin of crown-Porifera and the origin of its constituent classes, and therefore more time to accommodate the independent origin of Sponge skeletons in each class, rather than inheriting an ancestral siliceous skeleton.

The presence of a ‘metazoan toolkit’ of genes, and their associated networks of regulatory control and interactions, is now well established and its role in the origin of Animal body plans is becoming clearer. In the context of the evolution of Animal skeletons, can we establish which (if any) ancestral genomic regulatory elements were co-opted and if these are common to some, or all, biomineralising Animal lineages? In several cases gene sets and gene regulatory networks with a role in metabolism seem to have been co-opted for biomineralisation. One of the best-supported candidates for the ancestral repertoire of genes co-opted for biomineralisation is those involved in the calcium-regulated extracellular matrix system. Chitin synthase and bone morphogenetic protein signalling, along with calcium-binding and extracellular-matrix proteins have been found to play key roles in biomineralization in Molluscs, Vertebrates, and phosphatic Brachiopods. These observations are entirely compatible with the independent calcification of an inherited organic scaffold, utilising common metabolic pathways but employing different downstream biomineralisation-related genes. There is compelling evidence that chitin, and therefore chitin synthase genes, is a crucial component of such a scaffold. A chitin network forms at the onset of Brachiopod and Mollusc shell fields and has been found to be expressed in epithelial cells of Fish and Amphibians. Brachiopods and Molluscs have also possibly independently co-opted engrailed into shell formation Among the suite of 25 transcription factors are several involved in shell matrix protein production including pif, chitin-binding peritrophin-A domain gene, and chitin synthase. Carbonic anhydrases, which have a wide range of fundamental physiological roles, have also been shown to play a crucial role in biomineralisation in Calcareous Sponges and independently in different Mollusc lineages, also playing a role in calcification of organic precursors. In Vertebrates, an ancestral specific protein acidic and cysteine rich gene is thought to have given rise to a number of calcium-binding phosphoproteins via Vertebrate-specific gene duplication, with potential paralogs in Sea Urchin specific protein acidic and cysteine rich/osteonectin genes and even in some Lophotrochozoans. Cyclophilins, found to be integral to skeletogenic Sea Urchin primary mesenchyme cells, have similarities with proteins in Mollusc shell-secreting cells. Finally, some shell matrix proteins (hephaestin and hemicentin) of the Brachiopod Lingula are also found in the Coral skeletal organic matrix, but have a role in metabolism in other Metazoans. These lines of evidence all point towards independent co-option of similar gene sets involved in metabolism and, more specifically, calcium-regulationvia calcification of an organic scaffold. This would be consistent with a physiological response to changing seawater chemistry.

Intriguingly, the Echinoderm skeleton may provide an alternative model for co-option playing a role in the origin of Animal biomineralisation, with its origin in nutrient or blood collection and transport. A recent study identified five transcription factors and three signalling pathways involved in vascular endothelial growth factor signalling that are common to biomineralisation in Echinoderms and vascular tubulogenesis in Vertebrates. They find common cytoskeletal remodelling proteins essential for Echinoderm spicule formation and the formation of Vertebrate vascular tubes, structures with similar overall geometry but fundamentally different functions. This would represent a unique co-option of an ancestral tubulogenesis program for biomineralisation in Echinoderms. Although speculative, this may contribute to some of the unique properties of Echinoderm skeletal tissues, such as the remarkable conservation of the microstructure of stereom and the apparent singular origin of Echinoderm skeletons in contrast to multiple origins and progressive addition of complexity in virtually all other biomineralising phyla.

Mineralized tissues appear independently in different biomineralising Animal lineages. The numerous first appearances of skeletal tissues from the late Ediacaran to the early Ordovician are disparate in terms of mineralogy, phylogenetic distribution, functional ecology, and from across a range of time scales. This is also reflected in the modern distribution of biominerals suggestive of no common ancestry among skeletal taxa.

Biomineralised taxa are pre-dated by soft-bodied representatives. Although soft-bodied counterparts of biomineralising organisms are inherently less fossilisable, there is both direct and indirect evidence for them in the fossil record, such as in the terminal Ediacaran, a ‘naked’ Chancelloriid, and the apparent gap in the early Trilobite record filled with Rusophycus-type trace fossils.

The first skeletal tissues in Animals show greater disparity than their descendants, being subject to looser biological control prior to canalisation. There is a consistent pattern in the fossil record of early Animal skeletons of an organic scaffold inherited from a common ancestor, independent origins of similar laminar microstructures under loose control, followed by the evolution of more complex, more derived and increasingly entrenched microstructures. This is perhaps best documented in Cambrian Molluscs, but also apparent in, for example, Anabaritids, Namapoikia, bimineralic Sponge spicules, and Paraconodonts.

There is considerable evidence for the ‘biomineralisation toolkit’ hypothesis whereby a common set (or sets) of ancestral genes were independently co-opted for building skeletons in many animal groups at the same time. The skeleton evolved many times, both across and within phyla (and even classes). But biomineralising groups inherited an organic scaffold and a toolkit of genes from a common ancestor. Furthermore, there is a consistent pattern of increasing complexity and control in the early evolution of skeletons in different animal groups.

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Sunday, 31 May 2020

Soft-tissue preservation in Cloudinomorphs from the terminal Ediacaran Period of Nevada.

Commonly envisaged as a prelude to the Cambrian Explosion, the terminal interval of the Ediacaran Period (roughly 550–539 million years ago) chronicles several monumental events during the evolutionary dawn of Animal life. Among the most significant are the emergences of biomineralisation and active motility, which demarcate this interval from the rest of the Ediacaran Period. Toward the Period’s conclusion, the first Metazoan mass extinction event encompassed the downfall of the archetypal Ediacaran Biota. Their demise, however, was coincident with an ecological shift in which organisms such as Cloudina and other occupants of this novel tube-building morphotype become increasingly populous. Collectively, these 'Cloudinomorphs' (to avoid conflating unresolved phylogenetic relationships with shared morphologies) were small, sessile, and epibenthic, but they appeared with several key adaptations that may have enhanced their chances for ecological success. These attributes include: (i) the advent of macroscopic biomineralization in the form of shelly external tubes, potentially serving as an impediment to predation; (ii) the establishment of gregarious habits that may signal the onset of Metazoan ecosystem engineering behaviors; and (iii) the development of enhanced larval dispersal mechanisms, and presumably both sexual and asexual reproductive habits, versus stolon-like reproductive modes of some members of the enigmatic softbodied 'Ediacara Biota'. These compounded ecological innovations may have helped to place the Cloudinomorphs as central players in ushering in a phase of fundamental ecosystem reform and increased trophic complexity. Although its cause is equivocal at present, the changing of the ecological guard from largely sedentary Ediacara-type communities to much more dynamic syn-'Cambrian Explosion' ecosystems was well underway in the terminal Ediacaran. Indeed, as recently proposed, this interval possibly displays an even larger step-change in organismal and ecological complexity than at the Ediacaran–Cambrian boundary itself. Nonetheless, the most crucial task that remains is to untangle the potential relationships between the organisms of the Ediacaran Period and those well-defined as metazoans in the Cambrian Period. The Cloudinomorphs are one of the few groups known to span the Ediacaran–Cambrian boundary and thus understanding their phylogenetic position is key to unraveling the evolutionary and ecological relationships between the seemingly disparate biomes of the Ediacaran and Cambrian Periods.

In a paper published in the journal Nature Communications on 10 January 2020, James Schiffbauer and Tara Selly of the Department of Geological Sciences and X-ray Microanalysis Core at the University of Missouri, Sarah Jacquet, also of the Department of Geological Sciences at the University of Missouri, Rachel Merz of the Biology Department at Swarthmore College, Lyle Nelson of the Department of Earth and Planetary Sciences at Johns Hopkins University, Michael Strange of the Department of Geoscience at the University of Nevada, Las Vegas, Yaoping Cai of the Shaanxi Key Laboratory of Early Life and Environment, State Key Laboratory of Continental Dynamics, and Department of Geology at Northwest University, and Emily Smith, also of the Department of Earth and Planetary Sciences at Johns Hopkins University, provide a detailed report of internal soft-tissue preservation within Cloudinomorph fossils from the Wood Canyon Formation of Nye County, Nevada, and, moreover, one of the earliest reports of preserved internal anatomical structures in the fossil record. On the basis of the morphology and interpreted physiology of this soft-tissue structure, Schiffbauer et al. suggest that this feature holds significant potential to shed new light on the phylogenetic placement of the Cloudinomorphs.

Generalized stratigraphy of the Montgomery Mountains site. Ediacaran–Cambrian boundary denoted by the presence of Treptichnus pedum. Cloudinomorphs recovered from silty-shale below first dolostone marker bed in the lower member of the Wood Canyon Formation. Nye County indicated on map, with yellow star marking approximate sample locality. ZQ, Zabriskie Quartzite; Stirling Qtz, Stirling Quartzite. Schiffbaur et al. (2020).

The phylogenetic position of the Cloudinomorphs has yet remained unresolved, albeit not without effort. Previous attempts have used the only available information (to date) from the fossil record; their external tubes. Such features, specifically demonstrated by Cloudina, that have been employed to help constrain their phylogeny include (but are not limited to): (i) nested funnel-in-funnel tube construction; (ii) smooth inner tube wall lumen; (iii) presence of daughter-tube branching; iv) ovate tube cross-sections; (v) bulbous shape of the closed posterior bases; (vi) absence of basal attachment structures; (vii) calcareous composition in mineralized representatives; and (viii) microgranular tube wall ultrastructures. There are several caveats that should be considered, however. First and foremost, some of these features are not uniformly representative across all of the Cloudinomorphs, which should serve as a caution toward future attempts to resolve relationships within this morphotypic group. Moreover, at least some of these alleged diagnostic features (or lack thereof) may be taphonomic noise rather than primary biological signal. For instance, although a homogenous microgranular tube ultrastructure is commonly reported for Cloudina, lamellar construction has also been observed, raising the question as to the influence of diagenetic recrystallisation on retention of primary ultrastructure, or, for that matter, original composition. It thus follows that the degree of tube wall biomineralisation in addition to the original biomineral chemistry has been met with differing interpretations, likely compounded by varying preservational and diagenetic histories between localities. The absence of substrate attachment structures may be a consequence of displacement and transport during storm events, a common mode of deposition of Cloudinomorphs that yields fragmental tubes in detrital hash resembling biohermal or reefal buildups. Alternatively, if the attachment structures were originally soft tissue, they may have been taphonomically lost, in which case the absence of evidence should not be construed as evidence for absence. Ovate crosssections may result from compression of a modestly flexible tube during sediment compaction, which is almost certainly the case for tubes of some Cloudinomorph taxa that are interpreted to have been originally organic. As such, and as should be the case with all enigmatic fossils, attempts at phylogenetic assessment would be best suited to focus on taphonomically robust features or those that can be best determined to be biologically and taxonomically informative.

In conjunction with the influences of a complex and wide array of taphonomic histories, placement of the Cloudinomorphs is further confounded when we consider the diversity of modern tube-building organisms and their assumed convergence of tubedwelling habits. Most agree that the Cloudinomorphs are at least of 'lower; (phylogenetically earlier branching) Diploblastic Metazoan-grade organisation. However, differences in the value with which the aforementioned characteristics are weighted in comparison with polyphyletic modern tube-builders can yield a broad assortment of plausible affinities, ranging from Chlorophytes (Green Algae) to Triploblastic Metazoans (the group which includes all Animals other than Sponges, Ctenaphores, Cnidarians, Placazoans, and Flatworms). Although more antiquated interpretations have included presumably Poriferan-grade Archaeocyathids, recent discussion urged not to discount a Macroalgal affinity, owing to comparable annulated tubular morphologies observed in modern calcareous Dasyclad Algae. Extinct Microconchid Lophophorates have also been offered as a possible analog on the basis of tube structure and shape. Similarly, some PterobranchHemichordates produce dichotomous organic-walled tubes with reasonably comparable morphologies, and thus may also warrant consideration. Other authors have instead refused to wedge the Cloudinomorphs into any extant or extinct group, proposing otherwise that they occupy their own incertae sedis stem-metazoan family, the Cloudinidae. Satisfying the perceived majority of their exterior tube characteristics, however, most researchers currently fall into either Anthozoan Cnidarian or Polychaete Annelid camps, but further distinction has been hindered by the absence of preserved soft tissues.

The Wood Canyon fossil assemblage is dominated by Cloudinomorphic forms These fossils, as well as others from nearby units, have been taxonomically compared with the well-studied tubular fauna of the Gaojiashan Lagerstätte, South China and, more recently, to lesser-known Cloudinomorphs from the East European Platform. Systematic investigation of the Wood Canyon Cloudinomorph fossils has thus far formally described two new species, Saarina hagadorni and Costatubus bibendi, as the most abundant in this locality. Taphonomically, the Wood Canyon and Gaojiashan assemblages are highly comparable, with fossils from both units predominantly exhibiting three-dimensional pyritization. However, whereas the majority of  Cloudinomorph tubes from the Gaojiashan are completely pyritised (e.g., the full tube volume is filled by pyrite mineralisation), those from Nevada show pyritised external tube walls retaining three-dimensionality but without pervasive pyrite infilling. As a result, the Nevadan Cloudinomorphs offer a unique potential for capturing resolvable soft tissues, and x-ray tomographic microscopy provides an ideal method for non-invasive exploration of internal fossil features.

Wood Canyon Cloudinomorphs of the Montgomery Mountains site. (a) Holotype of Saarina hagadorni, sample USNM-E1636_009_B13. (b) Paratype of Saarina hagadorni, sample USNM-WCF_005_01. (c) Holotype of Costatubus bibendi, sample USNM-MS_DS_12. Samples reposited at the Smithsonian Institution. All scale bars are 1 mm. Schiffbaur et al. (2020).

Unlike some of the Cloudinomorphs that built more robust shelly tubes, the exterior tubes of the described Wood Canyon Cloudinomorphs are inferred to have been organic in original composition from indications of plastic deformation much like the Gaojiashan taxon Conotubus and East European representatives of Saarina. Generic and specific taxonomic identification of the Nevadan tubular fossils containing soft tissues is unfortunately muddied by a lack of substantive exterior tube detail, likely resulting from chemical limitation during preservation. The soft-tissue-bearing tubular fossils exhibit exterior tube diameters (approximately 2–4 mm) that generally fall within the observed range for the two described Wood Canyon Cloudinomorph genera (maximum diameters 3.92mm and 6.36mm for Saarina hagadorni and Costatubus bibendi, respectively), albeit greater than the median diameter for either genus (median diameters 0.74mm and 1.09 mm for Saarina hagadorni and Costatubus bibendi, respectively). Schiffbaur et al. interpret the annulation of the tubes observed both optically and by x-ray tomographic microscopy as a vestige of a 'funnel-in-funnel' tube construction, which supports the hypothesis of their Cloudinomorphic affinities.

Soft tissue-bearing Cloudinomorphs with schematic interpretation. 3D volume render from x-ray tomographic data shown in left image per frame (red-to-orange coloration indicates high density internal regions within exterior tube), with interpretive diagram in right image per frame. Examples here show (a) medial position and consistency (sample USNM-N1601_FL_018), (b) partial degradation/fragmentation (sample USNM-E1630_006), and (c) kinking and folding (sample USNM-N1601_FL_017). Soft tissue in sketches highlighted in red. Samples reposited at the Smithsonian Institution. All scale bars are 2 mm, sketches by Stacy Turpin Cheavens. Schiffbaur et al. (2020).

From three-dimensional reconstructions of x-ray tomographic data, internal structures were revealed within the external tubes from a smallsubset of the analysed specimens (roughly 11%; 4 of 35 analysed specimens), which Schiffbaur et al. interpret as preserved soft tissues. The soft-tissue feature manifests as a sub-millimetric to millimetric diameter, centrally positioned cylinder that largely follows the curvature of the sagittal external tube length. In three of four cases, the cylindrical feature is mostly continuous through nearly the full length of the external tube, and only fragmented taphonomically. One of these specimens shows significant kinking and sinuous bending of the internal cylinder relative to its external tube. The other specimen shows instead an incomplete internal cylinder broken at a fragmented section of the external tube and also assumed to be unpreserved at the apical/posterior end of the external tube. When viewing the x-ray tomographic data transversely to the tube length, the internal cylinder rests adjacent to the lower (with respect to bedding) internal surface of the tube wall.

3D reconstruction full movie of sample USNM-WCF_001. Movie progression: (1) sequential 2D tomograph slices moving through the host rock and fossil, bright regions indicate pyrite tube wall and gut. (2) Layered dissolve of host rock, revealing Avizo-segmented data. Red color = tube wall and disseminated pyrite. (3) Removal of disseminated and non-continuous pyrite around the tube wall structure. (4) Rotation and reveal of gut structure (gold/orange). Hints of tube wall funnel/transverse annulation structure can be observed during rotation. (5) Dissolution of tube wall for full gut reveal. Schiffbaur et al. (2020).

To better explore the transverse morphology and preservation of the internal cylinders, a portion of the fragmented specimen was selected for destructive preparation (via manual serial grinding) and subsequent scanning electron microscopic analyses. The sectioned soft-tissue cylinder was observed to be either infilled by sediment or fully mineralised, and verified to be pyritic in composition. The external tube was additionally confirmed to have been pyritised (mostly weathered to iron oxyhydroxides), within a fine-grained siliciclastic host rock matrix. In cross-sectional view, the external tube can be complete, but appears more robustly pyritised at the bottom edge, and tenuous at the upper edge, with respect to bedding. Where the interior tube directly abuts the exterior tube, the exterior tube may be very thin, but this appears to be a localized phenomenon and is not apparent in all of the x-ray tomographic- or scanning electron microscope-observed transverse cross-sections. The exterior tube shows marginal lateral compression. In portions where the internal cylinder is broadly sediment-filled, it displays ovate cross-sections comparable in shape to the compressed external tube. In some of these sediment-filled portions, pyrite does exist within the interior of the tube, potentially replicating an organic template. Portions of the internal cylinder that are fully mineralized, in contrast, show circular, uncompressed cross-sections. Where the internal cylinder is sediment-filled, pyrite mineralisation appears to extend both inward (towards the cylinder interior) and outward (into the lumen of the external tube) from a discernable cylinder wall.

Optical imaging and x-ray tomography of Cloudinomorph pyritised tube and soft tissue. (a) Light image of entire specimen (sample USNM-WCF_001) in planview, specimen partially obscured at rock surface. (b) Corresponding 3D volume render, showing soft tissue (orange) and tube wall (gray); boxes (d), (e) are marked in both (a), and (b) to help guide slight differences in orientation. (c) Close-up view of labeled box in (a), highlighting funnel rims (arrows) on external tube. (d) Close-up view of labeled boxes in (a), (b), 3D volume render showing partial soft tissue and funnel rims (arrows); (d) largely overlaps with (c), but includes also host rock encased portion of the fossil. (e) Partial soft tissue from labeled boxes in (a), (b), (f). Cross-sectional view of e showing relative position of soft tissue that has settled to the bottom of the external tube wall. Sample reposited at the Smithsonian Institution. All scale bars 2 mm. Schiffbaur et al. (2020).

Each case of soft-tissue preservation presents a balance between taphonomically constructive and destructive processes, wherein retention and replication of biological information necessitates that decay does not eradicate, and mineralisation does not overwrite, informative features. Impeding both decay and mineralization early in the taphonomic sequence of the Nevadan Cloudinomorphs created a 'goldilocks' scenario in which soft tissues may be distinguishably preserved, as opposed to their Gaojiashan contemporaries. Pyritisation proceeds because of a confluence of chemical and microbiological factors, including: (i) a limited source of organic material (usually the soft tissues of the deceased organism); (ii) focused degradation of that organic material by sulphate-reducing Bacteria; and (iii) anoxic pore waters rich in reduced iron along with available sulphate. While oxidizing the remnant organic material of the organism, sulphate-reducing Bacteria (in normal seawater pH) convert sulphate to bisulphide, which then serves as one of the building blocks of pyrite along with reduced iron as the other.

2D stacked tomograph, latitudinal cross-section slices through sample USNM-WCF_001. Movie progresses from upper part to lower part. Schiffbaur et al. (2020).

If any part of this process becomes chemically starved, fossil pyritisation will be halted. There are three paths that this can take, based on limitation of either organic matter, reduced iron, or sulphate. If Bacterial sulphate reduction proceeds uninhibited by sulphate availability, the organics of the decaying organism are likely to be entirely consumed. This process, limited only by the availability of organics, would leave no soft tissues to be preserved, and should result in authigenic, centripetal pyrite infilling. In the other two cases, pyritisation can cease relatively early in the taphonomic sequence once the burial environment becomes chemically limiting (assuming no replenishment). If the availability of reduced iron is limited, pyrite formation will discontinue, but further degradation of the organics by sulphate reducers could continue unrestricted. Where sulphate concentration is instead limited, decay by sulphate-reducing Bacteria would cease once the sulfate supply is expended. In turn, with no further generation of bisulphide, pyrite formation would be subsequently suspended once the available bisulphide is exhausted. Regardless which pathway is realized in the Wood Canyon burial environment, the necessary ingredient to preserve these soft tissues, and have them remain perceivable, is to terminate pyritisation before overgrowth can obscure or homogenise the features.

Cross-sectional morphology of preserved Cloudinomorph soft tissue. Cross-sections revealed by serial grinding of specimen USNM-WCF_001; portion of the fossil chosen for grinding shown. (a)–(e) Light and SEM images matched with approximately equivalent μCT tomographic slices (differences in obliquity imposed during serial grinding). Far right in a shows tube and gut pyritisation via EDS elemental mapping. (f) Position of slices (a)–(e) shown on μCT tomographic slice through the transverse plane. All scale bars 2 mm. Schiffbaur et al. (2020).

In the Gaojiashan, pyritisation likely proceeded uninhibited by sulphate or reduced iron. Thus, even though the external tube morphology may be faithfully replicated in this assemblage, any internal structures were homogenised or obliterated by the combination of continued decay and mineralisation. Conversely, we infer that pyritisation of the Nevadan Cloudinomorphs was abbreviated early in the taphonomic sequence by sulphate or reduced iron limitation. To briefly summarise taphonomy in the Wood Canyon: (i) The initial burial event emplaced the Cloudinomorphs within the sulphate reduction zone of the sediment (oriented prone to bedding, whether or not this was their in-vivo position). (ii) Decay by sulphate-reducing Bacteria commenced, producing bisulphide that initiated pyrite mineralisation. (iii) In a significantly sulphate-restricted local environment (with no sulphate replenishment), we infer that the rate of Bacterial sulphate reduction may have also been diminished once sulphate concentrations dropped below rate-independent levels. With tempered Bacterially mediated decay, the earliest stages of mineralisation focused on the two most histologically suitable loci for pyrite nucleation, the robust organic walls of the exterior tube and the presumably more labile internal soft-tissue cylinder. Schiffbaur et al. suggest that pyrite mineralisation of the external tube and internal cylinder occurred nearly simultaneously, as evidenced by the observed similarity in their compressed, ovate cross-sections from sediment compaction. (iv) Once structural integrity of supporting soft tissues was compromised through decay, the pyritizing soft tissues gravitationally settled to the imposed bottom of the external tube. Thus, both the ventral positioning of the internal cylinders within the recumbent external tubes and the distinction between bedding-respective dorsal and ventral coherency of exterior tube pyritisation (or perhaps ventral-inward pyrite infilling) serve as geopetal indicators. The gravitational slumping of the decaying soft tissue within the tube, as oriented recumbently, would have increased the distance for diffusion of bisulphide toward the upward-positioned wall of the exterior tube. If the reduced iron concentration was high in the burial setting, pyritisation would have therefore been focused more towards the decaying soft tissues, resulting in the observed preservational pattern. The kinked soft tissue observed in sample USNM-N1601_FL_017 may present a slightly different scenario, wherein the organism had died and slumped within its external tube prior to burial positioning or repositioning. And (v), either early sulphate exhaustion caused microbial decay by sulphate reducers to cease, or reduced iron was expended in the burial environment, thus halting continued pyritisation. The former chemical limitation may be more realistic. That is, if local sulphate concentrations instead remained sufficient to fuel continued (and less rate-restricted) bacterial sulphate reduction, it is probable that all of the soft tissues of the tube-dweller, including the internal cylindrical structure, would have been more rapidly exhausted. This taphonomic scenario likely would have yielded preservation of the exterior tube with more substantive detail, but leaving no soft tissues to be preserved. Schiffbaur et al. suggest that this is likely the norm for the majority of the specimens recovered from the Wood Canyon Formation.

2D stacked tomograph, longitudinal cross-section slicesthrough sample USNM-WCF_001. Sample in the reverse orientation (180°) as that from above. Schiffbaur et al. (2020).

In order to provide an improved phylogenetic resolution on the Cloudinomorphs, Schiffbaur et al. first consider which soft tissues are most likely to fossilise. Although they may be rare, there is no shortage of preserved internal soft-tissue structures reported from the fossil record. Fossilised internal soft tissues in the Ediacaran are limited to one possible occurrence of a muscular Cnidarian; on the other hand, Cambrian examples are much more numerous and diverse, including cardiovasculature, nervous and neurological tissues, musculature, and copious reports of digestive tracts. In Cambrian lagerstätten, guts are the most frequently preserved internal structures. Whereas fossil vasculature or nervous tissues are preserved as compressed or flattened features and musculature as bundled fibrous structures, fossil guts can reveal a broadly tubular nature where three-dimensionally preserved, and sometimes occur with the presence of associated digestive glands. Cambrian guts are typically preserved either as carbonaceous films, sediment infillings, or via phosphatisation, the latter of which is potentially reflective of the organism’s digestive physiology. However, there are limited (and perhaps contentious) examples of gut pyritisation as well as gut-content pyritisation. The consistent geopetal nature of the pyritised soft-tissue structures observed here supports the notion that they were originally centrally located structures in vivo, rather than adjacent to the exterior tube wall. At this stage, we can only speculate on the potential histological underpinnings that resulted in preferential pyritisation of these features. It is instead their cylindrical expression, propensity for preservation in Cambrian fossils, and consistent size, shape, and position within the external tube that most endorse a gut interpretation.

Additional detail of cross-sectional morphology. SEM backscattered electron micrographs (Z-contrast) of specimen USNM-WCF_001. Each row corresponds to a single slice at increasing magnifications from left to right, rows (a)–(c); dashed boxes in left and middle columns correspond to location for higher magnification images. Right-most frame in row (c) shows EDS elemental map of middle frame in row (c). Soft tissues in these slices are partially pyrite-infilled, increasingly so from (a) to (c), though distinct sediment grains can be observed. Note also distinct soft-tissue wall boundaries, indicated by black arrows in higher magnification views. White arrows in higher magnification views of rows (a), (b) indicate inferred direction of pyrite precipitation from soft-tissue wall, centripetally toward the interior and centrifugally from the exterior. Scale bars 200 μm for left-most column, and 100 μm for middle and right most columns. Schiffbaur et al. (2020).

Despite being soft tissues, the tendency for gut tracts to be preserved is likely amplified by several factors. Not only can portions of the digestive tract in some organisms be lined with decay-resistant cuticle, but guts are also segregated environments hosting their own microbiome and ions sourced from microbial metabolisms and ingested contents at the time of death. Guts can thus be isolated and accentuated taphonomic vessels, providing ideal conditions for self-contained mineralization. As observed by Schiffbaur et al., the presence of centripetally precipitated pyrite inward from an apparent soft-tissue cylinder wall suggests that their preservation did indeed proceed from the interior. The next key challenge is to identify, within reasonable Cloudinomorph assignments and from both morphological and taphonomic perspectives, which soft tissue structures, whether guts or otherwise, could conceivably leave comparably preserved cylindrical structures. Schiffbaur et al. detail the two primary but debated assignments for the Cloudinomorphs, Cnidarians and Annelids, and offer supplemental treatment on other possibilities (Hemichordates and Phoronids).

Proposed taphonomic sequence of the Wood Canyon Cloudinomorph soft tissues. (a) Cloudinomorph in hypothesised life position. External soft tissue hypothesised, modeled after Siboglinid Polychaete. (b) Burial by rapid sedimentation and initiation of decay. Sediment begins to enter tube cavity. (c) Burial compaction of the outer tube from weight of overlying sediment. Early pyritisation begins on interior surface of external tube and on both interior and exterior surface of soft-tissue cylinder. (d) Continued pyritization of exterior tube and soft-tissue cylinder. Inset of soft-tissue cylinder wall showing both inward and outward framboidal pyrite growth. (e) Remaining soft tissue decays, leaving pyritised exterior tube and interior soft-tissue cylinder. Gravitational settling of pyritised internal cylinder adjacent to lower external tube boundary. Stacy Turpin Cheavens in Schiffbaur et al. (2020).

Cnidarians, and more specifically Anthozoans, have probably received the most attention as a logical affinity for the Cloudinomorphs. Similarities reported between morphological characters of Anthozoans and Cloudina have served to propagate a cnidarian interpretation through the literature. On the other hand, Anthozoan internal anatomy is markedly disparate from the cylindrical structures observed by Schiffbaur et al. Cnidarians, regardless of class affiliation, are defined in part by the possession of a sac-like gastrovascular coelenteron; this simple two-way digestive system has a single orifice for the intake of food and expulsion of waste. Within the Anthozoans, the upper portion (the pharynx) can be broadly tubular, opening into a larger, mesentery-lined, and grossly tubular gastrovascular cavity with numerous outpocketings defined by septa, unlike anything observed herein. These numerous septa, which can be calcitic and thus easily preservable, provide structural support of the tubular pharynx and gastrovascular cavity, but such structures are not observed in any Cloudinomorphs.

Diagrammatic comparison of candidate taxa for Cloudinomorph affiliation. Sections of the tubes and body walls are removed to illustrate gut tracts (red). (a) Anthozoan coelenteron showing upper, tubular pharynx and lower, sac-like gastrovascular cavity with mesentery structure. (b) Polychaete Annelid with straight through-gut path. Stacy Turpin Cheavens in Schiffbaur et al. (2020).

Another possibility that should be considered is that our preserved soft tissues could represent the entire soft-tissue body, rather than an internal feature, of tube-dwelling Hydrozoan polyps. Although generally rare and somewhat contentious in the fossil record, Hydroid fossils have been reported dating back to the Cambrian. Many Hydrozoans live in colonial habits joined by an interconnected network of canals and exterior skeletal branches, for instance, perhaps akin to such modern calcareous examples as Millepora Fire Corals. The Cloudinomorph tube construction is strikingly different from the densely porous tubes of the Fire Corals, but a more important distinction may be found in the pattern of tube branching. If a colonial Hydrozoan assignment were fitting for the broader Cloudinomorphs, one may expect branching to be more common than observed. Although single-tube branching is known in Cloudina and presumed to indicate asexual budding behavior, it has not been observed in most other comparable tubiform Cloudinomorphs, such as those reported here from Nevada and elsewhere. At last, no indications of tentacles are found in the soft tissues reported by Schiffbaur et al., which have been considered diagnostic characters in a rigorous evaluation of putative fossil Hydrozoans. Although this may pose concern for such an interpretation here, rapid taphonomic loss of tentacles has been shown to be likely. Nevertheless, granting that features of Cloudinomorph external tubes have been deduced to be very generally Cnidarian as compared to other plausible affinities, the straight, sagittally continuous soft tissues, whether guts or not, are difficult to reconcile in favor of such an affinity.

The combination of straight, cylindrical soft tissues, and external tube structures may designate Polychaete Annelid Worms as the most fitting phylogenetic position for the Cloudinomorphs. Not only do Annelid through-guts express simple cylindrical morphologies, but the external tubes of the tube-building Annelids are also at least structurally comparable to the Cloudinomorphs, contrary to previous assertions. For instance, one of the features that has been used as a primary argument against a Polychaete affinity is the presence of closed posterior tube ends. Closed ends are known from some posteriorly complete Cloudinomorphs, notably Cloudina and Conotubus; although other Cloudinomorphs, like Saarina, may have had only partially closed or constricted posterior tube ends. This feature may therefore not be ubiquitous within the Cloudinomorphs without clear evidence for a closed basal tube end across all members. Perhaps more importantly, the previous claim that closed bases are absent in modern tube-dwelling Polychaetes is unsupported by zoological literature. For example, Siboglinids are known to have closed bases and many other tube-dwelling Polychaetes possess dedicated anatomical structures (ciliated fecal grooves) or other behavioral strategies to keep waste from accumulating in a closed posterior end of the tube. A second unsubstantiated argument is that Polychaete tubes are not composed of nested funnels, but such a tube construction is in fact found in Siboglinids like Oasisia. Finally, the mode of asexual reproduction by budding as inferred from branching in Cloudina tubes is sometimes thought to be more indicative of a Cnidarian affinity. Tube-dwelling Serpulids among other Polychaetes, however, are known to undergo comparable clonal reproduction, though not all cloudinomorphs, including those reported here Schiffbaur et al., show evidence of external tube branching. The point here is not to invalidate a valuable character evaluation of Cloudina, but instead to offer caution to its applicability to the broader Cloudinomorphs and limited comparisons with modern tube-dwelling Polychaetes. One previous study effectually compares morphological characters of Cloudina to broad-stroke Cnidarians, but makes a comparison with tube-dwelling Polychaetes, which much more narrowly focuses on three sessile, tube-dwelling families, Sabellids, Serpulids, and Cirratulids. The choice of these families clearly results from their calcareous tube-building habits in relation to the tubes of Cloudina, but information provided by the fossil record seems incompatible with such comparisons. The records of Sabellids and Serpulids extend only into the Carboniferous and Triassic, respectively, and the Cirratulids have a much younger appearance in the Oligocene, thus casting doubt on the appropriateness of these families as acceptable comparators.

 Scanning electron microscope image of exterior tube structure of Oasisia alvinae, a modern funnel-in-funnel tube-building Siboglinid Polychaete. Scale bar is 500 μm. Schiffbaur et al. (2020).

The overarching phylogenetic systematics of the ecologically diverse Annelids is complicated and controversial. They can be generally divided by life mode and feeding strategies into two reciprocal monophyletic major clades, the Errantia (free moving, predatory forms) and the Sedentaria (sessile, tube-dwelling forms), but they additionally include five basally branching lineages (Oweniidae, Magelonidae, Chaetopteridae, Amphinomidae, and Sipuncula). The lowest branching of these are tube-dwellers, the Oweniids and Magelonids. Together, these two families form a monophyletic sister
group to the other Annelids, the Palaeoannelida, followed by the basally branching, tube-building Chaetopterids.

Generalized phylogenetic scenario for divergence of stem-Lophotrochozoa and stem-Annelida with gut shape noted. Schiffbaur et al. (2020).

Outside of the three previously targeted Sedentarian families, placing the Cloudinomorphs within any other specific Polychaete designation may still impose a chronological gap, albeit likely more reconcilable, between the terminal Ediacaran and the earliest fossil record of readily identifiable Polychaete tubes. The earliest potential examples of Polychaete tubes previously reported are indeed Cambrian in age, including organophosphatic Chaetopterid tubes (Hyolithellus) from Greenland and calcareous tubes of Coleoides and Ladatheca from Newfoundland and England. Although it is important to note that a record of Polychaete tubes is ostensibly absent from exceptional Cambrian lagerstätten, such deposits do provide several plausible tube-free Annelid fossils, such as (among others) stem-Annelids from the Sirius Passet; Sipunculids, remarkably similar to recent examples, with preserved gut tracts from the Maotianshan Shale, and numerous Polychaetes from the Burgess Shale, most of which preserve gut tracts. Furthermore, moderate taphonomic survival of Annelid gut tracts has been demonstrated by decay experiments with Polychaetes. These fossils ultimately suggest the divergence of at least the basal-most Annelid branches (the Palaeoannelids and Chaetopterids) within the Cambrian Period. Schiffbaur et al. thus advocate an expanded investigation of the diversity of unresolved but comparable tubiform fossils across the Ediacaran–Cambrian transition in an effort to help potentially connect these records.

Although not a common interpretation for Cloudinomorphs, the tubicolous and vermiform Pterobranch Hemichordates do show some tubular similarities with organic-walled representatives of the morphoclade and thus have been previously considered1. The robust tubes of the Pterobranchs have left a considerable fossil record extending to the early Cambrian. They have additionally shown soft-tissue preservation, with a single example from the Chengjiang Lagerstätte. While no taphonomic details were reported, these soft tissues are presumed to have been pyritised but compressed (e.g., two-dimensionally pyritised). Commonly colonial, the Pterobranchs are stalked zooids with U-shaped guts that live within collared tubes. Although their digestive tract may not fit with the cylindrical morphology observed by Schiffbaur et al., their contractile stalks, on the other hand, may be a feasible non-gut interpretation; comparable in shape, position within the external tube, and with lengths that can extend through the entirety of the external tube. Pterobranch stalks, however, are densely muscular structures with a ventral nerve, and thus are reasonably difficult to reconcile with the sediment-infilled portions of the cylinders as observed by Schiffbaur et al..

 Colonial Pterobranch Hemichordates; zooid on right illustrates U-shaped gut path. Contractile stalk shown in tube cut-out below zooid on right, and contracted zooid shown on left. Stacy Turpin Cheavens in Schiffbaur et al. (2020).

It may also be appropriate to consider the sister class to the Pterobranchs, Enteropneust Hemichordates. The Acorn Worms are not tube-builders in the modern-day; although, with a few Cambrian tubicolous representatives, perhaps this was a more common life mode early in their evolutionary history. For instance, the well-known Burgess Shale fossil, Margaretia dorus, originally assigned to the Green Algae, has recently been shown to be a tubular dwelling structure of the vermiform Enteropneust Oesia disjuncta. As opposed to the U-shaped gut of the Pterobranchs, the Acorn Worms have an anterior mouth and posterior anus connected by a straight through-gut, which has been preserved in Cambrian representatives. These are decidedly more comparable to the cylinders reported by Schiffbaur et al., and the lack of hepatic sacs would suggest an affinity with modern Harrimaniid Worms, also similar to Cambrian representatives. Their external tubes, however, may pose the most significant obstacle for such an assignment of the Cloudinomorphs. The reported Cambrian tubes are solely organic in composition and their construction can be quite distinct, like the ornately perforated and anteriorly enclosed tube of Oesia.

Stylized Cambrian Enteropneust, showing Margaretia-like tube structure and straight through-gut path and reduced hepatic sacs. Stacy Turpin Cheavens in Schiffbaur et al. (2020).

Limited Cambrian examples of possible Phoronids have been reported. Much like the broader group Cloudinomorphs, these Horseshoe Worm fossils exhibited both ‘soft shell' and mineralised tubes, with calcareous tubes suggested as ancestral, counter to previous inferences regarding possible ancestral relationships in the Cloudinomorphs. Phoronids may also serve as the most reasonable extant analogue to the extinct Worm-like Tentaculitids, including the Microconchids, which have been offered as a potential interpretation for the Cloudinomorphs. Possible tabulae in Cloudina from Spain have been utilized to support the Microconchid reconstruction, but these structures are tenuous within a heavily recrystallised, sparry calcite-replaced specimen and thus may not be the most biologically informative of features. With regard to their internal anatomy, Phoronids and Microconchids, as other Lophophorates, should have a digestive tract that follows a U-shaped path with a superiorly positioned mouth and anus, again distinct from the morphology of the straight cylinders observed by Schiffbaur et al..

Phoronid with deep U-shaped gut path. Stacy Turpin Cheavens in Schiffbaur et al. (2020).

While a straight through-gut has typically been considered homologous in Bilaterians, recent discussion on Lophotrochozoan anatomical organization and evolution proposes instead that, in sessile forms, U-shaped guts may be the basal groundplan. This claim has roots in the Cambrian fossil record, with fossil U-shaped guts documented for instance in stem-Rhynchonelliform Brachiopods and Orthothecimorph Hyoliths. The ‘U-tube theory’ could imply that the Cloudinomorphs, if stem-Lophotrochozoans, would be expected to follow suit. There are, however, several caveats that may argue against this idea. Perhaps the most important of these stipulations is that not all sessile tube-dwellers possess a U-shaped gut. For instance, some Cambrian organisms like the problematic Hyolithellus have been inferred to possess a straight through-gut and are interpreted to be most likely Annelid-grade, potentially similar to Chaetopterid Polychaetes. If indeed guts, the soft-tissue structures observed here show no evidence of following a U-shaped path, which may call into question either the ‘U-tube theory’ on ancestral U-shaped guts or the suggestion of a basal Lophotrochozoan position for the Cloudinomorphs.

The structure and ingested contents of fossil guts hold significant potential to be behaviorally and ecologically informative. For instance, the preservation of digestive glandular structure and recognisable prey items in the gut contents of Cambrian Ecdysozoans have been used as verification of a predatory or scavenging life mode. These simple cylindrical Cloudinomorph soft tissues, however, are lacking any detail of differentiation or compartmentalization—which is not necessarily problematic for a Polychaete interpretation. Portions of the soft-tissue cylinder that are fully mineralised, as well as other sections that show sediment infill, can both be resolved with a gut interpretation. First, regions of pyrite infilling of the Cloudinomorph guts may tentatively represent mineralisation of ingested, non-descript, organic detritus, similar to gut-content/ cololite pyritisation observed in Cambrian Trilobites. Alternatively, these internal gut structures may represent pyritised internal gut folds like typhlosoles, which are known to occur in Annelids, though the taphonomic resolution and three-dimensional continuity of these features is unfortunately poor. Second, if the observed simple morphology is biologically faithful, in conjunction with their posited sessile habit, then we may be able to deduce that the Cloudinomorphs were likely detritivorous and presumably deposit-to-suspension feeders. The flexibility in feeding behaviors of modern-day tube-dwelling Polychaetes may provide insight on the presence of sediment encased within these fossil soft tissues. Specifically, Owenia and several Spionids are among species that can switch between suspension feeding and deposit feeding behaviors depending on external conditions. These organisms are normally suspension feeders in higher current flow, taking food from the water column with their tentacular palps. However, when water current is low and suspended food is unavailable, they tend to employ surface deposit feeding by placing their palps on the surface of the substrate, during which sediment is commonly ingested. This is not meant to suggest that other tube-dwellers could not have behaved similarly, but it is actualistic evidence provided directly by potential modern analogs. The potential feeding flexibility of the Cloudinomorphs adds diversity in Ediacaran feeding modes, for example, building on recent suggestions of macroscopic suspension feeding by Ernietta and scavenging by motile Bilaterians.

To our knowledge, the structures reported herein are not only the first recognisable soft tissues in Cloudinomorphs, but also the oldest guts yet described in the fossil record. As such, the Wood Canyon tubular fossil assemblage has provided a unique view into early animal anatomy. Nonetheless, for at least the cautions listed throughout the discussion above, Schiffbaur et al. choose to refrain from shoehorning the Cloudinomorphs into any explicit Polychaete family. However, it is the sum of their parts, including the external tube structure, internal soft tissues, and presumed behavioral considerations, that may best denote placement amongst the Annelida as the most plausible. The accord of sequencing-based phylogenies and the available fossil record indicates that stem-Annelids, regardless of whether they exhibited a tube-dwelling habit or not, had diverged by at least the Early Cambrian; and thus a placement of the terminal Ediacaran Cloudinomorphs within basal branches of the Annelids is very likely not unreasonable. If these structures are indeed guts, they are the earliest in the record, fortify a terminal Ediacaran presence of Bilaterians, demarcate the divergence of the Lophotrochozoa, and, perhaps, help to build a phylogenetic bridge across the Ediacaran–Cambrian boundary to the diversity of Annelids known from post-Cambrian Explosion lagerstätten. Nevertheless, when taken together, the novelties provided by the Cloudinomorphs in the terminal Ediacaran, including the advent of macroscopic biomineralisation, the establishment of plausible ecosystem engineering behaviors, the enhancement of larval dispersal mechanisms and sexual and asexual reproductive habits, plausibly novel feeding strategies, and direct soft-tissue evidence of a through-gut, signpost an immense ecological leap towards the rapid Metazoan diversification that transpired geologically soon after.

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