The two phases of the Cambrian Explosion

The dynamics of how metazoan phyla appeared and evolved – known as the Cambrian Explosion – remains elusive. We present a quantitative analysis of the temporal distribution (based on occurrence data of fossil species sampled in each time interval) of lophotrochozoan skeletal species (n = 430) from the terminal Ediacaran to Cambrian Stage 5 (~545 – ~505 Million years ago (Ma)) of the Siberian Platform, Russia. We use morphological traits to distinguish between stem and crown groups. Possible skeletal stem group lophophorates, brachiopods, and molluscs (n = 354) appear in the terminal Ediacaran (~542 Ma) and diversify during the early Cambrian Terreneuvian and again in Stage 2, but were devastated during the early Cambrian Stage 4 Sinsk extinction event (~513 Ma) never to recover previous diversity. Inferred crown group brachiopod and mollusc species (n = 76) do not appear until the Fortunian, ~537 Ma, radiate in the early Cambrian Stage 3 (~522 Ma), and with minimal loss of diversity at the Sinsk Event, continued to diversify into the Ordovician. The Sinsk Event also removed other probable stem groups, such as archaeocyath sponges. Notably, this diversification starts before, and extends across the Ediacaran/Cambrian boundary and the Basal Cambrian Carbon Isotope Excursion (BACE) interval (~541 to ~540 Ma), ascribed to a possible global perturbation of the carbon cycle. We therefore propose two phases of the Cambrian Explosion separated by the Sinsk extinction event, the first dominated by stem groups of phyla from the late Ediacaran, ~542 Ma, to early Cambrian stage 4, ~513 Ma, and the second marked by radiating bilaterian crown group species of phyla from ~513 Ma and extending to the Ordovician Radiation.

SciEntiFic REPORTS | (2018) 8:16656 | DOI: 10.1038/s41598-018-34962-y members are extinct. This therefore consists of the primitive relatives of the crown group, along the phylogenetic line up to, but not including, the last common ancestor of the crown group and their nearest living relatives 19 . The considerable number of characters that can define crown groups were often acquired incrementally over geological time 20 . Random, background extinctions will inevitably erode the base of a clade through time, whether or not basal members are particularly prone to extinction 19 . Hence, the older a fossil, the more likely it is to fall outside the phylum-level of classification. But mass extinctions may operate quite differently, as they can remove taxa selectively based on particular ecological or other traits 21 and lead to long-lasting changes in taxonomic composition and ecosystem functioning 22 .
Here we construct a high resolution temporal distribution of skeletal species (n = 1188) from the upper Ediacaran to the basal Cambrian Series 3 of the Siberian Platform in order to understand the evolutionary dynamics of the Cambrian Explosion (see Supplementary references). The Siberian Platform formed a separate province during the Ediacaran-Cambrian [23][24][25][26] , where the stratigraphy and age dating is relatively well known (Fig. 1) and the biota diverse. New coupled high-resolution δ 13 C and biostratigraphic data as well as improved U-Pb zircon dates suggests that terminal Ediacaran -early Cambrian sections on the northern and south-eastern Siberian Platform are more complete than previously thought, and also indicate that the Cambrian Explosion as shown by the record of skeletal biota may have been a more protracted event 12,27 . The first diverse skeletal assemblages of Cambrian type (including various halkieriids, chancelloriids, and hyoliths in addition to anabaritids and protoconodonts), occur between levels dated from 543.9 ± 0.24 to 529.7 ± 0.3 Ma which precede the strong basal Cambrian negative carbon isotope excursion (BACE), and in some areas even the basal Terreneuvian Trichophycus pedum ichnofossil assemblage 12,[28][29][30] . Additionally, Ediacaran shelly taxa (cloudinids) co-occur with some of the earliest Cambrian shelly taxa (anabaritids) on the south-eastern Siberian Platform, indicating a continuity of the skeletal fossil record around the Precambrian-Cambrian boundary 12 . The additional presence of late Ediacaran soft-bodied rangeomorphs, including their biomineralized holdfasts, as well as chambered palaeopascichnids and Nenoxites (=Shaanxilithes) trace fossils found in immediately underlying strata of the same sections 12,27 indicates that this record is, in turn, rooted in so-called "post-Kotlinian wormworld" (e.g. 13 ).
In particular we consider the distribution of stem and crown group Lophotrochozoa, which is a monophyletic clade of protostome animals within the Spiralia, consisting of Mollusca, Lophophorata, Nemertea and Annelida [31][32][33] . The Lophotrochozoa constitutes a third of all modern marine animals 34 , and was chosen as it is species-rich and represented mostly by skeletal taxa in Ediacaran-Cambrian strata. Deuterostome and cnidarian fossils are too scarce for quantitative analysis, and putative poriferans do not allow detailed character subdivision, due to either an absence of diagnostic spicules (e.g. Archaeocyatha) or the frequently disarticulated preservation of spiculate classes. More importantly, neither the temporal fossil record nor comparative characters of the Lophotrochozoa are reliant upon exceptional preservation (Lagerstätten), as has been noted in other significant groups of the radiation such as euarthropods. This taphonomic bias is exemplified by the fact that crown group euarthropods appear before (521 Ma) stem lineage euarthropods (518 Ma), due in part to differential skeletonisation 35 . Our study thus enables an understanding of how important phyla including the Mollusca, Brachiopoda and Annelida, may have been assembled, in turn informing likely selective pressures and ecological consequences.

Results
Proposed stem-group Lophotrochozoa. The soft-bodied Ediacaran taxon Kimberella (~560 to ~550 Ma) has been proposed to represent a stem group mollusc [36][37][38] , although this placement remains problematic 17 . We exclude this from our analysis given this controversy and the lack of skeletonized hard parts.
While both tommotiids (Fig. 2b,e) and halkieriids s.l. (Fig. 2d) possess multi-element shells (scleritomes), tommotiid sclerites form a narrow conical shell and penetrated by setal canals which can preserve phosphatized setae, and exhibit dense, and fine lamination. In some cases a bivalved larval protegulum with a colleplax-plate typical of the oldest linguliformean brachiopods is present [41][42][43][44][45] . Oymurania (Fig. 2f) has setigerous canals and two shell layers, one of which shows acrotretoid brachiopod columnar microstructure, and the other resembles the prismatic framework of paterinid brachiopods 46 . The former problematic fossil Tumulduria is now reinterpreted as a detached central portion of the ventral interarea of a paterinid brachiopod 47 .
Intact calcareous sachitid scleritomes are considered to belong to a bilateral motile organism that possessed a radula and sclerites with a branching, aesthete type of canal system found in some molluscs [48][49][50] . Complete sachitid scleritomes from the Early Ordovician are recognized as stem-group aculiferan molluscs 51 . Chancelloriid sclerites possess the same morphology and microstructure despite the presence of a markedly different scleritome of a sedentary radial-symmetrical animal [52][53][54] . Thus, a more basal position of sachitids among molluscs, or even lophotrochozoans, cannot be not excluded. Wiwaxiids, although being organic, show the same overall scleritome organization 55 .
The cup-shaped maikhanellids ( Fig. 2i) consist of merged sclerites identical to co-occurring sachitids 56 . Their cross lamellar microstructure is similar to that of some gastropods and the cap-shaped protoconch is typical of monoplacophorans 57 . Bivalved calcareous stenothecoids with their paired, serially arranged muscle scars on the inner surfaces of both valves represent a further group of mollusc-like fossils but with a set of features uncommon in crown group molluscs 54 .
The majority of Cambrian mollusc-like shells with essentially molluscan microstructures, protoconchs, and some features of torsia, are assigned to either the class Helcionelloida 58 , or subclass Archaeobranchia 59 . These mainly cup-shaped and low spiral, endogastrically coiled fossils are considered to be extinct lineages of the phylum Mollusca 58,59 . However, a helcionelloid affinity suggests that their untorted anatomy is due to aperture posterior emarginations, and the presence of a snorkel in some forms also suggests that helcionelloids (Fig. 2a,c) occupy a basal position within the phylum. The archaeobranchian hypothesis also emphasises a torted basic plan and ancestral gastropod affinities 59 . A stem group rather than crown group position for helcionelloids is further supported by the presence of paired bristle-like clusters extending from the aperture of the Pelagiella shell which have a striking resemblance to the parapodial chaetae of some polychaetes 60 . Additionally, helcionelloids are characterized by a different muscle system, densely porous shells that are more common in brachiopods than molluscs, and calcitic semi-nacre microstructures which are more typical of lophophorates [61][62][63] . These observations suggest that the Helcionelloida were stem-group molluscs that retained a number of shared basal features with lophotrochozoan ancestors. Pelagiella represents the most advanced branch of helcionelloids possessing a spirally coiled shell and asymmetric muscle scars suggesting at least partial torsion 59 .
Proposed crown-group Lophotrochozoa. Early Cambrian crown-group molluscs (Fig. 2h), are recognized among bivalves as well as gastropods of the Khairkhaniidae and Onychochilidae families, belonging to the Divasibranchia and Dextrobranchia orders, respectively 59,64 . Crown group Lophophorata are represented in the Cambrian by 13 orders of brachiopods (Fig. 2g), only one of which (Lingulida) survived beyond the Palaeozoic 45,65,66 .
Quantitative temporal distribution. Total skeletal species diversity on the Siberian Platform increases from the terminal Ediacaran to the middle of Cambrian Stage 2, then declines and rises again to reach a second peak at the beginning of Stage 4, followed by an abrupt and rapid decline at the end of Stage 4, followed by recovery around the Series 2/3 boundary (Fig. 3A). The Trilobita appears in Stage 3 and, as the most speciose group, mirrors this general trend. This is in contrast to the second most speciose group, the Archaeocyatha, which first appears in Stage 2 after which there is increase in diversity until the base of Stage 4 but then the group goes abruptly extinct shortly thereafter (Fig. 3A). Total skeletal lophotrochozoan species diversity likewise increases from the terminal Ediacaran to the middle of Cambrian Stage 2, but then declines until the middle of Stage 3, rises again to reach a second peak at beginning of Stage 4, followed by an abrupt and rapid decline until the middle of Stage 4, then followed by a further rise (Fig. 3B).
Of these, stem group lophophorates, brachiopods, and molluscs comprise a total of 354 species, and crown-groups a total of 76 species through the Ediacaran to Cambrian Stage 5 interval. Stem lophoporates,  (Fig. 3B). The first crown species are known from the late Fortunian (~537 Ma -gastropods; ~535 Ma -brachiopods and bivalves), and started to radiate later during the early Cambrian Epoch 2 (~522.5 Ma). Stem group species were devastated during the early Cambrian Stage 4 at ~513 Ma but crown group mollusc and brachiopod species, despite some changes in species composition, show no marked loss of diversity, and continued to diversify at a similar apparent rate (Fig. 3B).

Possible taphonomic and sampling biases. Taphonomic studies have shown that the fossil record can
test the proposition that marine community structure has changed over time 67,68 . Ediacaran to Cambrian skeletal lophotrochozoans are represented by taxa of comparable millimetric sizes, forming part of the small shelly fauna as shells and disarticulated sclerites. These fossils are generally either replaced by phosphate or present in the form of inner and outer moulds. Only lingulate brachiopods and tommotiids are preserved as original shells, and only rhynchonelliform brachiopods retain their original low-Mg calcite mineralogy. In the lower Cambrian of the Siberian Platform, such fossils are restricted to argillaceous limestones (mostly wackestones and packstones), and some grainstones, all of which accumulated onshore above either normal wave or storm wave base 69 . All fossils are extracted by the same method of dissolution in buffered acetic acid to isolate phosphatic and phosphatized shells, or moulds and steinkerns (e.g. 29 ). Worker bias is unlikely given that the assemblages reflect multiple different studies and no single worker or study dominates. We infer that taphonomic biases are minimized, and sampling biases present are shared by all small skeletal fossils.

Trends through time. Total lophotrochozoan biodiversity increases until the middle of Stage 2, but then
there is a notable decline that extends to approximately the middle of Stage 3 (Fig. 3B). This interval coincides in part with an expansion of anoxic sea floor around ~525 Ma inferred from U isotopes 70 . Stem-and crown group lophotrochozoan species show distinctly different temporal distributions, with stem group lophophorate, brachiopod and mollusc taxa originating and radiating first. The preferential extinction of stem group species in early Cambrian Stage 4, at ~513 Ma coincides with the well-known Sinsk Event, an episode of widespread shallow marine anoxia on the Siberian Platform and other locations globally, which also coincides with the major extinction of the Archaeocyatha 71 . It is probable that Archaeocyatha represent a poriferan stem group, and indeed a similar temporal separation of stem and crown group diversification is observed among other metazoans at phyla level, including the Porifera (where crown group demosponges are known by Cambrian Stage 3), Cnidaria and Echinodermata 54,72-74 . The first probable metazoan body fossils (rangeomorphs) appeared at ~570 Ma 75 . Rangeomorphs are complex, macroscopic eukaryotes, probably stem group metazoan taxa, although an affinity higher than Porifera has been proposed 76 . Rangeomorph-dominated assemblages were devastated by the Kotlin Crisis, which marks a turnover event 15 . After this we propose two phases of the Cambrian Explosion separated by the Sinsk Event extinction. The first was dominated by non-bilaterians (Porifera, Cnidaria and Ctenophora) joined by indeterminate bilaterian stem groups at ~ 560 Ma 18 and lasted until ~513 Ma. The general increase in diversity may have been interrupted by the global expansion of anoxic sea floor around ~525 Ma. Notably, this diversification started before, and continues across, the Ediacaran/Cambrian boundary and the Basal Cambrian Carbon Isotope Excursion (BACE) interval (~541 to ~540 Ma). The BACE has been ascribed to a possible global perturbation of the carbon cycle 12 .
The second phase was marked by radiating non-bilaterian and bilaterian (here determined as brachiopod and mollusc) crown group species, and started from ~513 Ma. This second radiation phase may have been interrupted or even terminated by the late Cambrian SPICE event, which marked a further minor extinction (Fig. 4). Crown groups brachiopod species continued to diversify during the remainder of the Cambrian and into the Ordovician. Bivalves and gastropods also formed a significant part of total global lophotrochozoan diversity and were joined by the appearance of bryozoans and cephalopods around the Cambrian/Ordovician boundary 77,78 . From that time onwards their diversity remained higher than stem group lophotrochozoans, which continued to decline dramatically during the Cambrian 51,59,71,79,80 . The last stem group taxa (a few hyolith genera) went extinct in the Permian 81 . The Sinsk Event might therefore be considered a mass extinction, which appears to have preferentially removed skeletal stem group lophotrochozoans at a point when diversity was high. This rapid removal is in contrast to background extinctions that are expected to erode the base of a clade gradually through time. We note that crown group brachiopod and mollusc do not show a marked increase in diversity after the removal of stem group lophophorate, brachiopod and mollusc taxa, but continue their former diversity trajectory. This suggests that their radiation was not dependent upon the removal of incumbent stem group taxa, but rather that crown group taxa were in some way more resilient to shallow marine anoxia or other coeval environmental perturbations. Like other mass extinctions, the Sinsk Event led to significant and long-lasting changes in taxonomic composition and ecosystems 22,79,82 .
A similar sequential faunal replacement pattern of Phanerozoic metazoans has been established in the form of evolutionary marine faunas 83 , which are in part bounded by mass extinctions. During the Ediacaran to Cambrian interval, further distinguished were the Tommotian, Cambrian s.s. and Palaeozoic faunas 84 . All these faunas were discriminated by empirical and statistical analysis of family diversity patterns only without reference to phylogenetic relationships. Their existence was, however, challenged 22 because their speciation/extinction trends could merely reflect replacement between major taxonomic groups that had coupled dynamics. But our analysis shows that evolutionary faunas may in fact be a manifestation of their composition, with the 'Tommotian' fauna being composed of mostly stem group lophotrophorates, molluscs and brachiopods, while the Cambrian s.s. and Palaeozoic faunas are dominated by crown group representatives of molluscs, brachiopods and many other phyla.
This pattern resembles the extinction of taxa at the Permo-Triassic boundary, when groups that originated in the early Palaeozoic either went extinct (tabulate and rugose corals, trilobites, cystoporates) or significantly declined (brachiopods, trepostomates, cryptostomates, conodonts) never to recover previous levels of diversity 85 . This is in contrast to the pattern shown by groups which appeared and diversified in the late Palaeozoic, such as gymnolaemates and new bivalve, gastropod and ammonoid orders [85][86][87] .
If ecological niches are relevant, the difference in maintaining the two phases of the Cambrian Explosion might be related to differences in ecospace that was actually "empty" for skeletal animals. During the earlier phase of stem taxa radiation (~543-513 Ma), speciation was most likely promoted by the lack of competition for existing niches. This is similar to the high rates of sympatric speciation, such as noted among modern benthic caenogastropods in lakes, where high phenotypic plasticity enables evolving ecophenotypes to diversify into different substrates (e.g. 88 ). A similar pattern of early diversification as a result of adaptations to different substrates is shown by both helcionelloid mollusc and archaeocyath sponge species during the first phase of the Cambrian Explosion inferred here. The helcionelloids underwent rapid morphogenesis 89 , and archaeocyaths display extremely high inter-habitat diversity (that is, beta-diversity) in reef communities on the Siberian Platform 90 , which may also reflect high speciation rates. Niche partitioning is not inferred, as the alpha-diversity Bilaterian diversification during the Ediacaran-Cambrian metazoan radaition, showing the fossil record of probable earliest metazoans (shown by a rangeomorph reconstruction), the Kotlin crisis, followed by two phases of Cambrian Explosion, separated by the Sinsk Event extinction (with a possible expanded interval of anoxia during Phase 1) and extending to the Ordovician Radiation through the SPICE extinction. Non-bilaterian stem group example is a stem group archaeocyath sponge; crown group is a crown group demosponge. Bilaterian stem group is shown by a tommotiid; crown group by a trilobite. (species number per community) remains consistently low 91 . A similar effect as a result of low competition, and also correlated with a rise in beta-diversity, has been observed to be the main driver of general diversity increase in the early Cambrian 92 . This dynamic creates the unusual situation when the boundaries of even major lower Cambrian subdivisions have not been established due to an absence of cosmopolitan species. By contrast, even though stem group diversity was significantly reduced during the later crown group brachiopod and mollusc diversification (~513-508 Ma), older niches were not completely eliminated. Thus, in the aftermath of the Sinsk extinction, crown groups were able to diversify via competition for existing niches in order to incorporate into existing communities.

Conclusions
This quantitative analysis of lophotrochozoan skeletal stem-and crown group temporal distribution suggests that the Cambrian Explosion sensu lato may be redrawn as two successive phases of morphological and functional innovation that started in the terminal Ediacaran and were separated by an extinction event. This in turn allows exploration of this phenomenon as an expansion of ecological repertoires that are tractable from the fossil record.

Methods and Data
We divide the terminal Ediacaran to Cambrian Series 2 Siberian record from ~545 to ~505 Ma based on radiometric dates into 16 temporal units based on either sub-division, or combination of one to three Siberian biostratigraphic zones to create broadly equivalent units of ~2.5 Myr each. Units start at the Ediacaran Cloudina-Namacalathus-Sinotubulites assemblage zone through transitional Ediacaran-lowermost Cambrian zones (informally named in ascending order Anabarites trisulcatus, Protohertzina anabarica, and Purella antiqua zones) through Terreneuvian and Cambrian Series 2 zones up to the basal Ovatoryctocara granulata Zone of the Cambrian Stage 5 (Series 3) (Fig. 1). We use the timescale for this interval from available radiometric dates from fossiliferous strata of Siberia, South China, and Avalonia 93-101 (see Supplementary data). We quantify the distribution of described skeletal species (n = 1188) from the upper Ediacaran to the basal Cambrian Series 3 on the Siberian Platform (see Supplementary references). This is derived from occurrence data of fossil taxa sampled in each time interval. In particular we quantify the temporal distribution of lophotrochozoan skeletal species (n = 430) (see Supplementary data). Chancelloriids, although they may belong to stem lophotrochozoans, are excluded from analysis due to their frequently disarticulated nature.