Cambrian petalonamid Stromatoveris phylogenetically links Ediacaran biota to later animals

Macro‐organisms of the Ediacaran period (635–541 Ma) were large and morphologically complex, with some living in aphotic habitats, presenting the possibility that they were early animals. However, ‘bizarre’ Ediacaran morphologies and mouldic preservation have frustrated comparison to later taxa. Consequently, both the positions of Ediacaran biota in the tree of life and the origins of the Metazoa have remained disputed. Here we provide phylogenetic evidence to identify Ediacaran macro‐biota as animals, based on 206 new fossils of Stromatoveris psygmoglena from the lower Cambrian Chengjiang Lagerstätte. Exceptionally preserved soft‐tissue anatomy shows that Stromatoveris was a soft‐bodied, radially symmetric animal with multiple, sub‐branched petaloids and a differentiated holdfast. Photo‐referenced morphological character analysis enables phylogenetic reconstruction of a monophyletic clade designated Petalonamae, that unites Stromatoveris with iconic Ediacaran genera (Rangea, Pteridinium, Ernietta, Swartpuntia, Arborea, Pambikalbae and Dickinsonia) and is placed as sister‐group to the Eumetazoa. Therefore, based on phylogenetic bracketing within the Metazoa, the Ediacaran petalonamids are established as animals. From these findings, it follows that petalonamids remained an important component of Cambrian marine ecosystems and that the metazoan radiation can be dated to a minimum age of between 558 and 571 myr.

D E S P I T E considerable debate, evolutionary relationships of the Ediacaran macro-biota have remained unresolved. Suggested affinities have ranged through protozoans, algae, fungi, lichens, basal opisthokonts and stem or crown-group animals (see reviews by Antcliffe & Brasier 2007;Budd & Jensen 2017). Their monophyly has also been extensively disputed. Of the two major taxonomic hypotheses, one scatters Ediacaran taxa across extant phyla (Budd & Jensen 2017) while the other proposes a distinct clade such as phylum Petalonamae (Pflug 1972a) (including Rangea, Arborea, Pteridinium and Ernietta) or the 'Vendozoa' or 'Vendobionta' (Seilacher 1989)  However, the presence of detailed anatomical similarities to Ediacaran taxa was subsequently questioned (Antcliffe & Brasier 2007) and Stromatoveris was listed as an animal of uncertain affinity in a recent review of Chengjiang fossils (Xian-Guang et al. 2017). Stromatoveris is here reinterpreted, based on 206 new fossils from the Chengjiang Konservat-Lagerst€ atte, Sanjiezi village, Erjie town, Jingning County, Kunming City, Yunnan Province, China, held in the collections of the Early-Life Institute, Northwest University, Xi'an, China. These fossils provide new insights into the comparative anatomy of Stromatoveris. Morphological phylogenetic analysis alongside 7 Ediacaran ingroup genera and 11, diverse outgroups then reveals that Stromatoveris links these members of the Ediacaran macro-biota to the animals of the Cambrian.

Phylogenetic analysis
Morphological phylogenetic analysis was conducted to test the relationship of the monospecific lower Cambrian genus Stromatoveris to 7 hypothesized petalonamid genera from the Ediacaran period and 11 outgroups, covering protozoa, fungi, algae and animals. The Ediacaran ingroup genera were Rangea (the type genus for the rangeomorphs; Dececchi et al. 2017;Sharp et al. 2017), Pteridinium, Ernietta, Swartpuntia, Arborea (using the specimen classifications of the South Australian Museum which incorporate some specimens previously classified as Charniodiscus; Laflamme et al. 2018), Pambikalbae (originally described as a member of Petalonamae; Jenkins & Nedin 2007) and Dickinsonia. These genera were selected because they represent intersecting sets of taxa previously suggested to fall within a single Ediacaran clade (Pflug 1972a;Seilacher 1989;Jenkins & Nedin 2007;Dececchi et al. 2017), cover a broad range of previously suggested Ediacaran groups and recovered clades (e.g. all named clades identified in the phylogenetic analysis of Dececchi et al. (2017): Rangeomorpha, Arboreomorpha and Erniettomorpha) and are represented by accessioned fossil specimens with excellent preservation (including three-dimensional anatomy), facilitating morphological character analysis alongside Stromatoveris.
A diverse range of 11 outgroup genera were also included to test ingroup monophyly robustly (all having been previously suggested as potential relatives of ingroup taxa) and to test a wide range of potential phylogenetic placements. Outgroup genera were Thectardis ( Morphological character analysis (the process of morphological observation and character coding for subsequent phylogenetic analysis) followed a best-practice protocol (Ramirez et al. 2007) including documentation of all 71 specimens on which coded morphological characters were specifically based, with a labelled photograph referenced to every character state. This yielded 42 morphological characters (40 parsimony informative) for 19 genera (8 ingroup genera; 11 outgroups). The photoreferenced morphological data matrix is available in Mor-phoBank (Hoyal Cuthill & Han 2018a) and in nexus format in Dryad (Hoyal Cuthill & Han 2018b). Seventy-four newly provided digital images (MorphoBank Media) are reusable under a CC BY creative commons licence. Duplicates of the project may be requested through Mor-phoBank for further research.
Phylogenetic character states pertinent to the hypothesis of ingroup monophyly (relative to the outgroup taxa) were coded at the level of observations on fossil morphology (for example, basal primary branch longer than apical primary branch), rather than interpretations which might follow from these observations (e.g. sub-apical primary branching during growth (Antcliffe & Brasier 2007;Hoyal Cuthill & Conway Morris 2014;Gold et al. 2015;Hoekzema et al. 2017)). Morphological characters which were quantitative in nature (e.g. width/length; Sperling et al. 2011) were coded based on measurements from digital photographs of documented fossil specimens (rather than qualitative assessments).
Character analysis and subsequent phylogenetic reconstruction had two primary aims. The first aim was to identify robust synapomorphies (shared derived character states) for the ingroup and the second was to establish ingroup phylogenetic positions relative to the outgroup taxa. Consequently, of the 42 total characters (Hoyal Cuthill & Han 2018a, b), 22 characters relate to the organization and structure of the petaloids and sub-branches (which make up the majority of the body in the ingroup taxa), 5 characters relate to basal attachment structures (e.g. basal stem and holdfast) and 15 characters represent fundamental morphological features (such as symmetry group and presence or absence of unit differentiation or an internalized body cavity) that resolve the relationships of the outgroups and are comparable to the ingroup fossils (with 14 out of 15 coded as non-missing for at least one ingroup taxon). The total number of petaloids per individual was not itself included as a phylogenetic character. This is because species represented by comparatively large numbers of fossils (e.g. Stromatoveris psygmoglena or Rangea schneiderhoehni; Vickers-Rich et al. 2013) show that the number of visible, preserved petaloids is highly variable among specimens, making it difficult to separate potential biological variation from preservational variability.
Parsimony analysis was conducted using the program PAUP version 4b10 (Swofford 2002) with default heuristic tree search settings. Phylogenetic analyses were conducted without any ingroup/outgroup monophyly constraint. Palaeopascichnus was set as the outgroup for rooting the tree (alternative rooting to the alga Bosworthia results in no change to the recovered phylogenetic topology). Tree comparisons were conducted in PAUP using the symmetric (Robinson-Foulds) distance, which counts the number of branches that must be contracted or decontracted to convert between two trees. Clade support values were calculated using PAUP. These were the bootstrap support (fraction of character samples which support a clade, over 500 replicates, with 100 indicating the highest possible support) and the decay index (increase in tree length required before the clade is no longer supported). Shared derived character states (synapomorphies) which supported specific major clades were identified using the program SplitsTree4 (Huson & Bryant 2006).
Minimum node dates for Petalonamae and Rangeomorpha were summarized from the literature based first on only fossil taxa included in this study, and second on combined clade membership information from this phylogenetic analysis (which analyses the position of rangeomorph type genus Rangea

Comparative anatomy of Stromatoveris psygmoglena
Among the new specimens of Stromatoveris, at least two and up to four branched petaloids (or 'fronds') are visible at the fossil surface. This multi-foliate arrangement is indicated by specimens exhibiting separated or strongly delineated petaloids ( Multiple petaloids can be distinguished from subbranches (e.g. primary lateral branches within a single petaloid) because petaloids are of equal and maximal size within the organism (Fig. 2) not part of an increasing size series. Petaloids are preserved in a variety of orientations, indicating flexibility and apical and lateral freedom, and are commonly longitudinally folded and 'furled' (curved). Multiple petaloids are arranged radially, often axis-to-axis ( Natural petaloid cross-sections ( Fig. 1E, G) show that body tissue of Stromatoveris is preserved at a width of approximately 0.05-0.1 mm (after sedimentary compaction). Relatively complete specimens, reaching up to 10.5 cm in length (Fig. 1A), show a blunt basal termination where petaloids join. In some specimens, the base is buried at a lower level than the petaloid apices (e.g.  fig. S1). There is, therefore, no evidence for a through-gut comparable to that of bilaterians, and little potential space for an internal digestive cavity comparable to the coelenteron of cnidarians or ctenophores (which extends through most of the body).
Stromatoveris shows striations on the petaloids, previously compared to the comb rows of ctenophores (Shu et al. 2006). New specimens instead show the repeatedly branched, 'feathered' organization ( Fig. 1)  dimensional relief (Fig. 1K). Occasionally, alternating primary branch originations are also visible (Hoyal Cuthill & Han 2018a, media M451664) at the petaloid axis (the 'stem', 'stalk' or zero order branch axis), although this is frequently concealed when petaloids are longitudinally folded (Fig. 1I, right). Alternate primary branching (alternating, left and right, from the central axis) is also indicated by interdigitated 'seams', where the apices of one row of primary branches are furled or folded over to meet another (Figs 1G-H, 2F). Higher order branching (secondary and above) is most often visible as a finely striated surface texture, which marks the longitudinal boundaries between sub-branches (e.g. Fig. 1K upper region). Exceptional Stromatoveris specimens retain areas of three-dimensional branching detail to at least tertiary level (Fig. 1I, K). However, some Stromatoveris specimens, or parts of specimens (e.g. Fig. 1A, upper region), are smooth (with entirely effaced surface texture) indicating that observed subdivision can be preservationally limited, as in Ediacaran taxa such as Rangea (Hoyal Cuthill & Han 2018a, media M451601). Sediment often infills furled petaloids and the spaces between them (Fig. 1E). These spaces vary in size and shape, indicating considerable petaloid flexibility (Figs 1, 2). Furled primary branches can also enfold a sediment filled, longitudinal space (approximately self-similar to that of the whole petaloid) for example, with roughly oval to tear-shaped cross-section (Fig. 1G).
These observations indicate that the branches of a given order (e.g. primary) met at their lateral margins to form a sheet-like structure (e.g. the petaloid at the lowest branching order). Frequently, this was then longitudinally folded at the central axis and furled so that the exterior lateral margins met at an interdigitating branch seam. Repetition at higher branch orders created a self-similar system of tube-like furled sheets, somewhat comparable to the 'quilted pneu' structure proposed by (Seilacher 1989). However, unlike closed tubes, branch seams were open to the external seawater at multiple locations and size scales, permitting through-flow of nutrient carrying fluid. Evidently, during burial, sediment could also enter through the open seams (Fig. 1E, G).
Stromatoveris specimens were occasionally found in close association with algae (Sinocylindra yunnanensis) and brachiopods (Lingulella chengjiangensis). In one case, the pedicle of a brachiopod contacts the margin of a Stromatoveris specimen, compatible with attachment as an epibiont (Hoyal Cuthill & Han 2018b, fig. S3).

Phylogenetic relationships of Stromatoveris and Ediacaran biota
Photo-referenced character analysis (Ramirez et al. 2007), with character coding based on 71 documented specimens and 102 digital media, produced a character-taxon matrix of 42 morphological characters (40 parsimony informative) for 19 genera. Twenty-seven characters describing details of petalonamid morphology are illustrated by photo-referenced examples in the main manuscript (Fig. 3). All character-taxon data and associated media are provided in Hoyal Cuthill & Han (2018a), where the photo-referenced character matrix, associated labels, specimen media, documentation and museum classifications can be viewed and downloaded. The character-taxon matrix is also provided in nexus format in Hoyal Cuthill & Han (2018b).
Morphological phylogenetic analysis using parsimony recovered a monophyletic clade, which we designate Petalonamae (following Pflug (1970a(following Pflug ( , b, 1972a, including both Cambrian Stromatoveris and seven iconic members of the Ediacaran biota (Fig. 4). Analysed alongside a wide range of outgroup taxa, the petalonamids were found to be monophyletic in both recovered trees