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Canadian Journal of Earth Sciences

Phylogenetic relationships among the Rangeomorpha: The Importance of outgroup selection and implications for their diversification.

Journal: Canadian Journal of Earth Sciences

Manuscript ID cjes-2018-0022.R2

Manuscript Type: Article

Date Submitted by the Author: 01-Jul-2018

Complete List of Authors: Dececchi, Thomas; University of Pittsburgh Johnstown, Biology Greentree, Carolyn; Monash University, School of Earth, Atmosphere and EnvironmentDraft Laflamme, Marc; University of Toronto - Mississauga, Chemical and Physical Sciences Narbonne, Guy; Queen's University, Geological Sciences and Geological Engineering

Keyword: , Phylogenetics, Rangeomorpha, Evolution

Is the invited manuscript for consideration in a Special Not applicable (regular submission) Issue? :

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Phylogenetic relationships among the Rangeomorpha: The importance of outgroup

selection and implications for their diversification.

Dececchi, T.A. 1*, Narbonne G.M.2, Greentree, C.3, and Laflamme, M.4

1- Queen's University, Department of Geological Sciences and Geological

Engineering, Bruce Wing/Miller Hall, Kingston, ON, CAN

* Current affiliation: Biology Department, Natural Sciences Division,

University of Pittsburgh Johnstown, Johnstown, Pennsylvania, 15904, U.S.A.

[email protected] Draft

2- Queen's University, Department of Geological Sciences and Geological

Engineering, Bruce Wing/Miller Hall, Kingston, ON, CAN.

[email protected]

3- Monash University, School of Earth, Atmosphere and Environment, Clayton,

VIC, AUS. [email protected]

4- University of Toronto Mississauga, Department of Chemical & Physical Sciences,

Mississauga, ON, CAN. [email protected]

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1 Abstract

2 The Rangeomorpha are the oldest, most diverse, and most disparate clade of

3 Ediacaran macrofossils. Easily identifiable by their self-similar branching pattern,

4 they occupied epibenthic niche space ranging from the lowest tiered and recumbent

5 taxa up to meter-long upright fronds. A phylogenetic analysis using the largest and

6 most complete character set known for this group scored for 14 separate taxa was

7 undertaken to resolve their internal relationships and test previous hypotheses of

8 their evolutionary and ecological history. Owing to the lack of consensus on the

9 relationship amongst Ediacaran clades, several permutations with different

10 potential outgroup taxa were performed. Across these analyses, there is a strong

11 signal for an upright frondose ancestralDraft state for this clade, likely displaying

12 primary branches that were double sided, non-rotated, with the lower tiered and

13 recumbent forms being derived members of a single subclade. This has implications

14 on the life history reconstruction as well as taxonomic implications for this clade

15 and the origins of large multicellular life in the late Ediacaran.

16

17 KEYWORDS: Ediacaran, Phylogenetics, Rangeomorpha, Evolution

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24 Introduction

25 The Rangeomorpha represent the most diverse Ediacaran clade (Dececchi et al. 2017).

26 They are characterized by a modular and self-similar “fractal” branching pattern that

27 spans at least 4 orders of subdivisions, ranging from primary branches that are several

28 centimeters in size all the way down to fourth order, sub millimetre branching (Narbonne

29 2004; Liu et al. 2016; Kenchington and Wilby 2017). These modular elements are

30 combined into a diverse array of forms, from flat-lying mats to erect fronds, and range in

31 size from a few centimetres to well over a meter in length. Rangeomorpha occupied a

32 range of ecological niches (Clapham et al. 2003, Ghisalberti et al. 2014, Liu et al. 2015,)

33 time slices (Xiao and Laflamme 2008; Laflamme et al. 2013) and water depths (Boag et

34 al. 2016), suggesting they representedDraft a successful group prior to their demise in the

35 latest Ediacaran. They are particularly abundant and diverse in the post-Gaskiers

36 Conception and St. John’s groups in Newfoundland (Hofmann et al. 2008; Narbonne et

37 al. 2009; Liu and Matthews 2017), age-equivalent sections in Charnwood Forest in

38 England (Wilby et al. 2011), but are also found in northwestern Canada (Narbonne et al.

39 2014), and younger occurrences in the Flinders Ranges in South Australia (Gehling and

40 Droser 2013), Siberia (Grazhdankin et al. 2008), southern Namibia (Vickers-Rich et al.

41 2013), and central China (Chen et al 2014) . This ubiquity has led to the Rangeomorpha

42 being one of the most well studied members of the Ediacaran paleocommunity, with

43 research focusing on aspects of their architecture (Narbonne 2004; Narbonne et al. 2009:

44 Brasier and Antcliffe 2009; Brasier et al. 2012), growth (Gehling and Narbonne 2007;

45 Antcliffe and Brasier 2007, 2008; Flude and Narbonne 2008; Bamforth et al. 2008; Hoyal

46 Cuthill and Conway Morris 2014; Dunn et al. 2017), ecology (Clapham et al. 2003; Liu et

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47 al. 2015; Boag et al. 2016), population structure (Darroch et al. 2013) and even potential

48 reproductive mode (Mitchell et al. 2015). Despite these studies, evolutionary

49 relationships amongst the Rangeomorpha remain contentious (Brasier et al. 2012; Liu et

50 al. 2016; Dececchi et al. 2017).

51 Phlylogenetic approaches have previously been employed to explore the natural history

52 of the Rangeomorpha (Brasier and Antcliffe 2009; Dececchi et al. 2017). The present

53 paper expands on these studies to explore the effects that character definition, selection,

54 and variation have on Rangeomorpha alpha taxonomy, including testing previous

55 proposals for defining higher-order rankings (i.e. genus and above). Furthermore, a series

56 of standards for character construction and taxonomic classification within this clade is

57 proposed and applied to investigate Draftthe relationships among the Rangeomorpha.

58 Establishing a cladistic-based hypothesis for the internal relationships among

59 Rangeomorpha will help guide the understanding of the diversity of life prior to the

60 explosion of complex metazoans (Erwin et al. 2011; Schiffbauer et al. 2016).

61

62 Methods

63 In order to create a well-supported phylogenetic hypothesis with a well-resolved

64 topology, it is recommended to incorporate characters derived from multiple axes of

65 information (development, growth, branch architecture, gross structural morphology,

66 etc.). In accordance with this proposal, and following the methodology of Dececchi et al.

67 (2017) in constructing a matrix of 19 distinct characters (Tables S1, S2) that fully

68 describe all morphological regions of known Rangeomorpha, and includes previously

69 proposed criteria for taxonomic differentiation (Brasier and Antcliffe 2009; Narbonne et

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70 al. 2009; Brasier et al. 2012).While this dataset is small compared to analysis of non-

71 Ediacaran taxa, it represents the most granular possible character resolution for these taxa

72 based on the available morphology. One major difference between this analysis and the

73 source data (Dececchi et al. 2017), beyond the addition of new taxa, is the modification

74 of several characters including changes in how growth polarity was previously defined by

75 Brasier et al. (2012) and expanding how growth is characterized and scored to reflect the

76 growth dynamics within the range of Rangeomorpha. The present paper uses the term

77 “polarity” as opposed to “terminal” from Dunn et al. (2017) due to the formers greater

78 prevalence in the literature, ease of use and the fact that the two do not differ in terms of

79 how one classifies the morphology of the taxa examined here. All characters were

80 unordered and unweighted in order toDraft reduce potential user bias. All phylogenetic

81 investigations were done in PAUP v. 4.0 (Swafford 2003) using the heuristic search

82 under default settings. Both the strict and majority rules consensus trees are presented to

83 illustrate both the most conservative topological reconstruction as well as one that are

84 found in the majority of trees, but due to the nature of the dataset with some taxa missing

85 data and the small number of OTU’s, may not be in 100% of reconstructions. This paper

86 uses parsimony over Bayesian approaches it may more accurately reflect how

87 morphological, as opposed to molecular data, functions (Goloboff et al. 2017, 2018).

88 All named taxa known from multiple specimens (5 or more) as well as several rare taxa

89 whose morphology may be informative for increasing topological resolution were

90 investigated and included in the analysis (Table S2). An expansive view of operational

91 taxonomic units (OTUs), including 14 named and 2 referred species as ingroup OTUs,

92 were included in the phylogenetic analysis in order to investigate recent proposals for

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93 synonymy/splitting in alpha taxonomy. The genus Fractofusus into F. misrai and F.

94 andersoni were differentiated based on the criteria identified by Gehling and Narbonne

95 (2007) as they show distinct branching architecture that may alter their phylogenetic

96 placement according to the architectural model. The single specimen

97 of Fractofusus from the Mackenzie Mountains (Narbonne et al. 2014) was scored as a

98 separate OTU in order to test its identification. Beothukis mistakensis and Culmofrons

99 plumosa were not synonymized and the so-called “MUN” frond (Liu et al. 2016) was

100 included as a separate OTU to test previous assertions of generic synonymy between

101 these three taxa (Liu et al. 2016; Dececchi et al. 2017).

102

103 To examine the effects of outgroup selectionDraft on internal relationships given the

104 uncertainty over the most likely sister taxon to the clade, the analyses was run using four

105 different outgroups: a member of the Arboreomorpha (Arborea arborea), two different

106 Erniettomorpha (Pteridinium simplex, Swartpuntia germsi), and two versions of an

107 artificial outgroup where the characters are set to the simplest state (as per Dececchi et al.

108 2017) that differed only in the presence of a surficial holdfast. This permits bracketing of

109 the possible effects of the still unknown sister group to Rangeomorpha, and also

110 examination whether differences in ancestral tiering strategy and method of attachment

111 altered the internal topology. Arborea and Swartpuntia are representatives of the upright

112 class of fronds (Narbonne et al., 1997; Laflamme et al., 2018), though not closely related

113 phylogenetically (Dececchi et al. 2017), with a well-developed holdfast and a petalodium

114 that would be placed in the water column while Pteridinium, though closer

115 phylogenetically to Swartpuntia represents distinct body form, a recumbent lower-tiered

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116 organism (Meyer et al. 2014; Darroch et al. 2015). To examine the level of support for

117 these relationships resampling was run without replacement or jackknifing (Siddell 2002)

118 for 10 and 20% of the character data to examine if a small number of characters were

119 having an extraordinary level of effect on the final topologies. The resulting figures, with

120 the exception of the Pteridinium permutation, strongly resemble the original trees (S1-5),

121 supporting the view that the tree topology is robust and that the character set is more than

122 adequate for the number of taxa analyzed.

123

124 have previously been subdivided into two major subgroups based on the

125 pattern of modular element arrangement along the stalk: the double-sided

126 rangid subgroup, in which each primaryDraft branch consists of secondary branches found

127 symmetrically on both sides of a central axis (strikingly similar to the “frondlets” of

128 Narbonne, 2004), and the single-sided charnids that consist of primary branches that are

129 asymmetrical (i.e. with secondary branches oriented downwards) (Narbonne et al. 2009;

130 Laflamme et al. 2012) or groupings based on branch rotation and furling (Brasier et al.

131 2012). It is our hope that these outgroups will help determine the robustness of the

132 proposed internal relationships among the Rangeomorpha, in addition to suggest a likely

133 plesiomorphic state with regards to water column subdivision and ecological tiering.

134

135 Results

136 Irrespective of outgroup chosen, there is consistently recover two distinct groupings

137 across the permutations: a group consisting of , Vinlandia, Trepassia and

138 Beothukis; and a group including Fractofusus, Bradgatia, Pectinifrons and the

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139 hapsidophyllid genera Hapsidophyllas and Frondophyllas. this unanimity reflects a

140 strong phylogenetic relationship among the taxa in these two clades. However, the

141 analyses also show that observed relationships between these two clades are critically

142 dependent on outgroup selection. The nature and significance of these findings are

143 discussed below.

144

145 Arborea as outgroup

146 A heuristic search of this taxonomic permutation resulted in 78 most parsimonious trees

147 (MPTs) of length 42 with a consistency index (CI) of 0.69 and a retention index (RI) of

148 0.764 (Fig. 1). A well-supported clade that corresponds to the traditional charnid group

149 (Narbonne et al. 2009 Charnia, Vinlandia,Draft Trepassia and Beothukis) with

150 Primocandelabrum as its most basal taxon. This group is characterized single sided

151 branching of the secondary branches and all taxa and furling of the first order branches in

152 taxa more derived than Primocandelabrum suggesting a shift in branching architecture

153 early on in this clade. The previously proposed rangid subgroup(Narbonne et al. 2009)

154 composed of double-sided primary branching fronds such as , ,

155 Bradgatia and Fractofusus is not supported however, as Avalofractus is recovered as the

156 basalmost member of the Rangeomorpha with Rangea representing the basal form of the

157 rangids, though that relationship is only poorly supported and thus uncertain.

158 Hapsidophyllas, Pectinifrons, Bradgatia, Fractofusus, and Frondophyllas are united into

159 a universally-supported clade that houses a diverse combination of recumbent to tiered

160 forms. This group is likely unified in 1) the presence of more than one degree of growth

161 polarity (i.e. bi or multipolar per Brasier et al. 2012), 2) distal inflation of the second

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162 order branching, 3) the absence of a basal disc (though it is unknown in Frondophyllas),

163 4) double-sided secondary branches, and 5) lacking a stem (although traits 4+5 have a

164 wider distribution among the Rangeomorpha).

165

166 Pteridinium as outgroup

167 Using Pteridinium produces 103 MPT of a similar length (44 steps) and support indices

168 (CI=0.659, RI=0.732) to the analysis. Under strict consensus there is little

169 resolution beyond the separation of the double-sided Hapsidophyllas, Pectinifrons,

170 Bradgatia and the Fractofusus species complex from the rest that remains in a polytomy.

171 Using majority rule consensus analysis, a more resolved topology with basal nodes

172 showing high, but not universal (99%)Draft support emerges (Fig. 2), the charnids are broken

173 up into a grade, not a single clade, with Charnia, Trepassia, Vinlandia at the base of

174 Rangeomorpha. A strongly-supported group was identified, with Beothukis at its base that

175 includes the traditional double-sided rangids as well as Culmofrons, MUN frond and

176 Primocandelabrum. Primocandelabrum appears to be their immediate outgroup to the

177 traditional rangids, whose basal members are Avalofractus and Rangea and a derived

178 grouping of primarily recumbent taxa. Beothukis is not united with the MUN frond and

179 Culmofrons in a single distinct clade, as these taxa are arranged as steps uniting the basal

180 charnids with the derived rangids.

181

182 Swartpuntia as outgroup

183 This permutations tree topology (Fig. 3) strongly resembles that of Arborea, differing

184 primarily in the placement of Rangea as the basal most rangeomorph, likely due to it

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185 having multiple petaloids similar to the outgroup. It displays a shorter tree (37 steps) for

186 28 MPT with similar support scores (CI of 0.676, RI of 0.739) than other permutations.

187

188 Artificial outgroup

189 Using the first artificial outgroup permutation (without a holdfast reconstructed),

190 produces 48MPT with a score of 40 (CI = 0.675, RI= 0.759). A separation of the upright

191 fronds (with the exception of Frondophyllas) from the recumbent or low tiered groups is

192 recovered, the latter of which is a polytomy under the strict consensus (Fig. 4). By

193 including the presence of a holdfast in the artificial outgroup (permutation 2) a strong

194 separation between the primarily upright fronds (charnids +Primocandelabrum + Rangea

195 + Avalofractus) and the recumbent/ Draftlower tiered double sided rangids (Fig, 5 a, b) is

196 shown. This gives similar tree scores to that using an artificial outgroup without a

197 holdfast (score 40, 139 MPT, CI = 0.675, RI= 0.759).

198

199 Discussion

200 A cladistic investigation into the internal subdivisions of the Rangeomorpha provides a

201 robust means of testing previous proposals more rooted in their gross phenotypes.

202 Interestingly, depending on the outgroup taxon used, one or both rangids and charnids are

203 either not recovered or recovered with some significant modifications. The selections of

204 outgroups permit evaluation of whether taxonomic or body plan differences play a

205 stronger influence on internal relationships within Rangeomorpha. When using a non-

206 upright frondose taxon, Pteridinium, produces a significantly different tree than do any of

207 the upright fronds. With Pteridinium the base of Rangeomorpha appears to comprise

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208 members of the charnids (Charnia-Trepassia-Vinlandia-Beothukis) which form a grade

209 into the traditional rangid (Fig. 2). This topology is likely driven by the fact that primary

210 morphology of the most basal members of charnids (Charnia-Trepassia-Vinlandia) have

211 constrained primary elements that are superficially convergent with the non-differentiated

212 primaries seen in the Erniettomorpha as well as having little to no recognized stem. This

213 convergence is not suspected to be homologous as Erniettomorphs and charnids are

214 constructed in significantly different ways (Laflamme and Narbonne 2008; Dececchi et

215 al. 2017) that are not captured by this analysis as it focuses on internal relationships

216 within the Rangeomorpha.

217

218 Outgroup selection appears to only Drafthave a minimal control on phylogenetic affinity and

219 final tree topology. Under the Arborea, Swartpuntia and both artificial outgroup

220 permutation, Avalofractus and Rangea are removed from association with other double-

221 sided forms (i.e. Fractofusus and Bradgatia) and placed either at the base of the chanrid

222 or the basal to the entire clade (Fig. 1,3-5). This challenges the recognition of the rangid

223 as a distinct group, especially as Avalofractus and Rangea are typically used as exemplar

224 taxa (Laflamme et al. 2012) and suggests instead that they represent a more

225 plesiomorphic state for the entire Rangeomorpha. Another possibility is that rangid is

226 restricted to those two taxa and all other double side taxa belong to a distinct clade that

227 has retained the ancestral double side morphology, though this would require redefining

228 the characteristics of rangid as presented in Laflamme et al. (2012). The strong similarity

229 between the topology of the Arborea and Swartpuntia and artificial permutations coupled

230 with Dececchi et al.’s (2017) finding that an upright frond is likely basal condition for

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231 Rangeomorpha, suggest than that the derived nature of the charnids seen in those

232 analyses is a more robust pattern then that seen using Pteridinium. Tellingly in no

233 analyses are the recumbent Fractofusus or Bradgatia reconstructed as basal members of

234 this clade contrary to previously suggestions that rangeomorphs were ancestrally lower

235 tiered, perhaps colonial, organism (Brasier and Antcliffe 2009). When coupled with the

236 finding that a holdfast (or similarly-enlarged basal structure) is found in members of

237 either potential sister clade Arboreomorpha (Jenkins and Gehling 1978) and the

238 Erniettomorpha (Darroch et al. 2015) it increases the likelihood that an upright, single

239 petalodium condition is ancestral for multiple groups of Ediacara biota.

240

241 The ancestral condition for RangeomorphaDraft is most parsimoniously reconstructed as an

242 upright frond with an exposed, double-sided (or unfurled per Brasier et al. 2012)

243 morphology to its branches. The presence Rangea and/or Avalofractus at the base of

244 either the entire clade or the base of the charnids in all but the Pteridinium permutation

245 implies that furled branches represent a derived feature within rangeomorphs. Although

246 this conclusion is not unanimous across all permutations, it is supported by the type

247 specimen of Trepassia (ROM 38628) that shows that new branches inserted at the apex

248 were originally double sided and poorly constrained, but that they rotated and became

249 more constrained in more mature branches towards the base of the frond (Narbonne et al.

250 2009). This pattern of growth potentially recapitulates the proposed evolutionary

251 continuum of branch rotation and constraint across the charnids subgrouping (Laflamme

252 et al. 2012). The recovery of the multibranched “network of leaves” taxa Hapsidophyllas

253 and Frondophyllas, which display a constrained branching pattern that resembles the one

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254 seen in Charnia (Bamforth and Narbonne 2009), nested well within a grouping of

255 double-sided branching argues for convergence in the evolution of single-sided branching

256 amongst the Rangeomorpha. This raises questions about the precise nature of the branch

257 rotation between these two groups.

258

259 The suggestion that the single-sided, constrained branching condition is a derived trait,

260 with a transitional series that includes Culmofrons through Beothukis and Trepassia,

261 agrees with previous workers (Narbonne et al. 2009; Laflamme et al. 2012). This

262 hypothesis does seem at odds with the first occurrence record, as the earliest known

263 Rangeomorpha are single-sided charnids, followed by the appearance of multiple double-

264 sided taxa 5 million years later (Liu Draftet al. 2012). However, the proposed reconstruction of

265 double-sided branching as ancestral for Rangeomorpha requires only a short ghost

266 lineage, far less than that observed among well-sampled vertebrate groups for example

267 (Clavin and Forey 2007; Benton et al. 2014; Brusatte and Carr 2016; Philips 2016). The

268 transition from an ancestral double-sided branching pattern towards a single-sided

269 branching architecture could be explained in many ways. The first is as a simplification

270 with the loss of the mirroring present in the ancestral double-sided element. Another

271 would be the evolution of a structureless primary branch from which rangeomorph

272 secondary branches sprout. If correct these two alternatives imply different evolutionary

273 trajectories. The first is simplification, a trend that is seen in other aspects of the anatomy

274 of these organisms such as the loss of holdfasts and stems in many derived rangids. The

275 other is the origin of a de novo branching style within the charnid subgrouping. Either

276 approach has implications for the growth, feeding, and tiering of the Rangeomorpha, and

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277 have implications for the ecological reconstructions as perhaps not all members of the

278 Rangeomorpha have the identical life histories.

279

280 Recent work has suggested that Ediacaran holdfasts are more complicated than

281 previously thought, possibly allowing for chemosymbiosis (Burzynski et al. 2017). Along

282 these lines, Dufour and McIllroy (2016) suggested that Fractofusus may have been at

283 least partially chemosynthetic. If correct this finding may have profound implications for

284 the clade as a whole since there is no major difference in morphology of the rangeomorph

285 branching pattern between Fractofusus and other Rangeomorpha with double-sided

286 primary branches including the basal members recovered here (Rangea and

287 Avalofractus). Nutrient acquisition Drafthas been suggested to have a role in the phenotypic

288 diversity of rangeomorphs (Laflamme and Narbonne 2008; Ghisalberti et al. 2014; Hoyal

289 Cuthill and Conway Morris 2017). Perhaps the high surface area to volume ratio of the

290 petalodium allowed for multiple feeding strategies ancestrally, which would have been

291 specialized in the derived recumbent taxa. Further work on this potential will impact

292 these reconstructions of the ecological complexity as different nutrient acquisition

293 methods may have been employed both inter- and potentially intraspecifically depending

294 on resource limitations.

295

296 These results also suggest that having more than one axis of enlargement (i.e. growth

297 axis) is derived from the ancestral unipolar state. Unipolar growth (i.e. growth along a

298 single plane) appears to be a common growth strategy for several Ediacaran groups

299 including Arboreomorpha (Laflamme et al. 2004), Erniettomorpha (Ivantsov et al. 2016;

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300 Dunn et al. 2017), Dickinsoniomorphs (Gold et al. 2015; Evans et al. 2017; Hoekzema et

301 al. 2017; Reid et al. 2017) and Bilaterialomorpha (Lin et al. 2006). However, given the

302 uncertainty in the evolutionary relationships between these groups (Erwin et al. 2011;

303 Dececchi et al. 2017), one cannot determine if this represents evolutionary convergence

304 or a deep homology across disparate clades. Furthermore, it should be noted that the

305 majority of rangeomorph taxa appear to have grown by branch/element inflation with

306 fewer examples of branch insertion/addition (Gehling and Narbonne 2007; Antcliffe and

307 Brasier 2007, 2008; Laflamme et al. 2007, 2012; Dunn et al. 2017). Thus, the term

308 polarity as used here reflects the direction of enlargement, either by inflation or insertion,

309 rather than necessarily the direction of new growth by segmental addition.

310 Draft

311 One interesting result is the implied pattern of early diversification soon after the rise of

312 the Rangeomorpha following the termination of the Gaskiers glaciation (Narbonne and

313 Gehling 2003; Liu et al. 2012; Narbonne et al. 2014), with some of the earliest known

314 specimens representing derived members of both branching strategies. It is of note that

315 younger Ediacaran assemblages (i.e. White Sea and Nama – Waggoner 2003; Boag et al.

316 2016) are characterized by a distinct loss in Rangeomorpha diversity and abundance,

317 especially in terms of lower-tiered and recumbent forms. This suggests a very rapid

318 radiation of Rangeomorpha bodyplans into an open (or minimally constrained) ecosystem

319 for macroscopic multicellular organisms (Narbonne et al. 2009; Cuthill Hoyal and

320 Conway Morris 2014). The loss of recumbent Rangeomorpha taxa could be explained by

321 several factors including competition with mobile metazoans found in the White Sea

322 (Paterson et al. 2017; Darroch et al. 2017) and Nama biotas (Darroch et al. 2015, 2016),

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323 bathometric factors as rangeomorphs migrated into shallow water settings (Grazhdankin

324 2004; Boag et al. 2016), are facies dependent (Wilby et al. 2015) or that the radiation of

325 recumbent Rangeomorpha represents a localized phenomenon with only a minor presence

326 beyond Avalonia.

327

328 Variation and taxonomy in Rangeomorpha

329 The extent to which interspecific variability should be accounted for both in character

330 state delineation and OTU selection is a source of concern in attempting to construct

331 characters for lineages of organisms whose affinities, physiologies, and developmental

332 biology are controversial. Two major sources of variation, ontogenetic and phenotypic,

333 have been suggested to be present inDraft these organisms (Liu et al. 2015; Kenchington and

334 Wilby 2017) and could influence multiple aspects of alpha taxonomy and ultimately

335 phylogeny. Building upon the work of Liu et al. (2016), the present paper proposes a

336 series of guidelines for the identification of these issues among the Ediacara biota, and

337 propose a series of best practices for how to account for them in future systematic and

338 phylogenetic studies.

339

340 The character set focuses strongly on features that do not appear to vary significantly

341 within complete specimens of a taxon across its known size classes or module counts,

342 taken here as signals for semiphorant categorisation amongst inflationary or insertion-

343 based growth taxa respectively. The exception to this guideline is the presence of a

344 holdfast (character 13), which, while variable between specimens, is thought to be driven

345 by preservation style rather than individual variation (Burzynski and Narbonne 2015,

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346 Burzynski et al. 2017). In this case if a single specimen for a given taxon was associated

347 with a holdfast it was scored as present for that taxon. The smallest known upright fronds

348 (Liu et al. 2012) and recumbent taxa (Gehling and Narbonne 2007; Flude et al. 2008;

349 Bamforth and Narbonne 2008) all show similar gross morphological features across size

350 ranges of a single taxon. One potential exception to this is Trepassia that shows variation

351 in branching pattern at their apex, showing double-sided morphology while the lower

352 (presumed) mature branches display a single-sided pattern (Narbonne et al. 2009). With

353 this possible exception, the characters used in this analysis do not show clear signs of

354 ontogenetic variation that could significantly influence the scoring or the topology of the

355 resulting trees.

356 Draft

357 Since ontogeny can lead to specimens with characters seen in more basal members of the

358 clade and lacking several derived features that would signal its true phylogenetic position,

359 it is important that different age classes of a single taxon not lead to different taxa once

360 investigated cladistically (Campione et al. 2012; Lamsdell and Selden 2013). This has led

361 some to suggest that paleontologists take a conservative approach to alpha taxonomy

362 (Benton 2008), however this can lead to overly broad attempts at synonymization

363 (“lumping”) and reduced phylogenetic resolution, especially in organisms with broad

364 geographic or stratigraphic ranges. In an attempt to minimize the potential for ontogeny

365 to overrun phylogeny, Longrich and Field (2012) put forth three testable criteria that need

366 to be met before suggesting synonymy based on ontogeny: 1) geographic/stratigraphic

367 overlap, 2) one taxon must be represented by more mature individuals than the other and

368 3) intermediates linking the two end members.

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369

370 There is little evidence that more than one purported species are simply members of an

371 ontogenetic continuum. One potential candidate for synonymization is Trepassia, but the

372 variation in frond architecture in the most distal primary branches of Trepassia

373 (Narbonne et al. 2009) is not supportive of this view. Trepassia grew by primary branch

374 insertion, which appears to be distinct from all other double-sided Rangeomorpha taxa

375 currently studied that instead display an inflationary mode of growth. Differences in

376 growth strategies appear early in development and are identifiable at even small size

377 classes and branch/module counts with size having very little to no relationship to branch

378 count (Gehling et al. 2000; Narbonne et al. 2009; Liu et al. 2012). This is because in

379 insertionary taxa branch number is aDraft factor in their indeterminate growth (Wilby et a.

380 2015; Dunn et al. 2017) while in inflationary taxa there is a cessation of addition of new

381 branches well before the largest size classes, indicating that while growth through

382 differentiation has ceased, growth through inflation has not. This early presence and

383 ubiquity across all size classes and localities for all taxa so far sampled suggest that this

384 trait is also independent of ontogeny, thus there is no evidence of switching between

385 growth styles with maturation. Therefore, currently there is little evidence that

386 ontogenetic variation has a significant influence on Rangeomorpha alpha taxonomy.

387

388 Another potential source of disparity is phenotypic plasticity leading to ecophenotypic

389 variation, where environmental differences cause non-heritable phenotypic divergence

390 between members of a single species (Whelan et al. 2012). This has been suggested to

391 occur in at least one lineage of Rangeomorpha (Liu et al. 2016) and may be applicable to

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392 many more of the currently recognized taxa (Hoyal Cuthill and Conway Morris 2017).

393 Thankfully there are several criteria that can be applied to Ediacaran systematic

394 descriptions before proposing synonymy of taxa through the invocation of ecophenotypic

395 variation. First off, specimens must be subjected to different environmental conditions.

396 Additionally, increased morphological discrepancy with ontogeny (Johnson 1981),

397 differentiation based on a single (or a select few) potentially correlated characters,

398 occurrence of intermediates across environmental gradients, and similar trends in other

399 closely related taxa should also ideally be demonstrated.

400

401 Following these criteria and the results of the cladistics analyses, the proposed synonymy

402 of Beothukis and Culmofrons (Liu etDraft al. 2016) is not supported. Importantly, both taxa are

403 found in close proximity on the same surfaces along the Mistaken Point Ecological

404 Reserve at Bristy Cove and Gull Rock Cove (Laflamme et al. 2012, Mason and Narbonne

405 2016). Furthermore, there is no evidence of increasing divergence between conditions

406 with increasing size, with features such as stem length showing increased differentiation

407 at smaller size classes. Using the data in Narbonne et al. (2009) and Laflamme et al.

408 (2012), stem length scales significantly differently (F=55.913, p(same)<0.0001). There is

409 no evidence for increased disparity with maturation as relative value differences is higher

410 at smaller size classes, with stems closer to 40% or more of total length, and invariably

411 remains above 29% of total length in Culmofrons while it remains below 5% in Beothukis

412 regardless of size class (Fig. 6). Additionally, the listed characters that distinguish

413 between Beothukis and Culmofrons, including significant disparity in stem length

414 proportions, the presence of a surficial holdfast (Burzynski and Narbonne 2015), and

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415 differences in the number of first order branches (and their arrangement) along the central

416 axis (Laflamme et al. 2012) are not suspected to be correlated (contra ecophenotypic

417 variation). The number of first order branches has been proposed to be an ontogenetic

418 feature (Liu et al. 2016) but as Beothukis and Culmofrons appear to grow by primary

419 branch inflation (not insertion; Narbonne et al. 2009; Laflamme et al. 2012), the number

420 of first order branches cannot represent an ontogenetically variable trait. Applying

421 previously recognized criteria across localities and taxa shows that the alpha diversity of

422 Rangeomorpha used here is not a by-product of phenotypic plasticity (contra Hoyal

423 Cuthill and Conway Morris 2017) and reflects real taxonomic differences.

424

425 Conclusions Draft

426 Rangeomorpha are among the most morphologically disparate and biologically diverse

427 Ediacaran clades. By characterizing and revising all known members of this clade with

428 more than 5 preserved specimens, it is possible to establish a robust dataset to study the

429 topology of the rangeomorph phylogenetic tree. Support is found for some existing

430 classification schemes (e.g. Narbonne et al. 2009; Brasier et al. 2012; Kenchington and

431 Wilby 2017), however, this study also demonstrates the need for caution when

432 interpreting evolutionary trends due to repeated convergences among divergent branches.

433 It is proposed that the ancestral condition for the Rangeomorpha was a double-sided

434 branched, unipolar frond, implying that the recumbent and multipolar Rangeomorpha are

435 derived members whose life habit may have differed significantly from basal frondose

436 forms. This study provides the framework for future analyses on evolutionary rates of

437 change as well as other biological traits that require a robust phylogeny. Both outgroup

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438 bodyplan and phylogenetic affinity in the outgroup taxa influence the arrangement of

439 internal nodal relationships of rangeomorphs, though the former may be a stronger factor

440 based on the current level of knowledge. Finally, this work helps establish the foundation

441 for methods in character construction to produce accurate and well-resolved trees. The

442 methods and best practice guidelines presented here should form the template for future

443 extensions of cladistic analysis of other Ediacaran groups.

444

445 Acknowledgements

446 We are grateful for funding through a William White Fellowship to T.A.D., NSERC

447 Discovery Grants to G.M.N. and M.L., and a Queen’ s Research Chair to G.M.N. We

448 also thank G. Burzynski for his helpfulDraft discussions.

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Figure captions

Figure 1: Phylogeny of Rangeomorpha using Arborea as the outgroup to polarize the

data. A) Strict consensus tree. B) Majority rule consensus tree. Numbers above nodes

represent percentage of trees supporting that node.

Figure 2: Phylogeny of Rangeomorpha using Pteridinium as the outgroup to polarize the

data. A) Strict consensus tree. B) MajorityDraft rule consensus tree. Numbers above nodes

represent percentage of trees supporting that node.

Figure 3: Phylogeny of Rangeomorpha using Swartpuntia as the outgroup to polarize the

data. A) Strict consensus tree. B) Majority rule consensus tree. Numbers above nodes

represent percentage of trees supporting that node.

Figure 4: Phylogeny of Rangeomorpha using an artificial outgroup, see text for details on

its composition. A) Strict consensus tree. B) Majority rule consensus tree. Numbers

above nodes represent percentage of trees supporting that node.

Figure 5: Phylogeny of Rangeomorpha using an artificial outgroup, though with the

presence of a holdfast included, though this is a derivate state in the matrix. A) Strict

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consensus tree. B) Majority rule consensus tree. Numbers above nodes represent percentage of trees supporting that node.

Figure 6: Regression of stem length versus total frond length, both in mm, based published specimens of both Culmofrons (closed circles) and Beothukis (open circles) from Narbonne et al. (2009) and Laflamme et al. (2012). Note the large number of specimens of Beothukis that show no stem development, recorded here as stem length of zero, across even the lowest size classes. This differs markedly from the condition in

Culmofrons where the smallest individuals tended to have proportionately longer stems, with stem length showing a slight negative allometric pattern.

Draft

Supplementary figure captions

S1: Jackknifed phylogeny of Rangeomorpha using Arborea as the outgroup to polarize

the data. A) 10% of characters dropped. B) 20% of characters dropped.

S2: Jackknifed phylogeny of Rangeomorpha using Pteridinium as the outgroup to polarize the data. A) 10% of characters dropped. B) 20% of characters dropped.

S3: Jackknifed phylogeny of Rangeomorpha using Swartpuntia as the outgroup to polarize the data. A) 10% of characters dropped. B) 20% of characters dropped.

S4: Jackknifed phylogeny of Rangeomorpha using an artificial outgroup to polarize the

data. A) 10% of characters dropped. B) 20% of characters dropped.

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S5: Jackknifed phylogeny of Rangeomorpha using using an artificial outgroup, though

with the presence of a holdfast included, to polarize the data. A) 10% of characters

dropped. B) 20% of characters dropped.

Draft

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Strict consensus tree Charnia

Vinlandia

Trepassia

Beothukis

MUN frond

Avalofractus

Culmofrons Draft Pectinifrons

Frondophyllas

Hapsidophyllas

Fractofusus andersoni

Fractofusus misrai

Fractofusus sp.

Bradgatia

Rangea

Primocandelabrum

Arborea

https://mc06.manuscriptcentral.com/cjes-pubs Page 35 of 48 Canadian Journal of Earth Sciences

Majority-rule consensus tree Charnia 100

100 Vinlandia

100 Trepassia

100 Beothukis

87 MUN frond

94 Culmofrons

Primocandelabrum Draft Avalofractus

Pectinifrons

74 Frondophyllas 100 77 Hapsidophyllas

Bradgatia 100

Fractofusus andersoni

54 68 Fractofusus misrai

Fractofusus sp.

Rangea

Arborea

https://mc06.manuscriptcentral.com/cjes-pubs Canadian Journal of Earth Sciences Page 36 of 48

Strict consensus tree Charnia

Trepassia

Vinlandia

Beothukis

Avalofractus

Culmofrons

Pectinifrons

Draft Frondophyllas

Hapsidophyllas

Fractofusus andersoni

Fractofusus misrai

Fractofusus sp.

Bradgatia

Rangea

MUN frond

Primocandelabrum

Pteridinium https://mc06.manuscriptcentral.com/cjes-pubs Page 37 of 48 Canadian Journal of Earth Sciences

Majority-rule consensus tree Charnia

Trepassia

Vinlandia

Beothukis

Avalofractus 66 Pectinifrons

77 Frondophyllas 100 83 84 Draft Hapsidophyllas

99 Bradgatia 100

Fractofusus andersoni

70 52 55 Fractofusus misrai

Fractofusus sp.

99 Rangea

Primocandelabrum 99

Culmofrons

MUN frond

Pteridinium https://mc06.manuscriptcentral.com/cjes-pubs Canadian Journal of Earth Sciences Page 38 of 48

Strict consensus tree Charnia

Vinlandia

Trepassia

Beothukis

MUN frond

Avalofractus

Culmofrons

Draft Pectinifrons

Frondophyllas

Hapsidophyllas

Fractofusus andersoni

Fractofusus misrai

Fractofusus sp.

Bradgatia

Rangea

Primocandelabrum

Swartpuntia https://mc06.manuscriptcentral.com/cjes-pubs Page 39 of 48 Canadian Journal of Earth Sciences

Majority-rule consensus tree Charnia 100

100 Vinlandia

100 Trepassia

100 Beothukis

70 MUN frond

85 Culmofrons

55 Primocandelabrum

Draft Avalofractus

Pectinifrons

91 76 Frondophyllas 100 82 Hapsidophyllas

Bradgatia 100

Fractofusus andersoni

55 Fractofusus misrai

Fractofusus sp.

Rangea

Swartpuntia https://mc06.manuscriptcentral.com/cjes-pubs Canadian Journal of Earth Sciences Page 40 of 48

Strict consensus tree Charnia

Vinlandia

Trepassia

Beothukis

MUN frond

Avalofractus

Culmofrons

Draft Rangea

Primocandelabrum

Pectinifrons

Frondophyllas

Hapsidophyllas

Fractofusus andersoni

Fractofusus misrai

Fractofusus sp.

Bradgatia

artificial outgroup https://mc06.manuscriptcentral.com/cjes-pubs Page 41 of 48 Canadian Journal of Earth Sciences

Majority-rule consensus tree Charnia 100

100 Vinlandia

100 Trepassia

100 Beothukis

71 MUN frond

85 Culmofrons

56 Primocandelabrum

100 Draft Avalofractus

Rangea

Pectinifrons

67 Frondophyllas 100 88 Hapsidophyllas

Bradgatia 62

Fractofusus andersoni

75 Fractofusus misrai

Fractofusus sp.

artificial outgroup https://mc06.manuscriptcentral.com/cjes-pubs Canadian Journal of Earth Sciences Page 42 of 48

Strict consensus tree Charnia

Vinlandia

Trepassia

Beothukis

MUN frond

Avalofractus

Culmofrons

Draft Pectinifrons

Frondophyllas

Hapsidophyllas

Fractofusus andersoni

Fractofusus misrai

Fractofusus sp.

Bradgatia

Rangea

Primocandelabrum

artificial outgroup with holdfast https://mc06.manuscriptcentral.com/cjes-pubs Page 43 of 48 Canadian Journal of Earth Sciences

Majority-rule consensus tree Charnia 100

100 Vinlandia

100 Trepassia

100 Beothukis

57 MUN frond

71 Culmofrons

Primocandelabrum

Draft Avalofractus

Pectinifrons

83 Frondophyllas 100 87 Hapsidophyllas

Bradgatia 100

Fractofusus andersoni

52 Fractofusus misrai

Fractofusus sp.

Rangea

artificial outgroup with holdfast https://mc06.manuscriptcentral.com/cjes-pubs Canadian Journal of Earth Sciences Page 44 of 48

Culmofrons y=0.25719x+0.84672

Draft

Beothukis y=0.067905x-1.6779

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Table 1 character list

1. Polarity (the number of apical growth tips within a single frondose organism): 0 unipolar 1 bipolar 2 multipolar

2. Petalodium number 0 single petalodium 1 multiple petalodia, each with a separate stalk or other division

3. Petaloid number (definition from Laflamme and Narbonne 2008) 0 2 petaloids, typically but not invariably creating a flat, single plane 1 multiple petaloids that are symmetrically arranged

4. inflation of first order 0 proximal 1 medial 2 moderate 3 distal Draft

5. Inflation of second order 0 proximal 1 medial 2 moderate 3 distal

6. Primary branches display branching pattern of "secondaries" rows (see Figure 3 in Laflamme et al. 2012), this arrangement shows: 0 no fractal divisions 1 rows displaying double-sided morphology 2 row displaying single sided morphology

7. Secondary branches display branching pattern of "tertiaries" rows (see Figure 3 in Laflamme et al. 2012), this arrangement is 0 secondaries not present or show no subdivisions 1 secondaries are double sided 2 secondaries are single sided

8. furled structure 1st order 0 no 1 yes

https://mc06.manuscriptcentral.com/cjes-pubs Canadian Journal of Earth Sciences Page 46 of 48

9. furled structure 2nd order 0 no 1 yes

10. growth axis 0 concealed 1 exposed

11. Subparallel (P) or radiate (R) or Irregular (I) 1st order 0 Subparallel 1 radiating 2 irregular

12. Subparallel (P) or radiate (R) or Irregular (I) 2nd order 0 Subparallel 1 radiating 2 irregular

13. presence of basal disc 0 absent Draft 1 present

14. Holdfast position (See Burzynski and Narbonne 2015 in relation to the sediment water interface and a larger discussion on differentiation this feature) 0 subsurface 1 surficial

15. Stem 0 absent/ minor, <5% total frond length 1 present >5% but less than 20% 2 >20%

16. Branches (primaries) constrained distally 0 absent 1 moderate 2 highly constrained-primaries bound to each other along length 3 distal tip of primary bound to marginal tubes or ridge

17. Branches (secondaries) constrained (box shape) 0 absent 1 present

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18. Presence of subsidiary frondlets (as per Gehling and Narbonne 2007) 0 absent 1 present

19. Growth pattern of new primaries 0 insertion 1 inflation 2 insertion then inflation

Draft

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19

18

17

2 - 0 1

15 15 16

14

1 0 1 0 0 1 0 0 1 0 0 1 0 1 0 0 0 0 ? 0 1 0 - 0 - 0 0 0 0 0 0 0 1 1 1 1 1 2 0 0 0 ?

0/1 0/1 0 1 1 2 0 0 0 1 0 0/1 1 0 1 0 0 0 1

11 11 12 13

0 0 0 0 0/1 0/1

? ? 1 1 1 0 1 0/1 0 1 ? 0/1 0 0 0 ? 0 ? - 0 ? 0 0 0 - 0 0 ? 0 0 0 0 1 0 0 - 0 2 1 ? 0 0 ? ? ? ? ?

8 1 9 1 1 10 1 1 0 1 1 0 0 0 1 0 0 0 1 0 0/1 0 0/1 1 0/1 0 0/1 1 0 0/1 0 0 0 0/1 0/1 0 0/1 2 1 1 0/1 1 0 0/1 2 0 0 0 0 0 0 1 0 1 0 0 0 0 0 1 - 0 1 0 0 - - 0/1 0 1 1 - - 0/1 1 - 0 0 - 0 0 - 0 0 0 0 1 0 - 3 0 ? 0 - 0 0 1 0 ? - 1 0 1 0 0 0 ? 0 1/2 - 1 0 1 0 1 0 - 1 1 2 1 0 2 - 0 0 - 0 0 0 0 0 0 0 ? 0 ? ?

7 2 2 2 1 1 1 1 2 2 1 1 ? Draft1 1 1 1 0 0 0 ? ?

2 2 2 2 1 2 1 2 Canadian Journal of Earth Sciences

5 1/2 6 1/2 1/2 1/2 1/2 1/2 ? 0 1 0 2 3 2 3 1 ? 1 0/1 1 3 1/2 1 1 - 2 - 0 - 0 - 0 - ? ? https://mc06.manuscriptcentral.com/cjes-pubs

4 0 2 1 1 0 0 ? 0 0 0 0 0 1 3 1 0 0 2 2 2 2

3 0 0 0 0 0 0 0 0 0 0 0 1 1 0 0 0 0 1 1 0 0

2 0 0 0 0 0 0 1 ? 1 0 0 0 0 1 0 1 0 0 0 0 0

0 1 0 0 0 2 0 0 0 0 2 0 0 0 0 1 1 0 0 ? 2

Taxa/character Taxa/character Charnia Trepassia Vinlandia Beothukis Avalofractus Culmofrons Pectinifrons Frondophyllas Hapsidophyllas Fractofusus andersoni Fractofusus misrai Fractofusus sp. Rangea 1 Bradgatia frond MUN Primocandelabrum Arborea Pteridinium Swartpuntia outgroup artificial holdfast outgroup artificial with