bioRxiv preprint doi: https://doi.org/10.1101/413450; this version posted September 10, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

Sea lettuce systematics: lumping or splitting?

Manuela Bernardes Batista1‡, Regina L. Cunha 2‡, Rita Castilho2 and Paulo Antunes Horta3*

1Postgraduate Program in Ecology, Universidade Federal de Santa Catarina, Florianópolis, SC 88040-900, Brazil 2Centre of Marine Sciences, CCMAR, Universidade do Algarve, Faro, . 3Phycology Laboratory, Department of Botany, Federal University of Santa Catarina, Florianópolis, SC 88040-900, Brazil

‡ These authors contributed equally to the work

*Corresponding author: [email protected]

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1 Abstract

2 Phylogenetic relationships within belonging to the Ulva is a

3 daunting challenge given the scarcity of diagnostic morphological features and the pervasive

4 phenotypic plasticity. With more than 100 species described on a morphological basis, an

5 accurate evaluation of its diversity is still missing. Here we analysed 277 chloroplast-encoded

6 gene sequences (43 from this study), representing 35 nominal species of Ulva from the

7 Pacific, Indian Ocean, and Atlantic (with a particular emphasis on the Brazilian coast) in an

8 attempt to solve the complex phylogenetic relationships within this widespread genus.

9 Maximum likelihood, Bayesian analyses and species delimitation tests support the existence

10 of 22 evolutionary significant units (ESUs), lumping the currently recognized number of

11 species. All individuals sampled throughout an extensive area of the Brazilian coast were

12 included within two distinct ESUs. Most of the clades retrieved in the phylogenetic analyses

13 do not correspond to a single nominal species. Geographic range evolution indicated that the

14 ancestor of Ulva had a distribution restricted to the temperate North Pacific. The dating

15 analysis estimated its origin during the Upper Cretaceous at 75.8 million years (myr) ago but

16 most of the cladogenetic events within the genus occurred in the last ten myr. Pervasive

17 human-mediated gene flow through ballast water and widespread morphologic plasticity are

18 the most likely explanations for the difficulty in establishing a reliable phylogenetic

19 framework for this conspicuous, widespred and many times abundant green

20 morphotype.

21

22

23 Keywords: sea lettuce; Ulva; ESUs, Evolutionary Significant Units; macroalgae; phenotypic

24 plasticity; rbcl, large subunit of the chloroplast-encoded RUBISCO gene; Ballast water;

25 Dispersion

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26 Introduction

27 The genus Ulva Linnaeus (, ) commonly known as sea lettuce is gaining

28 global relevance in coastal ecosystem management owing to their increased role in

29 generating green tides [1]. With the increasing need of targeting species for restoration

30 protocols, the precise identification of fast growing autochthonous species is critical for this

31 process. Ulva is a ubiquitous macroalgal genus showing species inhabiting a remarkable

32 variation of aquatic environments ranging from shallow rocky shores to the subtidal, up to

33 100 meters deep [2]. Due to their high salinity tolerance, some Ulva species may grow under

34 marine, estuarine or brackish conditions. They can either be found in areas strongly impacted

35 by anthropogenic activities or in pristine habitats [3,4]. Sea lettuce species are among the

36 most abundant organisms attached to ship hulls and ballast water [5]. Some species exhibit

37 invasive traits (e.g., high growth rates or efficient reproductive alternatives) that promote

38 dispersal abilities [6].

39 The systematics of this group of marine macroalgae remains challenging given their

40 morphological simplicity [7], widespread plasticity, intraspecific variation, and interspecific

41 overlap [8]. With more than 100 nominal species described on a morphological basis [9],

42 several are considered homotypic or heterotypic synonyms [10], and a precise evaluation of

43 current biodiversity within the genus Ulva is not yet available. This morphologic framework

44 with many potential cryptic species also precludes robust biogeographic interpretations and

45 fails to recognize connectivity and dispersal processes, essential for ecosystem management

46 [11].

47 Molecular tools have been an important ally not only for tracking potentially harmful

48 "green tide" species but also represent a more accurate procedure to evaluate the

49 and cryptic diversity within Ulva [12]. Thus far, molecular studies of the genus focused on

50 more restricted geographic areas (temperate and boreal waters of [9]; north-western bioRxiv preprint doi: https://doi.org/10.1101/413450; this version posted September 10, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

51 American coast [13,14,15]; New Zealand [16], southern Australia [17]; Hawaii [18,19];

52 western coast of India [12], and Japan [20]). Although the study of Hayden and colleagues

53 [13] includes 21 Ulva species, it is focused on the North American coast and a worldwide

54 phylogeny is still missing. Further, the genetic assessment of Ulva species from the

55 southwestern Atlantic, particularly of the Brazilian coast is completely absent. This region

56 may hold fundamental information to complete the systematic relationships within Ulva and

57 complete the biogeographical puzzle of the sea lettuce representatives, considering the global

58 processes of dispersion, invasion, and connectivity occurring in this area [21,22,23]. From a

59 global perspective, phylogeographic studies promote a better understanding on how

60 environmental processes along an ecological and evolutionary time scale influence complex

61 patterns of inter- and intraspecific genetic diversity, fostering discussions regarding lumping

62 or splitting systematics[24,25].

63 In this study, we analysed all available sequences (277 in total, 43 from this study) of

64 the large subunit of the chloroplast-encoded RUBISCO gene (rbcL), representing 35 nominal

65 species of Ulva with distromatic blade-form from the Pacific, Indian Ocean, and Atlantic

66 (with a particular emphasis on the Brazilian coast) in an attempt to better understand the

67 complex phylogenetic relationships within this widespread genus. We performed three

68 different species delimitation tests to better evaluate lineage boundaries, and estimated major

69 lineage splitting events within Ulva to infer its evolutionary history using a Bayesian

70 approach.

71

72 Results

73 Species delimitation tests

74 Species delimitation tests grounded on molecular data arrived at different results. GMYC

75 indicated the existence of 22 evolutionary significant units (ESUs) while mPTP and ABGD

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76 indicated 13 and 10 ESUs, respectively (Fig. 2). The northern hemisphere temperate regions

77 hosted the higher number of ESUs (endemic and non-endemic) and unique haplotypes.

78 Overall, species recovered by species delimitation tests coincided with well-supported clades

79 in both ML and BI analyses. Ulva limnetica was recovered as an ESU by the three methods

80 while Ulva arasakii was identified as a valid ESU by GMYC and mPTP, only. The GYMC

81 method is based on an ultrametric tree, here produced by BEAST, which does not

82 accommodate unresolved nodes, therefore, it is expectable that the recovered ESUs do not

83 correspond to the ML clades.

84 Ulva gigantea was recovered as a valid ESU by mPTP and GMYC. Samples from the

85 Brazilian coast morphologically identified as U. lactuca and U. ohnoi were assigned (in the

86 GMYC analysis) to the ESU1 dominated by U. fasciata or to ESU 2, respectively.

87

88 Phylogenetic analyses

89 ML analysis of the rbcL data set (277 individuals, 1141 bp) yielded the topology (-ln L =

90 3659.98) depicted in Fig. 2. BI analysis retrieved a similar tree (-ln L = 3883.26) with ESS

91 (Effective Sample Sizes) much larger than 200, indicating convergence of the runs.

92 Convergence diagnostic in Bayesian analyses indicates that as potential scaler factors (PSRF)

93 approaches 1.0, runs converge [26]. PSRF of our Bayesian analysis were all 1.00 indicating

94 run convergence.

95 The vast majority of the clades recovered by the ML analysis do not correspond to the

96 currently recognized nominal species within the genus but are congruent with results from

97 GMYC (Fig. 2). The first main clade (Bootstrap Proportions, BP = 78%), contains a group of

98 7 morphologically-defined taxa (Ulva fasciata / reticulata / lactuca / rotundata /

99 scandinavica / rigida / ohnoi) comprehending most of the Ulva’s distributional range in the

100 northern and southern hemispheres, as well as in tropical and temperate environments. This

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101 clade was identified as ESU 1 by GMYC. The second main clade (BP = 69%), corresponds to

102 the nominal species Ulva scandinavica / laetevirens / armoricana / rigida and was

103 recognized as ESU 3. The next clade (BP = 100%), corresponds to U. gigantea (ESU 5). The

104 following main clade (BP = 91%) comprehends most of the specimens assigned to U. linza

105 (ESU 6), except the two from New Zealand that group in a different clade (BP = 100%), and

106 were identified as a different ESU (ESU 7). Another clade (BP = 99%) includes three

107 specimens, one assigned to U. torta from Japan and two Ulva sp. from New Zealand,

108 classified as ESU 9 by GMYC. The clade including a specimen assigned to U. taeniata and

109 another to U. tanneri received high statistical support (BP = 96%) and was identified as ESU

110 10. The ESU 11 identified by GMYC corresponds to a clade including U. linza / flexuosa/

111 californica/ curvata/. The next clade (BP = 100%) comprehends U. compressa/

112 pseudocurvata/ mutabilis and was classified as ESU 14. Ulva rotundata and U. are

113 recovered as sister clades and identified as ESUs 16 and 17, respectively. The clade including

114 most of specimens assigned to U. pertusa and U. australis and one specimen identified as U.

115 lactuca (PB = 100%) was classified as ESU 19 by GMYC. The last main clade (PB = 100%),

116 included most of the specimens morphologically-identified as U. lactuca.

117 Ulva limnetica was recovered as the most basal lineage within the genus, and as a

118 valid taxon by the three species delimitation methods. The 47 specimens from Brazil,

119 previously identified as U. lactuca and U. ohnoi, were represented by six different haplotypes

120 that were all included within clades 1 and 2.

121

122 Date estimates

123 We estimated the age of the most recent common ancestor of the genus (MRCA) Ulva during

124 the Upper Cretaceous at 75.79 [59.6-93.8] myr. The estimated ages (in million years) for the

125 most recent common ancestor of each ESU determined by GMYC are the following: ESU 1 -

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126 6.91 [3.59-10.9]; ESU 2 - 8.97 [4.5-13.9]; ESU 3 - 7.23 [3.0-13.0]; ESU 4 - 20.11 [13.3-28.1];

127 ESU 5 - 8.14 [3.1-14.6]; ESU 6 - 9.61 [4.9-15.4]; ESU 7 - 2.01 [0.1-5.9]; ESU 8 - 36.07

128 [26.2-48.0]; ESU 9 - 3.75 [0.9-8.1]; ESU 10 - 2.02 [0.1-5.7]; ESU 11- 9.52 [4.3-16.0]; ESU

129 12- 17.54 [9.4-26.8]; ESU 13 - 58.41 [44.0-75.2]; ESU 14 - 4.89 [1.2-10.6]; ESU 15 - 44.63

130 [29.2-61.4]; ESU 16 - 2.09 [0.05-6.1]; ESU 17 - 1.83 [0.05-5.2]; ESU 18 - 7.41 [3.8-12.1];

131 ESU 19 - 10.97 [5.7-17.7]; ESU 20 5.96 [2.4-10.9]; ESU 21 - 26.66 [15.9-38.9], and ESU 22

132 - 75.79 [59.6-93.8]. The remaining estimates for the siphonous green algae included in this

133 analysis are congruent with a previous study [27] for the deeper nodes but are more recent for

134 the tips (Fig. 3).

135

136 Biogeographic analyses

137 The dispersal‐extinction‐cladogenesis (DEC) + “J” (jump dispersal or founder event

138 speciation) was selected as the best-fitting biogeographic model for Ulva (-ln L = 274.4;

139 Table 2). However, given the recent controversy involving the “J” parameter, we selected the

140 second-best model DEC (-ln L = 286.8; Table 2). “DEC+J model is a poor model of founder-

141 event speciation, and statistical comparisons of its likelihood with DEC are inappropriate”

142 [28]. Further, the parameter “J” artificially inflates the contribution of cladogenetic events to

143 the likelihood, and prevents of non-jump events at ancestral nodes [28].

144 The stem lineage of Ulva shows an ancestral area restricted to the temperate North

145 Pacific (Fig. 3). Some of the estimated ESUs (GMYC) are confined to

146 (ESUs 3 and 7), temperate North Pacific (ESUs 2, 8, 9, 10, 11, 12, 17, 19 and 21) or

147 temperate North Atlantic (ESUs 4, 5, 15, and 16), while ESU 1 showed the widest ancestral

148 range covering the northern temperate regions of the Pacific and Atlantic, eastern and

149 western Indo-Pacific and the tropical areas of the Atlantic and eastern Pacific (Fig. 3).

150

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151 Discussion

152 Previous studies focusing on relatively restricted geographic areas and a lower number of

153 species have recurrently reported inconsistent intra-generic phylogenetic relationships within

154 Ulva sea lettuce representatives [2,20]. Here, we revisited the systematics of the taxa with

155 this morphotype analysing rbcL sequence data from 35 ‘nominal’ species, defined as “taxa

156 named for our convenience” [29], representing almost its entire distribution.

157 Species delimitation tests indicated a variable number of ESUs depending on the

158 method (ABGD: 10; mPTP: 13; GMYC: 22). Most of the species described on a

159 morphological basis were not validated by mPTP and ABGD tests. For instance, ESU 1

160 identified both GMYC contains, among others, specimens assigned to U. rigida, U.

161 armoricana and U. scandinavica (Fig. 2), a group of nominal species already identified as

162 conspecific in a previous study based on rbcL sequence data and morphological information

163 [9]. This study also concluded that the use of morphological characters in Ulva is “largely

164 inconclusive and of limited value for the circumscription of species” [9]. Results presented

165 here reinforce that phenotypic plasticity and overlapping diagnostic characters produce a

166 complex scenario in which specimens with different morphoanatomical and structural

167 features are included within the same ESU (Fig. 2).

168 ML and BI analyses were mostly consistent with previous studies using a fraction of

169 our taxon sampling, but the inclusion of more species further complicated phylogenetic

170 relationships within the genus. Ulva gigantea is morphologically identical to U.

171 pseudocurvata but ML and BI analyses clearly separate both lineages (Fig. 2). On the

172 contrary, and regardless their morphologic distinctiveness, molecular analyses indicated that

173 specimens identified as U. compressa and U. pseudocurvata should be included within the

174 same species (Fig. 2), as reported in previous studies [9,13,30,31]. Moreover, U. mutabilis

175 shares the same haplotype as U. pseudocurvata (Fig. 2), and thus should be considered the

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176 same species. Our analyses showed that specimens identified as U. californica and U.

177 curvata are conspecific, although considered a valid species in a previous study [13].

178 The combination of species delimitation tests and molecular phylogenetic analyses

179 suggests the 22 ESUs indicated by GMYC, as a reasonable estimation of folios Ulva diversity.

180 Molecular data and crossing-test experiments identified U. fasciata and U. ohnoi [5], as well

181 as U. flexuosa and U. californica [32] as separate species. These findings corroborate results

182 from the GMYC that was the only species delimitation method splitting U. fasciata and U.

183 ohnoi into different ESUs (ESUs 1 and 2, respectively).

184 The inclusion of other molecular markers (e.g., nuclear ribosomal internal transcribed

185 spacers, ITS or the elongation factor tufA) does not seem to add any relevant phylogenetic

186 information to solve the complex relationships within Ulva. For instance, studies based on

187 ITS showed that the nominal species U. fasciata/U. armoricana/U. rigida/U. scandinavica/U.

188 taeniata [14,18] and U. linza/U. prolifera/U. californica [17] are found interspersed in the

189 recovered clades as shown in phylogenies based on rbcl sequence data (present study; [9]).

190 Another phylogenetic analysis of 17 Ulva species based on the elongation factor tufA

191 retrieved similar phylogenetic relationships within the genus [33].

192 Temperate seas host most of the diversity of this group and include ESUs that are

193 only present in one biogeographic region (e.g., ESUs 4, 5 and 16 from the North Atlantic; Fig.

194 3). This general pattern is supported by studies that characterise intermediate latitudes as

195 more diverse regarding [34]. However, there is some evidence of Chlorophyte high

196 species richness also in the tropics, particularly in the Indo-Pacific region [35].

197 Ancestral range estimation showed that the MRCA of Ulva was distributed along the

198 temperate North Pacific (Fig. 3). Most of the ESUs show ancestral distributions restricted to

199 northern temperate regions of the Pacific or Atlantic, with two exceptions: (1) ESU 1 that is

200 widely distributed in the northern temperate waters of the Pacific and Atlantic, temperate

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201 South America and Australasia, eastern and western Indo-Pacific, and (2) ESU 6 occurring

202 both in the southern (temperate Australasia) and northern (temperate north Pacific)

203 hemispheres. These findings suggest that Ulva ancestral lineage most likely adapted to

204 distinct biogeographic regions and environmental conditions, which may have promoted

205 diversification within the genus.

206 The crown group age of Ulva was estimated at 75.2 myr, but most of the cladogenetic

207 events within the genus took place between 10 and two myr ago (Fig. 3), most likely

208 experiencing the changes that took place in the northern hemisphere. The centre of origin

209 (speciation) located in the North Pacific that comprehends most of the cold-temperate faunas

210 was formed c. 40 myr ago, but only after the opening of the Bering Strait about 12 myr ago

211 started what is called the Great Trans-Arctic Biotic Interchange [36]. The continuous biotic

212 flow between the North Pacific and the Arctic-North Atlantic was fully established at about

213 3.5 myr and dramatically changed the ecosystems of the latter region [36]. When a major

214 glaciation started between 2.9 and 2.4 myr ago, the cold-temperate biotas moved southward

215 in both oceans (Pacific and Atlantic). The Great Trans-Arctic Biotic Interchange may explain

216 the close phylogenetic affinity we found between ESUs belonging to the temperate-north

217 Pacific and Atlantic biogeographic regions.

218 Current geographic distribution of the 22 identified ESUs reflects the existence of

219 physiological barriers that may represent the main constraint for their distribution. However,

220 there are ‘nominal species’ belonging to the same ESU that are from distinct biogeographic

221 regions and environmental conditions, sharing the same haplotype (e.g., HAP01: U.

222 scandinavica from the UK and U. lactuca from Australia), which suggests that temperature is

223 not the only driver of Ulva distribution (Fig. 2). Natural dispersal in Ulva is mostly driven by

224 the oceanic drifting of propagules or mature thalli detached from the substrate, or using

225 floating substrates [37,38]. Therefore, the existence of individuals belonging to the same ESU

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226 and occurring in disjunct biogeographic regions (e.g., ESU 1; Fig. 3), suggests that

227 anthropogenic transport may be playing an important role in their distribution.

228 The conflicting intra-generic phylogenetic relationships and the wide non-overlapping

229 geographic distributions of some of the ESUs almost certainly result from the extensive

230 human-mediated gene flow that can potentially confound species boundaries in more recently

231 derived species, and an ancient age of the stem lineage of Ulva that may hinder phylogenetic

232 information due to homoplasy. There is an increasing body of evidence showing that ballast

233 water is an important factor on macroalgal transport [39]. Our reconstructed phylogenetic

234 patterns with clades without any geographic correspondence and grouping samples from

235 distant locations is consistent with a scenario of human-mediated dispersal. Clade 3, for

236 instance, includes nine nominal species and groups samples from New Zealand, Portugal,

237 Vancouver, Finland or Japan, which represent the world’s top trade routes for container

238 shipping (https://geopoliticalfutures.com/top-container-ship-trade-routes/). Because Ulva is

239 recognized as a suitable biological model to address topics such as tissue morphology,

240 complex multicellularity and developmental biology [1] a well-established lineage phylogeny

241 is pivotal to a sound foundation of those studies.

242

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243 Materials and Methods

244 Sampling

245 The study area included an extensive area of the coast of Brazil (states of Santa Catarina,

246 Parana, Bahia, and Fernando Noronha archipelago). We followed [40] for morphological

247 identification. Species with tubular morphology were not sampled. Twenty individuals were

248 randomly collected from the tidal region of each sampled locality, except in the Fernando de

249 Noronha archipelago, Brazil where only two specimens were found. Samples were placed in

250 individual plastic bags and transported to the laboratory in a refrigerated container. For the

251 molecular analysis, a 2 x 2 cm fragment of the largest leave of each individual was removed

252 and inserted in small tea bag to avoid crumbling and subsequently sealed in zip-lock plastic

253 bag, filled by dry silica gel. All vouchers of the collected material are currently retained in

254 the Laboratory of Phycology of the Federal University of Santa Catarina and will be

255 deposited in the FLOR Herbarium. The list of specimens, sample location and GeneBank

256 accession numbers are shown in the supplementary material_S1.

257

258 DNA extraction, PCR amplification and Sequencing

259 Species identifications were based on both DNA sequence data and morphological

260 information. About 10 mg of dried Ulva tissues were used for DNA extraction from the

261 samples obtained in the Brazilian coast. Samples were placed in a 1.5 ml Eppendorf tube,

262 frozen with liquid nitrogen and grounded for 2 minutes with a plastic pestle. DNA was

263 extracted using a commercial kit [NucleoSpin® II, Macherey-Nagel, Düren, Germany]

264 following the manufacturer's instructions. The chloroplast-encoded rbcL gene was amplified

265 in 47 specimens using the PCR profile described in [9] and the published primers pair RH1

266 and 1385r [41] that amplify the large subunit of the chloroplast-encoded RUBISCO gene

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267 (rbcL). After amplification, PCR products were purified using the kit PEG 8000

268 (Polyethyleneglycol 8000) [42].

269 The purified PCR products were subsequently sequenced by the chain termination

270 method (Sanger et al., 1977), with the Big Dye Terminator v 3.1 kit (Thermo Scientific,

271 Carlsbad, CA, USA) following the protocol specified by the supplier. Resulting cycle

272 sequence reaction were purified with ETOH/EDTA precipitation and were either sequenced

273 at the Universidade Federal de Santa Catarina, Laboratório de Fisiologia do Desenvolvimento

274 e Genética Vegetal on a 3500 XL - Applied Biosystems (Thermo Scientific) or at the ABI

275 3730 - Applied Biosystems. Resulting chromatograms were assembled using the software

276 Sequencing Analysis v6.0 (Thermo Scientific).

277 278 Species delimitation tests

279 We used three different approaches to delimit evolutionary significant units (ESUs) within

280 Ulva: (1) the Automatic Barcode Gap Discovery (ABGD) method [43]; (2) the single-rate

281 Poisson Tree Processes method (PTP, [44]), and (3) the general mixed Yule-coalescent

282 (GMYC) model. ABGD detects a gap in the distribution of pairwise distances and uses this

283 information to partition the sequences into groups of hypothetical species. This analysis was

284 performed through the web server of ABGD

285 http://wwwabi.snv.jussieu.fr/public/abgd/abgdweb.html), using three distance options (Jukes-

286 Cantor, Kimura 2-parameter and Simple) and the remaining parameters were used as default.

287 PTP considers that every species evolved at the same rate, using the within-species number of

288 substitutions that are modelled with a single exponential distribution and the multi-rate

289 Poisson Tree Processes (mPTP, [45]), which takes into account the different rates of

290 branching events within each delimited species, assuming that the intraspecific coalescence

291 rate may vary significantly even among sister species. GMYC uses a “threshold time before

292 which all nodes reflect diversification events and after which all nodes reflect coalescent

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293 events” [46]. We used the single-threshold GMYC model as implemented in SPLITS, code

294 written by T. Ezard, T. Fujisawa and T. Barraclough in R v.3.5 [47]. The required ultrametric

295 tree was obtained with BEAST2 v2.5 [48] as described above.

296 All analyses were run on CCMAR computational cluster facility (http://gyra.ualg.pt)a

297 and on R2C2 computational cluster facility (http://rcastilho.pt/R2C2/R2C2_cluster.html) both

298 located at the IT Services of the University of Algarve.

299 300 Phylogenetic Analyses

301 All DNA sequences were aligned using MAFFT v.7.245 (Multiple alignment using Fast

302 Fourier Transform) [49] using the --auto option that automatically selects the appropriate

303 strategy according to data size. The Akaike information criterion [50] as implemented in

304 MODELTEST function of phangorn R-package [51] selected the GTR+I+ (I=0.6927; =

305 0.8877) as the model that fits better the data. These setting were used in the maximum

306 likelihood (ML) analysis that was performed with PhyML v3.0 [52]. The robustness of the

307 inferred trees was tested by nonparametric bootstrapping (BP) using 1000 pseudoreplicates.

308 Bayesian inferences (BI) were performed with MRBAYES v.3.1.2 [53] by Metropolis coupled

309 Markov chain Monte Carlo (MCMCMC) sampling for 20x106 generations (four simultaneous

310 MC chains; sample frequency 1000). Length of burn-in was determined by visual

311 examination of traces in TRACER v.1.6 [54]. According to prior information [9],

312 olivascens was selected as outgroup in both analyses.

313

314 Dating Analysis

315 BEAST v.1.8.4 [55] was used to date the origin of the genus Ulva. We included several species

316 of siphonous green algae from the order that have paleontological record to

317 allow the calibration of the phylogeny. First calibration refers to the earliest occurrence of the

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318 fossil Uncatoella verticillata from the Lower Devonian [419.2-397.5] myr [56]. This fossil

319 has the thallus and reproductive structures comparable to those of extant [57]

320 here represented by the stem lineage of the clade occidentalis, B. oerstedii and

321 Chlorocladus australasicus. This calibration was modeled with a normal distribution

322 (mean=408.35, stdev=10.85). The second calibration refers to the fossil heberti

323 from the Danian [66-61.1] myr representing the stem lineage of the Acetabularieae [58] here

324 represented by acetabulum, A. crenulata and A. dentata. This calibration was

325 modeled with a lognormal distribution (LogN mean=1.14, stdev=1.0, Offset=61.1). We ran

326 the analysis for 150,000,000 generations, sampling every 1000 generations. Length of burn-in

327 was determined by visual inspection of traces in TRACER v.1.6 [54]. The final tree was

328 produced by TREEANNOTATOR using the “maximum clade creditability” option and mean

329 node height, after burn-in of 1500 generations. The convergence to the stationary distribution

330 was confirmed by inspection of the MCMC samples and of effective sample sizes (ESS

331 should be > 200) using TRACER v1.6.0.

332

333 Biogeographic analyses

334 We used the R package BIOGEOBEARS (https://cran.r-

335 project.org/web/packages/BioGeoBEARS/index.html) [59,60] to estimate the ancestral

336 ranges within Ulva. BIOGEOBEARS calculates maximum likelihood estimates of the

337 ancestral states (range inheritance scenarios) at speciation events by modeling transitions

338 between discrete states (biogeographical ranges) along phylogenetic branches as a function of

339 time. Available models include a likelihood version of DIVA ([Dispersal ‐ Vicariance

340 Analysis 61]), Lagrange’s DEC model ([Dispersal-Extinction-Cladogenesis 62]) and

341 BayArea [63]. Additionally, it implements the parameter “+J” that describes founder-event

342 speciation, which is fundamental in oceanic settings [60,64].

15 bioRxiv preprint doi: https://doi.org/10.1101/413450; this version posted September 10, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

343 We defined 10 geographical areas: (1) Central Indo-Pacific (CIP); (2) Eastern Indo-

344 Pacific (EIP); (3) (TAT); (4) Temperate Australasia (TAU); (5) Temperate

345 Northern Atlantic (TNA); (6) Temperate Northern Pacific (TNP); (7) Temperate South

346 Americ (TSA); (8) Western Indo-Pacific (WIP); (9) (TEP) and (10)

347 Temperate South (TSAf). According to the algae data base

348 (http://www.algaebase.org/), the maximum number of areas that any species may occur was

349 set to seven.

350 351 Acknowledgments

352 MBB was supported by the Fundação Grupo Boticário de Proteção à Natureza (1051-20152),

353 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq 306917/2009-2 to

354 PAH) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES/PNPD

355 02828/09-0 and CAPES/PNADB 2338000071/2010-61 to PAH), Brazilian Research

356 Network on Global Climate Change and Fundação de Amparo à Pesquisa e Inovação do

357 Estado de Santa Catarina (FAPESC). RLC was supported by a post-doctoral fellowship

358 (SFRH/BPD/109685/2015) from FCT (Fundação para a Ciência e Tecnologia, Portugal). We

359 thank the Portuguese FCT for CCMAR Strategic Plan PEst-C/MAR/LA0015/2011and

360 UID/Multi/04326/2013 (RC).

361

362 References

363

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529 Figure Legends

530 Figure 1. Biogeographic realms according to [65].

531

532 Figure 2. Phylogenetic relationships of Ulva based on maximum analysis of a portion of the

533 large subunit of the chloroplast-encoded RUBISCO gene (rbcL) produced by PhyML.

534 Numbers at the nodes represent bootstrap proportions. Assignments of each Ulva sample to

535 delineated entities from mPTP, ABGD and GMYC species delimitation tests are shown by

536 coloured rectangles. The estimated Evolutionary Significant Units (ESUs) by GMYC are

537 depicted in grey rectangles. Biogeographic realms according to [65] assigned to each sample

538 are shown by colored squares (CIP, Central Indo-Pacific; EIP, Eastern Indo-Pacific; TAT,

539 Tropical Atlantic; TAU, Temperate Australasia; TNA, Temperate North Atlantic; TNP,

540 Temperate North Pacific; TSA, Temperate South America; WIP, Western Indo-Pacific.

541

542 Figure 3. a. BEAST maximum clade credibility chronogram showing main cladogenetic

543 events within the genus Ulva (in the cartoon) and several species of the order Dasycladales.

544 Numbers at the coloured squares indicate present-day distribution of species at the tips, and

545 ancestral ranges on the nodes. The corresponding 95% highest posterior density intervals

546 (blue bars) are depicted; b. Detail of the cartoon showing main cladogenetic events within the

547 genus Ulva estimated by BEAST with ancestral estimation inferred with BioGeoBEARS.

548 Numbers at the coloured squares indicate present-day distribution of Ulva species at the tips,

20 bioRxiv preprint doi: https://doi.org/10.1101/413450; this version posted September 10, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

549 and ancestral ranges on the nodes. Map of the biogeographic realms according to [65] is also

550 shown.

551

552

21 bioRxiv preprint doi: https://doi.org/10.1101/413450; this version posted September 10, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

Arctic

Temperate Northern 50 Temperate Northern Atlantic (TNA) Temperate Paci c (TNP) Northern Paci c (TNP)

Tropical Tropical Atlantic (TAT) Latitude 0 Eastern-Paci c Central (TEP) Western Indo-Paci c (CIP) Indo-Paci c (WIP) Eastern Temperate Indo-Paci c (EIP) South-Africa Temperate Temperate (TSAf) Australasia −50 South-America (TSA) (TAU)

Southern Ocean

−100 0 100 Longitude GMYC

mPTP ABGD single GMYC- ESU CIP EIP TAT TAU TNA TNP TSA WIP HAP24 fasciata New Zealand HAP23 fasciata New Zealand bioRxiv preprint doi: https://doi.org/10.1101/413450; this version posted September 10, 2018. The copyright holder for this preprint (which Japão/South Korea/Hawaii/Azores/Australia was not certified by peer review) is the author/funder, who has grantedHAP21 bioRxivfasciata a license to displayIndia the preprint in perpetuity. It is made fasciata available under aCC-BYHAP22 4.0 Internationalfasciata license. India lactuca Hawaii/India/Australia/Brazil 62 HAP06 lactuca Brasil ohnoi India HAP01 reticulata India 1 HAP63 reticulata India rigida Ireland 78 HAP03 lactuca Brazil rotundata Ireland HAP02 lactuca Brazil { scandinavica UK HAP20 fasciata HAP05 lactuca Brazil HAP53 ohnoi Florida beytensis NA 76 67 HAP04 ohnoi Gulf of /India/Hawaii/Japan/Brazil HAP61 reticulata NA { reticulata Hawaii 67 HAP73 spinulosa Japan 2 HAP62 reticulata Philippines 82 HAP75 taeniata fasciata California HAP19 taeniata California 97 HAP37 laetevirens Australia { HAP69 scandinavica UK armoricana France/Japan/New Zealand 69 HAP09 armoricana New Zealand laetevirens Australia HAP08 cf. rigida USA (Rhode Island) 51 3 86 HAP64 rigida Ireland/New Zealand/Chile rigida Ireland/Netherlands/China/South Korea 82 HAP65 rigida New Zealand { scandinavica Ireland/Netherlands HAP66 rigida New Zealand HAP51 muscoides 4 64 HAP29 gigantea Ireland 100 HAP28 gigantea Ireland HAP30 gigantea UK 5 HAP48 linza China HAP60 prolifera China linza China/South Korea/Japan 84 HAP42 prolifera Japan 72 { 56 HAP46 linza China 6 91 72 HAP45 linza China 85 HAP44 linza China/South Korea/Japan 87 HAP47 linza South Korea HAP39 linza New Zealand 100 HAP41 linza New Zealand HAP40 linza New Zealand stenophylla Oregon 7 HAP74 taeniata California 8 68 HAP72 sp. New Zealand { 99 69 HAP71 sp. New Zealand 9 68 HAP78 torta Japan 96 HAP77 taeniata Australia HAP76 tanneri Japan/California 10 61 HAP43 linza Portugal 60 HAP27 flexuosa Japan 74 HAP14 californica Vancouver californica California/Oregon/Ireland 11 67 HAP13 { curvata Virginia HAP15 californica Japan/South Korea HAP70 simplex Japan 12 76 HAP26 flexuosa Finland mutabilis Portugal 13 74 HAP52 pseudocurvata California 100 HAP17 compressa Ireland { 14 69 HAP18 compressa Japan HAP31 intestinalis Sweden 15 100 HAP68 rotundata Ireland 60 HAP67 rotundata Ireland 16 100 HAP50 lobata Oregon HAP49 lobata California 17 57 HAP32 lactuca NA australis Spain/China/Australia 18 HAP11 pertusa California/Japan/China/New Zealand HAP57 pertusa China { HAP56 pertusa China 100 100 HAP55 pertusa New Zealand HAP12 australis Australia 19 53 HAP10 australis Spain HAP54 pertusa California HAP59 pertusa South Korea HAP58 pertusa South Korea HAP33 lactuca Ireland HAP36 lactuca 65 HAP35 lactuca California 100 HAP34 lactuca Oregon lactuca Northwestern Pacific/Japan/Ireland/UK 20 68 HAP25 { fenestrata Northwestern Pacific HAP16 cf. lactuca USA (Rhode Island) HAP07 arasakii Japan 21 HAP38 limnetica Japan 22 1 2 3 a 4 5 6 bioRxiv preprint 8 was notcertifiedbypeerreview)istheauthor/funder,whohasgrantedbioRxivalicensetodisplaypreprintinperpetuity.Itmade 5

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