A Three-Genome Five-Gene Comprehensive Phylogeny of The

Home , Amaryllidoideae, Lapiedra, Narcissus assoanus, Narcissus asturiensis, Narcissus bujei, Narcissus bulbocodium

1 A three-genome five-gene comprehensive phylogeny of the

2 bulbous genus Narcissus (Amaryllidaceae) challenges current

3 classifications and reveals multiple hybridization events

5 Isabel Marques1,2*, Javier Fuertes Aguilar3, Maria Amélia Martins-Louçao4, Farideh

5 3 6 Moharrek , Gonzalo Nieto Feliner

7 1Department of Agricultural and Environmental Sciences, High Polytechnic School of

8 Huesca, University of Zaragoza, Carretera Cuarte km 1, 22071 Huesca, Spain.

9 2UBC Botanical Garden & Centre for Plant Research and Department of Botany,

10 University of British Columbia, 3529-6270 University Blvd, Vancouver BC V6T 1Z4,

11 Canada.

12 3Real Jardín Botánico, CSIC, Plaza de Murillo 2, Madrid 28014, Spain.

13 4Centre for Ecology, Evolution and Environmental Changes. Faculdade de Ciências.

14 University of Lisbon. Lisbon, Portugal.

15 5Department of Plant Biology, Faculty of Biological Sciences, Tarbiat Modares

16 University, Tehran 14115-154, Iran.

17 *Corresponding author: Isabel Marques ([email protected])

19 Running title: A three-genome phylogeny of Narcissus

21 Abstract

22 Besides being one of the most popular ornamental bulbs in western horticulture, the

23 Mediterranean genus Narcissus has been the subject of numerous studies focusing on a

24 wide scope of topics, including cytogenetics, hybridization and the evolution of

25 polymorphic sexual systems. Phylogenetic hypotheses based on chloroplast data have

26 provided a backbone for the genus but a detailed phylogenetic framework is still

27 lacking. To fill this gap, we present a phylogenetic study of the genus using five

28 markers from three genomes: ndhF and matK (chloroplast DNA), cob and atpA

29 (mitochondrial DNA), and ITS (nuclear ribosomal DNA). In addition, we use

30 chromosome counts from 89 populations representing 69 taxa. All analyses confirm that

31 Narcissus is monophyletic with two main lineages largely corresponding to subgenera

32 Hermione and Narcissus, but with incongruences between organellar and nuclear

33 ribosomal phylogenies. At the infrageneric level, our phylogenetic results challenge

34 well-established taxonomic groups, such as sections Jonquillae, Bulbocodii and

35 Pseudonarcissi, each of which contains at least two distinct lineages that do not

36 constitute monophyletic groups, and highlight the influence of allopolyploid species in

37 the monophyly of sections within subg. Hermione. The type species of the genus and its

38 section is also nested within sect. Pseudonarcissi supporting new nomenclatural

39 changes. Our results also confirm the intersubgeneric hybrid nature of several hybrids

40 including allopolyploids (e.g., N. dubius, N. tortifolius, N. miniatus). Morphological and

41 cytogenetic evidence independently support the hypothesis that some of the

42 incongruence can be attributed to hybridization, such as the splits of sections Bulbocodii

43 and Pseudonarcissi or the disparate phylogenetic placements of sections Ganymedes,

44 Aurelia and the southern lineage of sect. Bulbocodii (sect. Nevadensis). Together, this

45 indicates a significant role for reticulate evolution in shaping the diversity of this genus.

46 A Bayesian divergence time analysis suggests that the major diversification events took

47 place during the Neogene and provides younger estimates for the main nodes than

48 previous studies, which fit paleoclimatic and paleotectonic reconstructions of the

49 western Mediterranean during this period.

51 Key-words: hybridization; ITS; Mediterranean region; Narcissus; organellar DNA; phylogeny.

55 INTRODUCTION

56 Narcissus L. (Amaryllidaceae) is a medium-sized genus of geophytes mainly

57 distributed in the Mediterranean basin. The center of diversity lies in the Western

58 Mediterranean area, particularly in the Iberian Peninsula (Blanchard, 1990; Mathew,

59 2002). The genus has been reported to have originated in the Late Oligocene to Early

60 Miocene eras, around 29.3–18.1 Ma (Santos-Gally & al., 2012) and is currently one of

61 the most important commercial bulbous plants. Moreover, it is one of the oldest groups

62 of cultivated bulbs, with more than 27000 names registered (Hanks, 2002; Kington,

63 2008).

64 Following Fernandes’ (1934,1951) pioneering cytogenetic research, Narcissus has

65 been a model for karyotype evolution involving not only chromosome number changes

66 but also significant structural chromosomal rearrangements (Fernandes, 1968, 1975;

67 Fernandes & Neves, 1941; Díaz Lifante & al., 2009) and genome size changes

68 (Zonneveld, 2008; Marques & al., 2012). Since the early reports of heterostyly by

69 Fernandes (e.g., 1935, 1964), initially questioned by Bateman (1952) and Baker (1964),

70 the reproductive biology of Narcissus species has also attracted much attention. In

71 particular, a considerable number of studies have focused on the evolution of

72 heterostyly in this genus (e.g., Arroyo & Dafni, 1995; Barrett & al., 1996, 2004; Barrett

73 & Harder 2005; Pérez & al., 2004, 2006; Medrano & al., 2005). Other subjects of plant

74 evolution that have been addressed using Narcissus species include natural

75 hybridization (Marques & al., 2007, 2011, 2012, 2016) as well as differentiation and

76 other processes at the micro-evolutionary level (Jiménez & al., 2009; Medrano & al.,

77 2014). In addition to being a model system for basic research, Narcissus has also been

78 the subject of drug screening focused on diseases such as Alzheimer’s (Heinrich & al.,

79 2004; Vrondeli & al., 2005; Rønsted & al., 2008; Berkov & al., 2014).

80 However, despite all this progress, the taxonomy of Narcissus remains unsettled

81 due to a number of factors, including the apparent importance of natural hybridization,

82 the phenotypic variability of some morphological traits and the possible naturalization

83 of some cultivars (Fernandes, 1968; Blanchard, 1990; Mathew, 2002). Historically, both

84 wide and narrow species concepts have been proposed and, therefore, estimates of the

85 number of species in Narcissus have varied extensively through time, from between 16

86 to nearly 160 (eg., Haworth, 1831; Herbert, 1837; Spach, 1846; Baker, 1875). Much of

87 the disagreement lies in the recognition of closely related taxa either as different

88 subspecies, varieties or simply as the same species adapted to different environments, a

89 disparity that is still visible in modern classifications (Table S1). By 1968, Fernandes

90 had accepted 63 species divided into 10 sections (Fernandes, 1968), which were later

91 reduced to 27 species in 11 sections by Webb, who prioritized morphology over

92 cytological data (Webb, 1980). The subsequent classifications mostly followed the

93 approach of Fernandes but incorporated newly described species (eg, Blanchard, 1990:

94 65 species; Mathew, 2002: 87 species; RHS, 2016: 81 species). However, synthetic

95 approaches continue to be published (Zonneveld, 2008: 36 species; Aedo, 2016: 25

96 species) with discrepancies occurring at the sectional level or even in the recognition or

97 not of more than one subgenus (Table S1).

98 The phylogeny of Narcissus also remains relatively unsettled. The first small-

99 scale phylogenetic analysis including species from all sections based on chloroplast

100 markers, found only one section to be clearly monophyletic (Graham & Barrett, 2004).

101 It also supported the division of the genus into two clades, largely corresponding to

102 subgenera Hermione and Narcissus (Graham & Barrett, 2004). An extended

103 phylogenetic analysis with 39 species, also using chloroplast markers (Rønsted & al.,

104 2008), confirmed the topology of the previous phylogeny. Using nuclear DNA content,

105 Zonneveld (2008) proposed a new classification eliminating sections and creating new

106 ones, solving most of the paraphyly found in those phylogenies. However, the most

107 recent phylogenetic work found even subg. Hermione to be non-monophyletic (Santos-

108 Gally & al., 2012). A more comprehensive phylogenetic framework based on a wide

109 sampling and using differently inherited markers is thus needed, in particular because

110 hybridization between Narcissus species has been frequently reported (Fernandes, 1967;

111 Graham & Barrett 2004; Marques & al., 2007). For instance, 40% of specific and

112 subspecific names of naturally growing plants in the latest list of accepted botanical

113 names of Narcissus have a suspected or explicitly attributed hybrid origin

114 (https://www.rhs.org.uk/plants/pdfs/plant-registration-forms/daffalpha).

115 Generating a phylogenetic hypothesis that serves as a sound evolutionary

116 framework in which hypotheses can be tested, requires that several issues be addressed.

117 A representative sampling of species, geographic areas, and sequences from genomic

118 regions with different modes of inheritance should ideally go along with accounting for

119 the main processes responsible for the differences between gene trees and species trees

120 (i.e., hybridization; incomplete lineage sorting, ILS) and for those causing mixed

121 phylogenetic signals (recombination). In addition, exploring divergence times and

122 diversification rate shifts, together with cytogenetic data and morphological characters,

123 may help in our understanding of the topological patterns retrieved from phylogenetic

124 analyses and ultimately in interpreting the diversity and systematic implications.

125 Therefore, our general goal was to construct the first multilocus phylogeny of

126 Narcissus that samples three genomes, specifically DNA sequences from the nuclear

127 ribosomal internal transcribed spacer (ITS) regions (ITS1+5.8S+ITS2), two chloroplast

128 regions (ndhF and matK), and two mitochondrial regions (cob and atpA). To achieve

129 this in this genus, we focus on the following issues that critically influence the

130 phylogenetic signal and its relevance to a natural classification: (1) integrating

131 cytogenetic evidence (our own and that available in the literature); (2) assessing

132 hybridization and its consequences on the systematic arrangement in the genus; (3)

133 estimating divergence times and diversification rate shifts; and (4) testing currently

134 recognized infrageneric taxonomic groups (subgenera, sections).

135

136 MATERIALS AND METHODS

137 Plant Sampling for phylogenetic and karyological studies. — For the

138 phylogenetic study, we collected 1770 specimens of Narcissus belonging to 103 taxa

139 and 277 populations covering the whole geographic distribution of Narcissus, the range

140 of morphological variation and all of the currently recognized sections (Tables S2, S3):

141 Apodanthi A.Fern., Braxireon (Raf.) Valdés [=Tapeinanthus (Herb.) Traub], Bulbocodii

142 DC., Ganymedes (Salisb. ex Haw) Schult. & Schult. fil., Jonquillae DC., Narcissus,

143 Pseudonarcissi DC., Aurelia (Gay) Baker, Serotini Parl. and Tazettae DC. Specimens

144 were identified based on the taxonomic keys of Fernandes (1968) except for additional

145 species described later (Table S3). Twenty-one putative hybrid taxa were included in

146 this study: two species suggested to have a hybrid origin (N. dubius Gouan, N.

147 tortifolius Fern.Casas), fourteen accepted hybrids (e.g., N. ×rozeirae Pérez-Chisc. &

148 Fern.Casas), plus six taxa collected in the wild and having clearly intermediate

149 morphological characters between two sympatric species (e.g., N. fernandesii Pedro ×

150 N. papyraceus Ker Gawl.). Based on their close position to Narcissus, four

151 Amaryllidaceae species were used as outgroups: Lapiedra martinezii Lag., Leucojum

152 aestivum L., Sternbergia lutea (L.) Ker-Gawl. ex Spreng. and Nerine bowdenii Watson

153 (Meerow & al., 1999; Ito & al., 1999; Lledó & al., 2004; Meerow & Snijman 2006;

154 Meerow & al., 2006). Samples were preserved as bulbs, silica-gel dried leaves and/or

155 stored at –80° C until DNA isolation.

156 Of the 277 populations included in this study, chromosome numbers were

157 obtained from 89 of them covering 69 of the 103 taxa sampled. For each taxon, root tips

158 were collected from 8 to 15 different individuals (Table S3), either using radicles from

159 germinated seeds or directly from bulb roots. Roots were prepared according to

160 Fernandes (1975). Five to 10 different metaphase plates per individual were observed in

161 a total of 2990 observations performed from 2005 to 2010. Previous reports in the

162 literature were also considered in cases where either a voucher specimen or a confident

163 geographic origin could be traced.

164

165 DNA isolation, PCR amplification, cloning and sequencing. — DNA was

166 isolated using the DNeasyTM Plant Minikit (Qiagen, Hilden, Germany) following the

167 manufacturer’s instructions. DNA concentration was measured spectrophotometrically

168 using a GeneQuant RNA/DNA calculator with a capillary cuvette (Pharmacia,

169 Cambridge, UK) and subsequently preserved at –80ºC. Two chloroplast regions (ndhF,

170 matK), two mitochondrial regions (cob, atpA) and one nuclear region (ITS including

171 ITS1+5.8S+ITS2) were amplified using primers and conditions summarized in Marques

172 & al. (2010). Since direct sequencing recurrently produced multiple sequence signals,

173 purified PCR products of the ITS region were all cloned following Marques & al.

174 (2010) using the One Shot TOP10 protocol (Invitrogen, San Diego, California, USA).

175 However, because of the high number of specimens included in this study and to assure

176 that all ITS sequences were cloned, we were only able to sample one individual per

177 population. Competent TOP10F’cells were chemically transformed and the resulting

178 colonies were screened for plasmids with inserts by PCR, isolating 10 single colonies

179 from each reaction. Purified PCR products were sequenced in both directions on a 3730

180 DNA Analyzer (Applied Biosystems) at the Centro de Genómica y Proteómica, Parque

181 Científico in Madrid (Spain). In total, we obtained 1770 sequences (1750 of Narcissus

182 plus 20 sequences for the outgroups) from each of the four organellar regions

183 representing all specimens collected for this study. For the ITS region, we obtained

184 1640 cloned sequences (1600 from Narcissus and 40 from the outgroups) from 163

185 individuals (Table S3). DnaSP version 5.10.1 (Rozas, 2009) was used to characterize

186 DNA polymorphism and nucleotide diversity, π.

187

188 Sequence data quality tests. — Checking the reliability of sequence data is

189 always necessary but particularly so when using large data sets. Thus, we (1) tested the

190 existence of recombinants in all datasets (Schierup & Hein, 2000a,b; Posada &

191 Crandall, 2002; Supplementary Methods), (2) we looked for possible pseudogenes in

192 the ITS dataset, which if undetected would result in analyzing paralogous sequences

193 (Bailey & al., 2003; Mayol & Rosselló, 2001; Supplementary Methods), and (3) we

194 performed distance-matrix rate tests to graphically explore the disparities in the

195 evolution of different genes from the same organisms without previous knowledge of

196 the time of divergence between species or their phylogenetic relationships (Syvanen,

197 2002; Hao & Golding, 2006; Supplementary Methods). We also constructed parsimony

198 networks of the chloroplast and mitochondrial datasets using TCS 1.21 (Clement & al.,

199 2000) for obtaining detailed information and visualization of the haplotype distribution

200 across species with underlying taxonomic information (Supplementary Methods).

201

202 Phylogenetic analyses. — Consensus sequences from electropherograms were

203 assembled and sequences were aligned manually using BioEdit 7.0.0 Sequence

204 Alignment Editor (Hall, 1999). Three matrices were obtained (chloroplast,

205 mitochondrial and nuclear) and analyzed to detect variable sites and informative

206 positions. Since no phylogenetic incongruence was found between any of the four

207 organellar regions (see Supplementary Methods; Table S4), they were all combined into

208 a single matrix. In contrast, the topological conflict between the ITS and the organellar

209 regions involving well-supported lineages meant that these datasets could not be

210 combined (see Supplementary Methods; Table S4). For efficiency in the phylogenetic

211 analyses, we minimized redundant sequences, eliminating identical sequences from the

212 same population in the organellar dataset. In the case of ITS, in which only one

213 individual per population was sampled, one copy of each of the different cloned

214 sequences from each individual was retained; in practice, this meant one in most cases

215 and two in a few cases. As a result, from all 1770 organellar sequences, we obtained a

216 non-redundant matrix containing 289 organellar sequences, and from all 1650 cloned

217 individuals, we obtained a non-redundant matrix with 169 ITS sequences. These

218 sequences were deposited in Genbank (Table S3). To check for the influence of

219 allopolyploid taxa on the non-monophyly of clades, N. tortifolius, N. dubius and N.

220 miniatus were excluded to create an organellar non-redundant matrix without

221 allopolyploids. For ITS, sequences from the hybrids N. ×alleniae and N. ×pujolii were

222 also excluded to create a non-redundant matrix without allopolyploids. In addition, a

223 smaller matrix containing only chloroplast sequences (the reduced chloroplast ndhF-

224 matK dataset) was used for estimating divergence times and diversification rate shifts.

225 This was obtained by trimming the non-redundant chloroplast matrix to include only

226 one sequence per species (or subspecies if any) and when possible retaining the earliest

227 diverging sequence. Sequences from non-established hybrids were also excluded from

228 this reduced matrix that contained 105 sequences, but two extra outgroup species

229 (Asparagus cochinchinensis (Lour.) Merr. and Nothoscordum dialystemon (Guagl.)

230 Crosa) were added to calibrate the trees.

231 Maximum parsimony (MP) analyses were conducted with PAUP version

232 4.0a152 for Macintosh (Swofford, 1999) using the heuristic search option with 100

233 replicate searches, each with random addition of sequences and tree bisection

234 reconnection (TBR). In each replicate, 50 parsimonious trees were saved with length

235 equal to or shorter than the most parsimonious trees obtained in a single addition

236 heuristic search. Clade support was estimated using 104 fast stepwise-addition replicates

237 (Mort & al., 2000).

238 The best available model of molecular evolution for each DNA region was

239 selected using hierarchical likelihood ratio tests (hLRT) and Akaike information

240 criterion (AIC) as implemented in MrModeltest2.3 (Nylander, 2002). GTR+I+G (Yang,

241 1996), the symmetrical model with some sites assumed to be invariable and variable

242 sites assumed to follow a discrete gamma distribution, was the best fit for all datasets.

243 This model was used to perform Bayesian analyses using MrBayes 3.2.6 (Huelsenbeck

244 & Ronquist, 2001; Ronquist & al., 2012). Four Markov chain Monte Carlo (MCMC)

245 simulations were run simultaneously for 50 x 106 generations and sampled every 100

246 generations. We confirmed that the likelihoods had reached a steady phase with Tracer

247 1.3.1 (Rambaut & Drummond, 2007), and 25% of the total number of trees was

248 discarded as burn-in. The remaining sample was used to create a 50% majority-rule

249 consensus tree. The program FigTree 1.4 (http://tree.bio.ed.ac.uk/software/figtree/) was

250 used for visualization of trees obtained from parsimony and Bayesian analyses.

251

252 Molecular dating. — Absolute lineage divergence times in Narcissus were

253 estimated in BEAST (Drummond & al., 2012) using a Bayesian relaxed clock-model.

254 We used the reduced chloroplast ndhF-matK dataset (N=105) because the level of

255 variation of the mitochondrial data is too low and we opted not to use ITS sequences

256 since generation times and polyploidy may affect rates of homogenization (Álvarez &

257 Wendel, 2003) and thus the molecular clock. We used the following settings: a non-

258 partitioned dataset with GTR+I+G as a substitution model, birth-death speciation

259 model, and uncorrelated lognormal relaxed clock (UCLD). Two replicate MCMC

260 searches of 30 million generations each were run under these settings and their results

261 pooled using LogCombiner v. 1.7.2 (after removing 25% of samples as burn-in). We

262 used Tracer 1.6 (Rambaut & al., 2014) to determine stationarity of the Markov chain

263 and to verify that all parameters had effective sampling sizes (ESSs) >200.

264 TreeAnnotator v1.4.2 and FigTree v. 1.3.1, respectively, were used to produce and

265 visualize the resulting maximum clade credibility (MCC) tree. Two external calibration

266 points based on secondary metacalibration of Angiosperm clades (Magallón & al.,

267 2015) were used to obtain absolute divergence times: (a) The root node, i.e., the crown

268 age of Amaryllidaceae or the divergence between Asparagus and the rest of the tree,

269 was constrained to 62.49 Ma (49.18 -76.72) and (b) the stem age of Amaryllidoideae, as

270 inferred from the divergence time estimated between subfamilies Allioideae and

271 Amaryllidoideae, represented by Nothoscordum L. and Crinum L., respectively, in

272 Magallón & al. (2015), was set at 55.21(40.23-70.17) Ma. In our dataset, this node is

273 represented by the divergence between Nothoscordum and all Amaryllidoideae genera:

274 Nerine, Lapiedra, Leucojum, Sternbergia, and Narcissus.

275

276 RESULTS

277 Sequence data quality tests. — Tests for recombination in ITS sequences

278 detected significant events only for thirteen accessions (Supplementary Methods; Table

279 S5), ten of them falling in the same clade in the BI ITS tree. No recombination events

280 were detected in the chloroplast or mitochondrial sequences. With respect to the

281 possible occurrence of pseudogenes, inspection of conserved motifs, secondary models,

282 sequence integrity of the 5.8S gene, methylation sites (Fig. S1) and GS contents (Fig.

283 S2) revealed that only three sequences of N. tortifolius1 could be identified as potential

284 pseudogenes (Supplementary Methods). Since the exclusion of these three sequences in

285 preliminary analyses did not affect the results (see Fig. 1 and 2), they were maintained

286 in the final matrices. The mutation rate in ITS differs from that of the organellar

287 sequences, which was particularly evident when comparing sequences of subg.

288 Hermione among themselves and against sequences of subg. Narcissus (Fig. S3).

289

290 Nucleotide characteristics and variation of organellar sequences. — The

291 aligned organellar DNA matrix was comprised of 3285 bp, 1285 from the chloroplast

292 regions and 2000 from the mitochondrial regions (Table 1). Of 178 variable sites, 83

293 were parsimony informative (including outgroups). The two chloroplast regions

294 contained more informative characters than the mitochondrial regions

295 (ndhF>matK>cob>atpA), but the latter were particularly useful for discriminating

296 sequences between subgenera Hermione and Narcissus. Nucleotide diversity (π) was

297 higher in the chloroplast than in the mitochondrial regions. No gaps were required for

298 the alignment of the sequences. Average GC content was higher in the mitochondrial

299 than in the chloroplast regions (Table 1). Fig. S4 illustrates the distribution of plastid

300 DNA haplotypes across species in a statistical parsimony network constructed using the

301 algorithm described in Templeton & al. (1992).

302

303 Phylogenetic analyses of organellar sequences. — BI analyses based on 289

304 sequences generated 24010 trees for the combined non-redundant organellar DNA

305 matrix with a log likelihood (lnL) score of –10239.23. Values of the Potential Scale

306 Reduction Factor (PSRF) were always equal or close to 1. MP analysis yielded 283

307 equally parsimonious trees of 352 steps with a consistency index (CI) of 0.74 excluding

308 non-informative characters and a retention Index (RI) of 0.86. Because BI and MP

309 analyses of the organellar sequences yielded identical topologies and comparable levels

310 of branch support, only BI trees are shown.

311 Narcissus is recognized as a monophyletic lineage with high support values

312 (100% PP, 98% BS) and two large, highly supported lineages (Fig. 1). The first lineage

313 (100% in both analyses) largely corresponds to subg. Hermione and included taxa with

314 a main basic chromosome number x = 5 and a likely derived one of x = 11 (Fig. 1). The

315 second lineage corresponds to subgenus Narcissus (100% PP, 100% BS) and contained

316 all the remaining taxa with the main basic chromosome number x = 7 and a likely

317 derived one of x = 13 (Fig. 1).

318 Hermione is divided into two moderately supported groups (I and II in Fig. 1A).

319 Group I (100% PP, 78% BS) includes most species of sect. Tazettae with the exception

320 of N. elegans (Haw.) Spach and some specimens of N. tazetta L. Group II (77% PP,

321 67% BS) includes three highly supported subgroups: (1) the monospecific sect. Serotini

322 (100% in both analysis) including N. serotinus L. and the hybrid N. ×alentejanus

323 Fern.Casas (N. cavanillesii × N. serotinus); (2) a heterogeneous group (100% in both

324 analyses) containing the remainder of the accessions of N. tazetta L. plus N. elegans, the

325 allopolyploid N. miniatus Donn.-Morg., Koop. & Zonn. (N. elegans × N. serotinus), and

326 two hybrids, N. ×alleniae Donnison-Morgan (N. miniatus × N. viridiflorus) and N.

327 ×perezlarae Font Quer (N. cavanillesii × N. miniatus); and (3) the monotypic sect.

328 Aurelia (100% in both analyses) with N. broussonetii Lag., which was placed in a

329 trichotomy with the two other groups in the Bayesian analysis (99% PP), but was

330 resolved as sister to them in the parsimony analysis (79% BS).

331 The second main lineage (subg. Narcissus) is divided into six major groups (III

332 to VIII). Group III (Fig. 1A; 100% in both analyses) is sister to the other groups (IV-

333 VIII; 100% PP, 82% BS) and includes only sect. Apodanthi. Groups IV-VIII are placed

334 in two large clades (IV-V and VI-VIII) (Fig. 1A-B). The first clade (groups IV-V, 100%

335 PP, 82% BS) includes a single accession of N. bulbocodium subsp. quintanilhae2

336 A.Fern., isolated from the other accessions of N. bulbocodium L. (100% in both

337 analyses), which is sister to a clade (100% in both analyses) showing a trichotomy at its

338 base consisting of (1) one supported clade (100% in both analyses) including N.

339 viridiflorus Schousb. (sect. Jonquillae;) plus one accession of the hybrid N. ×alleniae

340 (N. miniatus × N. viridiflorus); (2) one moderately supported clade (82% PP, 67% BS)

341 containing two subgroups with some samples from sect. Jonquillae (100% PP, 71% BS)

342 and those of N. dubius and N. tortifolius from sect. Tazettae (83% PP, 67% BS; note

343 that they are classified as subg. Hermione) plus the hybrid N. ×pujolii (N. assoanus ×

344 N. dubius). These two groups, (1) and (2), are collectively labeled as IV (Fig. 1A). (3)

345 The third, well-supported, lineage (100% PP, 82% BS; labeled as Group V; Fig. 1A)

346 includes most specimens from sect. Bulbocodii.

347 The second large clade of subg. Narcissus (groups VI-VIII; 68% PP, 57% BS;

348 Fig. 1B) includes two weakly supported clades (VI and VII-VIII). Group VI includes

349 the remainder of the species of sect. Bulbocodii (76% PP, 54% BS; Fig. 1B). The clade

350 encompassing groups VII and VIII is weakly supported (66% PP, 55% BS) and has a

351 trichotomy at its base. Two of the three branches of that trichotomy are named as group

352 VII, the first including sect. Braxireon with N. cavanillesii Barra & G. López and its

353 two hybrids (100% in both analyses), and the second branch containing two clades, one

354 encompassing the remainder of the species of sect. Jonquillae (100% PP, 69% BS) and

355 the other including accessions of sect. Pseudonarcissi from southern Spain (100% PP,

356 85% BS; Fig. 1B). Group VIII is a polytomy (100% in both analyses) composed of the

357 two species of sect. Narcissus (not forming a clade), the monophyletic sect. Ganymedes

358 (100% in both analyses) and the remaining taxa of sect. Pseudonarcissi arranged in two

359 different placements (Fig. 1B). Several subspecies (and varieties) do not fall together

360 and should be further investigated.

361

362 Nucleotide characteristics and variation of ITS sequences. — Total length of

363 the ITS1+5.8S+ITS2 sequences varied from 841 to 870 bp ITS1 and ITS2 showed a

364 comparable length and number of variable and informative characters (Table 1).

365 Alignment of the matrix required the introduction of gaps, especially in sequences

366 belonging to subg. Hermione. The largest indels required a 12-bp deletion in ITS 1 in

367 six accessions of N. elegans, N. serotinus, N. miniatus and N. ×alleniae and an 8-bp

368 insertion occurring in the ITS2 of 39 sequences of subg. Hermione and sect. Apodanthi.

369 The number of 1-bp indels was higher in ITS2 than in ITS1 (Table 1) because 8 of the

370 11 indels needed for outgroup alignment are concentrated in that region. The 5.8S

371 region had a total length of 163 bp except in three sequences of N. tortifolius1

372 (Supplementary Methods). Average GC content was 63.74%. Sequences of subg.

373 Hermione had higher values of nucleotide diversity than those of subg. Narcissus

374 (0.07433 vs. 0.00554). Only six of the 163 individuals sampled rendered more than one

375 different ITS sequence, four of them corresponding to hybrids or species of hybrid

376 origin (N. tortifolius, N. ×pujolii, N. ×alleniae, N. bulbocodium subsp. obesus1, N.

377 ×rozeirae, N. segurensis Ríos, D.Rivera, Alcaraz & Obón). Conversely, the same

378 cloned sequence was found in different taxa in twenty cases; in sixteen of them one of

379 the individuals sharing the sequence was a hybrid (Table S6). Despite their shorter

380 length, ITS sequences yielded higher nucleotide diversity than the organellar sequences

381 (0.065 vs. 0.007).

382

383 Phylogenetic analyses of ITS sequences. — Bayesian analyses of 169 ITS

384 cloned sequences produced 17537 trees with a lnL score of –12413.67. PSRF values

385 were always close to 1. MP analysis yielded 25 equally parsimonious trees of 528 steps

386 with a consistency index (CI) of 0.85 excluding non-informative characters and a

387 retention index (RI) of 0.99. Since the topology of the BI tree is the same as that of the

388 MP tree, only the BI tree is shown. As in the organellar tree, two major, strongly

389 supported lineages were recovered that largely correspond to subgenera Hermione and

390 Narcissus (Fig. 2), although the composition of the groups indicated in roman numerals

391 differ between the two trees.

392 In the subg. Hermione lineage (100% PP, 96% BS), two major groups were

393 retrieved (labeled as Group I and II in Fig. 2A) that were not congruent with those

394 found in the organellar DNA tree. Group I (100% in both analysis) included sect.

395 Serotini in addition to N. elegans, N. miniatus and the hybrid N. ×alleniae (Fig. 2A).

396 Group II (100% in both analysis) included the remainder of the species of sect. Tazettae

397 and sect. Aurelia (Figs. 1, 2).

398 The subg. Narcissus lineage (100% PP, 87% BS) was divided into five major

399 groups (III -VII). Group III included all species of sect. Apodanthi and, as in the

400 organellar tree, was sister to the remainder of the groups (84% PP, 75% BS; Fig. 2A).

401 Group IV (100% PP, 90% BS; Fig. 2A) included some but not all species of sect.

402 Bulbocodii (100% PP, 88% BS) and was sister to a large poorly supported clade (Group

403 V to VII; 82% PP, 88% BS) that involved a polytomy at its base in which most

404 branches correspond to the remaining species of sect. Bulbocodii (Group V). The two

405 remaining branches of the polytomy corresponded to groups VI and VII (Fig. 2A-B).

406 Group VI is poorly supported (66% PP, 56% BS) and includes two lineages: one formed

407 by sect. Braxireon (100% in both analyses) and by some species of sect. Jonquillae

408 (100% PP, 85% BS), and the other containing the remainder of the species of sect.

409 Jonquillae together with sect. Ganymedes (100% PP, 98% BS) nested within them.

410 Group VII included all species of sect. Pseudonarcissi (86% PP, 88% BS; Fig. 2B),

411 unlike the organellar tree where they were distributed in clades VII and VIII (Fig. 1B).

412 Removing potential pseudogenes or recombinant sequences did not affect the

413 topology of the ITS tree or the main results as shown in the two inserts (Fig. 2A-B).

414 Interestingly, of 163 cloned individuals only six contained more than one ITS copy (see

415 sample names shaded in Fig. 2A-B). Some of these copies fell into different clades and

416 mostly corresponded to taxa of hybrid origin (with the exception of N. bulbocodium

417 subsp. obesus1).

418

419 Incongruence between ITS and organellar trees. — The comparison of the

420 topologies of the organellar and ITS trees revealed that several well-supported groups

421 differ in their phylogenetic placement (Fig. 3). The most evident discordance is the

422 placement of N. tortifolius and N. dubius which fell into different subgenera in the two

423 trees. These two species, classically considered part of sect. Tazettae will be discussed

424 below. Five other supported groups differed in their placement between the two

425 phylogenies: sect. Serotini, sect. Ganymedes, sect. Jonquillae, sect. Bulbocodii and sect.

426 Pseudonarcissi (Fig. 3).

427 To sort out the incongruence between the ITS and organellar trees we

428 distinguish between discrepancies in taxon integrity and in phylogenetic position. The

429 incongruence that is mainly due to phylogenetic position is arranged into different

430 categories depending on two criteria. First, the extent of the incongruence, i.e., whether

431 it affects a single individual or most samples from one species or a group of species

432 forming one lineage. Second, the degree of the incongruence estimated in terms of the

433 disparity, or phylogenetic distance, between the positions where the element in question

434 (sample, species, multi-species lineage) falls in the two phylogenies. We refer to deep

435 incongruence when the positions in the two phylogenies are in different major lineages

436 (subg. Hermione or Narcissus). Medium incongruence refers to a disparate position of

437 the element in question in the two phylogenies that involves different small or medium

438 lineages within the same major lineage. Our phylogenetic study of Narcissus includes

439 cases that can be referred to those categories, and are noted in the discussion section.

440

441 Karyological study. — Chromosome counts confirmed two basic chromosome

442 numbers: x = 5 for subg. Hermione and x = 7 for subg. Narcissus with several derived

443 numbers (Table S3). Within subg. Hermione, diploid, tetraploid and hexaploid levels

444 were recorded. A derived basic number x = 11 was found in N. papyraceus Ker Gawl.,

445 s.l. (2n = 22) and N. broussonetii Lag. (2n = 22, 44); the two species have a similar

446 chromosome structure differing only in two pairs of chromosomes. Within subg.

447 Narcissus, polyploidy was detected in all sections except Apodanthi and Ganymedes

448 which are diploid throughout (2n = 14). Nevertheless, diploid cytotypes (74%)

449 outnumbered polyploid cytotypes (Table S3). Up to four different chromosome numbers

450 were found in a single population of N. bulbocodium, which shows the highest

451 chromosome number (2n = 72) in this study. Derived chromosome numbers also occur

452 in several populations of N. bulbocodium subsp. obesus (Salisb.) Maire (2n = 26, 39). B

453 chromosomes were found in several individuals from different sections (Table S3).

454 Remarkably, N. poeticus L. and N. radiiflorus Salisb. are diploid with chromosomes

455 similar to the diploid species of sect. Pseudonarcissi.

456 Putative hybrids have either the same chromosome number as their parents (e.g.,

457 N. asturiensis (Jord.) Pugsley × N. bulbocodium subsp. obesus, 2n= 14) or an

458 intermediate chromosome number (e.g., N. bulbocodium subsp. obesus × N. calcicola

459 Mendonça 2n= 26), but allopolyploids have their own chromosome numbers: N. dubius

460 (2n = 50), N. tortifolius (2n = 36), N. miniatus (2n=30).

461

462 Molecular dating. —The Bayesian divergence time analysis performed in

463 BEAST provided time estimates of 16.05 Ma (10.56–23.51, 95% HPD) for the stem age

464 of Narcissus, dating the origin of the genus to the Middle Miocene (Fig. S5). The age

465 for the split into two subgenera (crown age) is 13.80 Ma (8.97–19.08, 95% HPD; node 3

466 in Fig. S5) whereas the initial diversification within subgenera Narcissus and Hermione,

467 i.e. their crown ages, is 9.99 Ma (6.54–14.12, 95% HPD; node 5) and 5.98 Ma (3.31–

468 9.24, 95% HPD; node 4), respectively (Fig. S5 and Supplementary Methods). These

469 estimates for early diversification in Narcissus lie between the Middle and Late

470 Miocene. Within subg. Narcissus, the majority of the most diversified lineages,

471 including both groups of sect. Jonquillae (Fig. S5, nested within node 12, and node 7),

472 both groups of sect. Pseudonarcissi (Fig. S5, nested within node 10 and 12), and both

473 groups of sect. Bulbocodii (Fig. S5, nodes 13 and14), diverged in the Pliocene, whereas

474 the less diversified lineages (sections Ganymedes, and Apodanthi, Fig. S5, nodes 6 and

475 11) have their crown age in the Pleistocene or late Pliocene. In subg. Hermione, the

476 diversification of its two main groups occurs at two different times. The crown age for

477 the first lineage (Fig. S5, node 9), which includes sections Aurelia and Serotini and

478 some species of sect. Tazettae, can be dated to the Late Pliocene, while the age inferred

479 for the second group formed exclusively by the remainder of the species of sect.

480 Tazettae is Early Pleistocene (Fig. S5, node 8 and Supplementary Methods). We did not

481 detect any significant diversification rate-shift in the plastid data (Fig. S6, Table S7,

482 Supplementary Methods).

483

484 DISCUSSION

485 Two consistent lineages within Narcissus. — Our phylogenetic analyses, based

486 on a comprehensive sampling of sequences from the three genomes, confirms the

487 monophyly of Narcissus, anticipated in Meerow & al. (2006), and the existence of two

488 lineages largely corresponding to subgenera Hermione and Narcissus (Fig. 1, 2), also

489 reported in recent works (Graham & Barrett, 2004; Pérez & al., 2004; Rønsted & al.,

490 2008; Santos-Gally & al., 2012). These two lineages were also suggested in Fernandes’

491 (1968) classical cytogenetic studies revealing that Hermione has a main basic

492 chromosome number of x = 5 along with chromosomes of heterogeneous length,

493 including acrocentric chromosomes. Narcissus has a main basic chromosome number of

494 x = 7 along with a karyotype generally composed of long chromosomes and no

495 acrocentric chromosomes. Apart from the allopolyploids N. dubius and N. tortifolius

496 (and other established hybrids), which show more complex patterns due to hybridization

497 (see below), all species consistently fell either in the Hermione or the Narcissus lineage

498 in both the nuclear and organellar phylogenies,.

499 There are also differential morphological, phenological and distributional

500 patterns. In general, species of the Hermione lineage flower in autumn or late winter and

501 have tubular flowers with white tepals and a short corona, while representatives of the

502 Narcissus lineage are mostly spring-flowering species with cup-shaped or tubular

503 flowers, yellow tepals and a medium to large corona (Fernandes, 1968). Subgenus

504 Narcissus is both morphologically more diverse and more species-rich than Hermione.

505 In addition, species of subg. Hermione frequently span large ranges extending from the

506 Atlantic coast of Morocco throughout the Mediterranean basin. Subgenus Narcissus has

507 diversified in the western Mediterranean and many species are restricted to the Iberian

508 or Moroccan mountains (Santos-Gally & al., 2012). Thus, based on all this evidence, we

509 recommend classification of Narcissus into the two subgenera Hermione and Narcissus.

510

511 Major systematic conclusions from phylogenetic analyses. — At the

512 infrageneric level, there is good agreement between taxonomic assignment and

513 phylogenetic lineages in some groups (Fig. 1-3). Besides the two subgenera, sections

514 Apodanthi, Serotini as well as the monotypic Braxireon appear as well-defined lineages

515 each with a single origin. The case of sect. Ganymedes is worth noting since, although it

516 is monophyletic in both the organellar and ITS trees, unexpectedly its position in the

517 two trees is not the same. It is close to sections Pseudonarcissi and Narcissus in a

518 polytomy at the base of clade VIII in the organellar tree (Fig. 1B), and is nested within

519 sect. Jonquillae in the ITS tree (Fig. 2A). A similar situation occurs in sect. Aurelia,

520 which is sister to sections Serotini and Tazettae in the organellar tree (Fig. 1A) but is

521 nested within sect. Tazettae in the ITS tree (Fig. 2A).

522 In contrast, our phylogenetic analyses identified unexpected lineages that

523 question the integrity of four important sections (Bulbocodii, Jonquillae, Pseudonarcissi

524 and Tazettae) and defy the current morphologically based classifications. Two large

525 sections, Jonquillae and Bulbocodii, consist of two different lineages that are not closest

526 relative to each other, both in the organellar and nuclear trees; a third, Pseudonarcissi, is

527 only monophyletic in the ITS tree; and Tazettae is split into three lineages in the

528 organellar and into two in the ITS trees (Figs. 1-3).

529 The two clades of sect. Jonquillae are consistent – except for one uncertain

530 placement (see below) – with the previous cytogenetic identification of two different

531 types of karyotypes that were used to define subsections Jonquillae and Juncifolii

532 (Fernandes, 1939). The former grouped karyotypes with long chromosomes including

533 N. jonquilla L., N. fernandesii, N. cordubensis Fern.Casas, N. willkommii (Samp.)

534 A.Fern., N. viridiflorus and N. cerrolazae Ureña, while the latter grouped karyotypes

535 with short chromosomes including N. assoanus s.l., N. baeticus Fern.Casas and N.

536 gaditanus Boiss. & Reut. The only uncertainty in the matching between the two

537 subsections and our phylogenetic results is the placement of the only sample of N.

538 baeticus within subsect. Jonquillae instead of Juncifolii in the ITS tree (Fig. 2A).

539 Overall, our results support Fernandes’ groups (Fernandes, 1939) and question the

540 integrity of sect. Jonquillae.

541 Section Bulbocodii, encompassing the hoop petticoat daffodils, also consists of

542 two main groups in the two phylogenies (Fig. 1-3). There are some indications of a

543 geographical pattern for these two groups in the organellar tree. The group labeled as V

544 in Fig. 1A includes yellow-flowered species widespread in the Iberian Peninsula and

545 northern Morocco, with several ploidy levels. The second group of sect. Bulbocodii,

546 labeled as VI in Fig. 1B, includes southern Iberian and Moroccan, mostly white or

547 creamy-yellow-flowered species endemic to mountains regions. It is likely that these

548 two groups reflect two previously undetected differentiated lineages of hoop petticoat

549 daffodils, but an alternative explanation involving hybridization cannot be totally

550 rejected. Therefore, despite the clear identification of two lineages, more taxonomic

551 work is needed to fully understand the diversity, and particularly species delimitation,

552 within sect. Bulbocodii.

553 Section Pseudonarcissi, encompassing the trumpet daffodils, is the third

554 important group of species in which an unexpected result was obtained, calling into

555 question its taxonomic cohesiveness. The pattern and interpretation are less clear than

556 for sections Bulbocodii and Jonquillae because in the ITS phylogeny, all trumpet

557 daffodils fall together within the same lineage whereas in the organellar phylogeny the

558 pattern is more complex. In the latter tree, a group of species from southern Spain forms

559 a clade sister to a part of sect. Jonquillae (Fig. 1B, group VII) and separate from the

560 northern trumpets section.. This phylogenetic split between southern and northern

561 species of Pseudonarcissi is also supported by the fact that the two groups have distinct

562 environmental niches (Marques & al., 2008). The exception among the southern species

563 in the organellar tree is N. bujei Fern.Casas of which the sequences fall into two

564 different clades. Some of the sequences are part of a polytomy at the base of a clade

565 with most northern trumpet daffodils while the rest are sister to N. alpestris Pugsley in

566 another subclade within clade VIII (Fig. 1B). An old divergence between the northern

567 and southern groups and ILS could explain this pattern in which N. bujei would have

568 originated at more or less the same time when the northern and southern lineages

569 diverged. However, this hypothesis is compromised by the fact that ILS is less likely to

570 occur in organellar trees than in nuclear trees due to lower effective population sizes.

571 Alternatively, a hybridization event between the southern trumpet lineage and sect.

572 Jonquillae after the divergence of N. bujei, with subsequent chloroplast capture, is also

573 a plausible scenario for explaining the data. Based on genome size data, Zonneveld

574 (2008, 2010) proposed a new sect. Nevadensis to include N. bujei, N. nevadensis

575 Pugsley, N. longispathus Pugsley, N. alcaracensis Ríos, D.Rivera, Alcaraz & Obón, N.

576 segurensis and N. yepesii Ríos, D.Rivera, Alcaraz & Obón and also suggested an

577 ancient hybrid origin for N. bujei with N. longispathus and N. hispanicus Gouan as

578 parents. However, the molecular evidence provided here for N. bujei, which does not

579 easily fit Zonneveld’s taxonomic proposal (Zonneveld, 2008; 2010), and the

580 inconclusive morphological evidence recommend further studies to elucidate the origin

581 of N. bujei (Zonneveld, 2010).

582 For sect. Tazettae, the organellar-nuclear discordance related to N. dubius and N.

583 tortifolius is easy to interpret and to accommodate taxonomically since these two

584 species are intersubgeneric allopolyploids (see below). In contrast, the other distinct

585 lineages of sect. Tazettae do not allow a straightforward interpretation. The fact that

586 subg. Hermione contains several allopolyploid species makes it difficult to obtain

587 monophyletic lineages unless excluding those allopolyploids. To assess how

588 allopolyploids could influence the monophyly of sections in subg. Hermione, we carried

589 out parsimony and Bayesian analyses of the organellar and nrDNA ITS matrices

590 excluding N. miniatus, N. tortifolius and N. dubius. In the case of ITS, we also excluded

591 the hybrids N. ×alleniae and N. ×pujolii (Figs., 1, 2, S7, S8). The results show that

592 three sections (Serotini, Tazettae, and the monotypic sect. Angustifolii Fernandes

593 containing N. elegans; see below) constitute monophyletic groups provided that we

594 disregard the likely hybridogenous sect. Aurelia (see below).

595 Our study also has implications for the composition of a fourth section,

596 Narcissus, which is nested within sect. Pseudonarcissi which questions its taxonomic

597 integrity; in the organellar tree the samples of sect. Narcissus do not even form a

598 monophyletic group. From a karyological and molecular point of view it is not possible

599 to separate sect. Narcissus from sect. Pseudonarcissi, but morphologically they are

600 quite distinct. Species of sect. Narcissus usually have single flowers, pure white tepals

601 with a discoidal to cup-shaped, intensely red or orange corona (Blanchard, 1990), which

602 is similar to the floral morphology of members of subg. Hermione, although no

603 molecular evidence supports this relationship. Other similarities between N. poeticus

604 and species of sect. Pseudonarcissi are found in the karyotypes (Fernandes, 1968; 1975)

605 and genome size values (Zonneveld, 2010), which are often identical. Therefore, the

606 evidence available indicates merging of these two sections. Yet, the fact that N.

607 poeticus, along with the other members of this section, has a long history of cultivation

608 and artificial selection, requires caution when discussing relationships.

609 The fact that several subspecies (and varieties) often do not group together (e.g.,

610 N. bulbocodium subsp. obesus; N. nobilis var. leonensis (Pugsley) A.Fern., among

611 others) should be further investigated because this may reflect poor taxonomic

612 placement.

613

614 Evidence of hybridization in the evolution of Narcissus. — In addition to

615 questioning the current circumscription of the four sections, we found support for the

616 existence of hybridization, including allopolyploid speciation. Incongruence due to

617 discordant placement of the same taxa in the nuclear and organellar trees is a significant

618 finding, which has systematic and evolutionary implications. Incongruence between tree

619 topologies from genes with different inheritance can be due mainly to hybridization or

620 ILS. Telling apart these two phenomena is complex both for recent and old divergence

621 and hybridization events. A number of useful approaches have been proposed to tackle

622 this (Maureira-Butler & al., 2008; Kubatko, 2009; Joly & al., 2009; Pelser & al., 2010;

623 Escudero & al., 2014; Payseur & Rieseberg, 2016). However, these approaches tend to

624 generate an excess of false positives, identifying lineages as hybrids when they are not

625 (Heled & al., 2013). Additionally, they have mostly been tested on simulated data

626 and/or only on datasets of very limited size (Jones & al., 2014). Because of the size and

627 complexity of our Narcissus data sets, we analyzed the phylogenetic results together

628 with our own independent evidence based on cytogenetic data, morphology and

629 reproductive biology (Marques & al., 2010; 2016), as well as with previous evidence

630 from various sources (see introduction) in order to identify the causes of the underlying

631 incongruence (Wendel & Doyle, 1998). Below we only present highly likely cases of

632 hybridization in Narcissus revealed by phylogenetic incongruence and supported by

633 independent data, but further specific studies are needed to support other potential

634 ‘minor’ cases of hybridization (e.g., N. ×hannibalis, N. ×tuckeri, N. subsp. obesus × N.

635 calcicola, among others). To facilitate discussion, we group them into deep and medium

636 incongruences following the categories described in the results. A general observation

637 resulting from this work is that putative cases of hybridization are not restricted to

638 closely-related species.

639

640 Deep incongruence— Narcissus dubius (2n = 50; distributed from southeastern

641 Spain to southern France) and N. tortifolius (2n = 36; endemic to a few populations in

642 southeastern Spain; Barra & López-González, 1982; Barra, 1999; Sánchez Gómez & al.,

643 2000) have classically been assigned to sect. Tazettae based on morphological

644 characters (e.g., Fernandes, 1969, 1975; Webb, 1980), or recognized as a separate sect.,

645 Dubii (Fernández Casas, 1983; 1984). However, in our study only the ITS tree supports

646 the relationships with sect. Tazettae (subg. Hermione, Fig. 2A, group II), whereas in the

647 organellar tree, they are placed with other species of sect. Jonquillae (subg. Narcissus;

648 Figs. 1A, group IV). This pattern supports the hypothesis that both species are

649 intersubgeneric allopolyploids. Furthermore, our results suggest that plants of sect.

650 Jonquillae (either an ancestral tetraploid or an unreduced form of N. jonquilla) might

651 have acted as female parent and N. papyraceus (or a close species) as male parent. This

652 parentage is reciprocal to that hypothesized earlier based on the close karyological and

653 morphological similarity with N. tazetta (Fernandes, 1937; 1969; 1975; Koopowitz &

654 al., 2002; Romero & al., 1983). The fact that these two allopolyploids are completely

655 fertile (Koopowitz & al., 2002) suggests that their origin is not recent.

656 Narcissus ×alleniae (N. miniatus × N. viridiflorus), N. ×alentejanus (N.

657 cavanillesii × N. serotinus), N. ×perezlarae (N. cavanillesii × N. miniatus) —

658 Hybridization, supported by morphological and experimental independent evidence

659 explains these three examples of deep incongruence. These hybrids have organellar

660 sequences identical or very similar to their female parent (Fig. 1A), and in the ITS

661 phylogeny they also appear with one or, in the case of N. ×alleniae, two progenitors

662 (Fig. 2A). Preservation of both the maternal and paternal ITS sequences is probably

663 facilitated by the different genomic location of ribosomal loci in the parental genomes,

664 which belong to different subgenera, that can hamper homogenization of ITS sequences

665 (Nieto Feliner & Rosselló, 2012), although stabilized hybrids such as N. ×perezlarae

666 can have distinct ITS copies (Marques & al., 2010). Our chromosome counts support

667 the hybrid nature in these cases since N. ×alleniae, N. ×alentejanus and N. ×perezlarae

668 have chromosome numbers intermediate between their parents (Table S3; see also

669 Marques & al., 2010 for the latter two examples).

670 Another example of an intersubgeneric hybrid confirmed by our phylogenetic

671 study is N. ×pujolii originating from hybridization between N. assoanus and N. dubius.

672 The positions where the cloned sequences of this hybrid fall in the ITS phylogeny

673 reflect the placement of its parental species, i.e., with N. dubius in the subg. Hermione

674 lineage and with N. assoanus in the subg. Narcissus lineage (Fig. 2A, group II), whereas

675 in the organellar phylogeny it groups with N. dubius and N. tortifolius (Fig. 1A, group

676 IV).

677

678 Medium incongruence —In the ITS tree, sect. Ganymedes groups with sect.

679 Jonquillae, while in the organellar tree it groups with several trumpet daffodils (sect.

680 Pseudonarcissi) (Figs. 1B, 2A, 3). An ancient hybridization event to explain this

681 incongruence is plausible for morphological reasons. The nodding flowers and reflexed

682 tepals of N. triandrus L. and its allies resemble some trumpet daffodils, such as N.

683 cyclamineus, and suggest a member of sect. Pseudonarcissi as one progenitor of sect.

684 Ganymedes. The close position in the ITS tree together with its karyotype suggests that

685 the maternal progenitor was N. jonquilla s.l.

686 Narcissus miniatus falls into different lineages within subg. Hermione in the

687 organellar and nuclear phylogenies (Figs. 1A, group II; Fig. 2A, group I) because of its

688 allohexaploid nature (2n = 30), its progenitors being N. elegans (2n = 20) and N.

689 serotinus (2n = 10) (Díaz Lifante & al., 2009; Marques & al., 2010). Despite the earlier

690 interpretation that this species is a synonym of N. serotinus (Fernandes, 1951, 1968;

691 Phytos & Kamari, 1974), recent cytogenetic evidence (Díaz Lifante & al., 2009) is

692 consistent with the phylogenetic results provided here (Figs. 1, 2) and in Marques & al.,

693 (2010). In the ITS tree, sequences of N. serotinus, N. elegans and N. miniatus fall

694 together in the same branch, while in the organellar phylogeny, N. elegans and N.

695 miniatus share the same sequence while N. serotinus falls into another branch.

696 The monotypic sect. Aurelia (N. broussonetii) has a chromosomal structure close

697 to N. tazetta, differing by two pairs of chromosomes, which is consistent with its

698 placement in the ITS tree where it is nested within this group (Fig. 2A). In contrast, in

699 the organellar DNA tree, N. broussonetii grouped with N. elegans and N. serotinus (Fig.

700 1A). Consistent with this finding, it presents isobrachial long chromosomes which are

701 also present in N. serotinus and some species of sect. Apodanthi (Fernandes, 1940;

702 Fernandes, 1975). There is considerable evidence for a hybrid origin of N. broussonetii

703 and our phylogenetic results point towards N. tazetta s.l. and N. serotinus as possible

704 parents.

705 Narcissus calcicola1 (Table S3) represents another likely case of hybridization

706 or introgression, falling within its own sect. Apodanthi in the organellar tree (Fig. 1A,

707 group III) but together with N. gaditanus, within sect. Jonquillae, in the ITS tree (Fig.

708 2A, group VI).

709

710 Divergence age estimates inferred from molecular dating — The divergence

711 time estimates inferred from the chloroplast tree in BEAST suggest Neogene ages for

712 the main internal nodes (Fig. S5; Supplementary Methods). The early diversification of

713 Narcissus (13.80 Ma) occurred in the Middle Miocene in the transition between the

714 Langhian and Serravallian periods. During this period at the end of the Middle Miocene

715 Climatic Optimum (MMCO) c. 17–15 Ma, when the African and West European plates

716 were close, a drier and cooler climate was rapidly established that eliminated the

717 dominance of subtropical forests in the Mediterranean region. This event dramatically

718 affected the composition of Western Mediterranean flora and vegetation (Barrón & al.,

719 2010) resulting in extinction of a large number of Angiosperm lineages (Postigo Mijarra

720 & al., 2009). As a consequence of the climatic and biotic changes, the vegetation in the

721 Western Mediterranean (Iberian Peninsula and NW Africa) evolved towards open

722 grassland ecosystems (Jiménez-Moreno & al., 2010; Wolfe, 1975; Zachos & al., 2001).

723 In a climatic context of aridification, with dry summers and cold winters coupled with

724 bimodal rain seasons (spring and autumn), associated with increasing numbers of

725 grazing ungulate species in open woodlands (Domingo & al., 2014), the adaptive

726 advantage of geophytes, such as daffodils and other monocots, is evident (Fragman &

727 Shmida, 1997; Noy-Meir & Oron, 2001). The same timing for the colonization of the

728 Mediterranean region, often from African sources, has been inferred for other monocot

729 groups characterized by geophytic life-forms, like Androcymbium Willd. (Caujapé-

730 Castells & al., 2001) and subfamily Hyacinthoideae (Ali & al., 2013).

731 The distinct ages for the diversification of the two main lineages of Narcissus

732 (subgenera Hermione and Narcissus) suggest different biogeographic scenarios. Our

733 results date the early diversification of subg. Narcissus in the mid-Tortonian (9.99 Ma)

734 with a subsequent diversification in the Iberian Peninsula throughout the progressive

735 onset of the Mediterranean climate. Geologically, this took place after the break of the

736 arc connecting the East Iberian Peninsula and North Africa, before the final Pliocene

737 accretion of the Betic–Rif Cordilleras and the opening of the Alboran Sea (Lonergan &

738 White, 1997, Rosenbaum & al., 2002). It is tempting to hypothesize that the

739 predominant spring-flowering in this subgenus evolved as an innovation within the

740 climatic context of increasing seasonality and progressive onset of the Mediterranean

741 climate. The autumnal flowering of close relatives of Narcissus, such as those

742 represented by three of the four outgroups (Lapiedra martinezii, Sternbergia lutea,

743 Nerine bowdenii) and even species of Leucojum other than L. aestivum as well as in

744 other bulbs related to South African lineages, makes this hypothesis conceivable.

745 In contrast, the age estimate for the crown node of subg. Hermione is 5.98 Ma,

746 which corresponds to diversification starting in the late Messinian. During this period,

747 successive sea desiccation resulted in land connections between Mediterranean islands,

748 archipelagos and the mainland. This period has frequently been invoked to explain

749 lineage diversification, speciation and dispersal across the Mediterranean region, e.g., in

750 Androcymbium (Caujapé-Castells & Jansen, 2003), Smilax L. (Chen & al., 2014),

751 Festuca L. subg. Schenodorus (P.Beauv.) Peterm. (Inda & al., 2014). This scenario is

752 consistent with the widespread distribution of species of subg. Hermione in the

753 lowlands of the entire Mediterranean region, including some islands, e.g. Malta, Crete,

754 Cyprus (Mifsud & Caruana, 2010; Hand & al., 2011; Turland & al., 1993) that were

755 connected to the mainland only at that time (Krijsman & al., 1999).

756 Our mean age estimates are substantially more recent than those obtained by

757 Santos-Gally & al. (2012), i.e., 23.6, 21.6, 19.6 and 13.2 Ma for the stem ages of

758 Narcissus and the crown ages of Narcissus, as well as of subgenera Narcissus and

759 Hermione, respectively. Only in the first case (stem age of Narcissus), their mean

760 estimate falls within the 95% confidence intervals provided here. The reasons for these

761 discrepancies are likely the calibration, ours being based on the most recent and

762 complete dating of angiosperm lineages published thus far (Magallón & al., 2015), and

763 on the speciation model prior, ours being a birth-death process, which is more

764 appropriate when using phylogenetically distant calibration points to account for lineage

765 extinction.

766

767

768 TAXONOMIC IMPLICATIONS

769 According to the results discussed above, we consider that some taxonomic

770 adjustments are needed to provide formal recognition for those lineages that are strongly

771 supported in this study and by independent evidence, but that were not recognized by

772 most previous infrageneric classifications (see in Fig. S9 how our schematic proposal

773 matches the phylogenetic results). In three cases (sections Jonquillae, Pseudonarcissi,

774 Bulbocodii) these adjustments consist of subdividing sections into two to reflect natural

775 groups. Due to the disparate placement of the resulting segregated groups in the

776 phylogenetic hypotheses generated, they cannot be placed together in a single section

777 without introducing radical taxonomic changes in the infrageneric classification of the

778 entire genus. In comparison, the accommodation of lineages of hybrid origin in the

779 infrageneric treatment is more difficult, no matter whether they are monophyletic in the

780 two trees, since they could be placed in the section corresponding to either the maternal

781 or paternal lineage. This is aggravated when the hybrid lineage involves progenitors

782 from the two subgenera, as in the allopolyploids N. dubius and N. tortifolius. For this

783 reason, we have been conservative in maintaining those hybrid lineages as separate

784 sections, to reflect their dual placement and to preserve the monophyly of the maternal

785 and paternal sections. This is the case for sections Ganymedes, Dubii and Aurelia.

786 Another taxonomic change is needed because of the placement of the type

787 species of the genus, N. poeticus and its relative N. radiiflorus, in sect. Pseudonarcissi.

788 This finding requires changing the circumscription of sect. Narcissus to coincide with

789 what was, to date, sect. Pseudonarcissi. However, to maintain the recognition of the

790 morphologically characteristic trumpet daffodils, we propose the recognition of two

791 subsections even if subsect. Pseudonarcissi is paraphyletic.

792 For subg. Hermione, we are proposing a conservative infrageneric treatment

793 because the eastern Mediterranean and further eastern areas have not been sufficiently

794 sampled for N. tazettae and because of the suspected effects of allopolyploid taxa on the

795 phylogenetic trees. Further studies might eventually recommend recognizing just a

796 single section within subg. Hermione. However, we prefer to be conservative at this

797 point trying to reconcile the evolution of the genus with morphological characters.

798 The placement of each species in the newly proposed infrageneric taxonomic

799 treatment can be seen below and the correspondence between different treatments is

800 shown in Table S1 (see also Fig. S9).

801

802 Narcissus L., Sp. Pl.: 289. 1753 – Type N. poeticus L.

803 Subgenus Narcissus

804 Nine sections are recognized:

805

806 Narcissus sect. Braxireon (Raf.) Valdés in Lagascalia 12(2): 274. 1984 – Type

807 N. cavanillesii Barra & López

808 Species included in our phylogeny: Narcissus cavanillesii (Cav.) Barra & G.

809 López

810

811 Narcissus sect. Apodanthi A.Fern. in Bol. Soc. Brot. ser. 2 40: 241. 1966 – Type

812 N. rupicola Dufour

813 Species included in our phylogeny: Narcissus albimarginatus D. Müll.-Doblies

814 & U. Müll.-Doblies, N. atlanticus Stern, N. calcicola, N. cuatrecasasii Fern.Casas, N.

815 cuatrecasasii var. segimonensis Fern.Casas, N. marvieri Jahand. & Maire, N. rupicola,

816 N. scaberulus Henriq., N. watieri Maire.

817

818 Narcissus sect. Jonquilla DC. in Redouté, Liliac. 8: 126. 1816 – Type N.

819 jonquilla L.

820 Species included in our phylogeny: Narcissus cerrolazae, N. cordubensis, N.

821 fernandesii, N. fernandesii var. major A.Fern., N. jonquilla, N. jonquilla var. henriquesii

822 Samp., N. viridiflorus, N. willkommii.

823

824 Narcissus sect. Ganymedes (Salisb. ex Haw.) Schult. & Schult. fil. in Roem. &

825 Schult. in Syst. Veg. 7: 952. 1830 – Type N. triandrus L.

826 Species included in our phylogeny: Narcissus triandrus, N. triandrus var.

827 loiseleurii (Rouy) A.Fern., N. triandrus subsp. lusitanicus (Dorda Alcaraz &

828 Fern.Casas) Barra, N. triandrus subsp. pallidulus (Graells) Rivas Goday

829

830 Narcissus sect. Juncifolii (A.Fern.) Zonn., Pl. Syst. Evol. 275: 122. 2008 – Type

831 N. assoanus Dufour

832 Species included in our phylogeny: Narcissus assoanus, N. assoanus subsp.

833 praelongus Barra & G. López, N. baeticus*, N. assoanus subsp. rivas-martinezii

834 (Fern.Casas) Barra, Díez & Ureña*, N. gaditanus

835 We recognize the division of sect. Jonquilllae into two different sections based

836 on cytological, genomic and our own phylogenetic results. Zonneveld (2008) had

837 already proposed the placement of N. assoanus and N. gaditanus in sect. Juncifolii due

838 to substantial differences in genome size (< 20 pg in sect. Juncifolii and >30 pg in the

839 remaining species of sect. Jonquilllae). Zonneveld (2008) accepted the previous

840 criterion of Fernandes (1966, 1969) who split this section into two subsections based on

841 substantial karyotype differences.

842 *Narcissus baeticus requires further work since, as discussed above, it grouped

843 in sect. Juncifolii in the organellar tree but fell into sect. Jonquillae in the ITS tree. Our

844 phylogenetic results support the recent transfer of subsp. rivas-martinezii from N.

845 fernandesii to N. assoanus (Barra & al., 2016) since this subspecies fell within sect.

846 Juncifolii in both the organellar and the ITS trees.

847

848 Narcissus sect. Bulbocodii DC. in Redouté, Liliac. 8: 126. 1816. – Type N.

849 bulbocodium L.

850 Species included in our phylogeny: Narcissus bulbocodium, N. bulbocodium

851 subsp. graellsii, N. bulbocodium subsp. quintanillae*, N. jacquemoudii Fern.Casas, N.

852 jeanmonodii Fern.Casas, N. bulbocodium var. citrinus Baker, N. bulbocodium var.

853 nivalis (Graells) Baker, N. tingitanus Fern.Casas

854 *Note: This taxon requires further work to determine its correct placement,

855 although we include it here at this point since most of our populations grouped with the

856 remaining members of this section.

857

858 Narcissus sect. Meridionalis I. Marques, Fuertes, Martins-Loução, Moharrek, &

859 Nieto Fel., sect. nov. – Type N. cantabricus DC.

860 Species included in our phylogeny: Narcissus bulbocodium subsp. obesus*, N.

861 cantabricus, N. hedraeanthus, N. peroccidentalis Fern.Casas, N. romieuxii Braun-

862 Blanq.

863 This newly described section encompasses several species related to, and

864 traditionally included in, sect. Bulbocodii, which have here been revealed to constitute

865 an independent lineage. Morphologically, these species usually have creamy-yellow to

866 white concolor flowers, with usually exerted anthers and some degree of

867 zygomorphism, and are narrowly distributed in mountainous regions in Southern Iberia

868 and Morocco. The name Meridionalis (southern) refers to such a geographic pattern

869 compared to sect. Bulbocodii.

870 *Note: This taxon requires further study since different samples fell into the two

871 hoop petticoat daffodils groups (sections Bulbocodii and Meridionalis).

872

873 Narcissus sect. Narcissus

874

875 Narcissus Subsect. Narcissus

876 Species included in our phylogeny: Narcissus poeticus, N. radiiflorus

877 The inclusion of this group within sect. Pseudonarcissi had already been

878 detected in the phylogenetic study of Graham & Barrett (2004). Fernandes (1968) had

879 also remarked on the similarity of karyotypes. The genome size value measured in N.

880 poeticus (26 pg) is also within the range of values reported for sect. Pseudonarcissi by

881 Zonneveld (2008) who also stated that “it is the deviating morphology of the flower,

882 with a very short and orange coloured corona, that has prevented so far to unite these

883 sections”. Based on all evidence, we now include it as subsection of Narcissus.

884

885 Narcissus subsect. Pseudonarcissi (DC.) I. Marques, Fuertes, Martins-Loução,

886 Moharrek, & Nieto Fel., stat. nov. ≡ Narcissus sect. Pseudonarcissi DC. in Redouté,

887 Liliac. 8: 126. 1816 – Type N. pseudonarcissus DC.

888 Species included in our phylogeny: Narcissus abscissus (Haw.) Schult. &

889 Schult.f., N. alpestris, N. asturiensis, N. bicolor, N. bujei*, N. confusus Pugsley, N.

890 cyclamineus, N. hispanicus, N. hispanicus subsp. perez-chiscanoi (Fern.Casas)

891 Fern.Casas, N. jacetanus Fern.Casas, N. macrolobus (Jord.) Pugsley, N. minor L., N.

892 moleroi Fern.Casas, N. nanus (Haw.) Spach, N. nobilis, N. nobilis var. leonensis, N.

893 pallidiflorus Pugsley, N. primigenius (Fern. Suárez ex Laínz) Fern.Casas & Laínz, N.

894 pseudonarcissus, N. pseudonarcissus subsp. eugeniae (Fern.Casas) Fern.Casas, N.

895 pseudonarcissus subsp. munozii-garmendiae (Fern.Casas) Fern.Casas, N. portensis

896 Pugsley, N. pumilus Salisb.

897 *Note: Further work is necessary to determine its correct placement.

898

899 Narcissus sect. Nevadensis Zonn. in Pl. Syst. Evol. 275 (1–2): 125. 2008 – Type

900 N. nevadensis Pugsley

901 Species included in our phylogeny: Narcissus alcaracensis, N. longispathus, N.

902 nevadensis, N. segurensis, N. yepesii

903 Our results support the earlier subdivision of sect. Pseudonarcissi proposed by

904 Zonneveld (2008) into his new sect. Nevadensis based on substantial genome size

905 differences (30-39 pg in sect. Nevadensis vs. 22-26 pg in sect. Pseudonarcissi with the

906 exception of N. bicolor that has 68 pg).

907

908 Subgenus Hermione (Haw.) Spach

909 Five sections are recognized:

910

911 Narcissus sect. Serotini Parl., Fl. Ital. 3: 157. 1858 – Type N. serotinus L.

912 Species included in our phylogeny: Narcissus serotinus

913

914 Narcissus sect. Aurelia (Gay) Baker, Handb Amaryll.: 2. 1888 – Type N.

915 broussonetii Lagasca

916 Species included in our phylogeny: Narcissus broussonetii

917 In addition to the cytological and phylogenetic evidence for a hybrid origin, this

918 monotypic section has a distinct morphological feature: a vestigial corona with exerted

919 anthers whose filaments are attached to the floral tube just below the tepals (Blanchard,

920 1990).

921

922 Narcissus sect. Dubii Fern.Casas in Fontqueria 6: 39, 41. 1984 – Type: N.

923 dubius Gouan

924 Species included in our phylogeny: Narcissus dubius, N. tortifolius

925

926 Narcissus sect. Tazetta DC. in Redouté, Liliac. 8: 126 (1816) – Type N. tazetta

927 L.

928 Species included in our phylogeny: Narcissus tazetta*, N. pachybolbus

929 Durieu, N. papyraceus, N. pannizianus Parl., N. polyanthus Loisel.

930 *Note: As stated above, a deeper study of this species is needed to fully

931 understand its variation.

932

933 Narcissus sect. Angustifolii (A.Fern.) Fern.Casas in Anales Jard. Bot. Madrid

934 55(1): 174. 1997 – Type N. elegans (Haw.) Spach.

935 Species included in our phylogeny: Narcissus elegans, N. miniatus

936 This group was created by Abilio Fernandes as a monotypic subsection to

937 accommodate the tetraploid N. elegans, which he thought to be an ancient tetraploid that

938 should be differentiated from N. serotinus and the remaining species of section

939 Tazettae. Our phylogenetic results reveal different placements for N. elegans and N.

940 serotinus in the two trees (Fig. 1A, 2A) especially in the analyses without allopolyploids

941 (Fig. S7, S8). Therefore, we think it is appropriate to follow a conservative criterion and

942 recognize Fernandes’ section including the allopolyploid derivative N. miniatus.

943

944 ACKNOWLEDGEMENTS

945 The authors wish to dedicate this paper to the memory of Abílio Fernandes and Rosette

946 Batarda, whose pioneer work established the grounds for evolutionary studies in

947 Narcissus. The authors thank A. González Fernández de Castro, A. Susanna, A.

948 Tribsch, Á. Bueno, C. Herrera, C. Sérgio, C. Garcia, C. Lefebvre, C. Tauleigne-Gomes,

949 D. Draper, E. Triano, F. Domínguez, G. Kamari, G. Bacchetta, H. Nava, J.C. Moreno, J.

950 Feise, J. Mota, J. Pedrol, J. Pérez, J. Paiva, M. Luceño, M. Vincens, M. Lizana, M.

951 Serrano, M. Medrano, N. Membrives, P. Escobar, V. Valcárcel, and S. Jury for

952 supplying material, and I. Sanmartín for guidance on the dating analyses. The authors

953 also thank the helpful comments of four reviewers, M. Pirie and J. Kadereit, who have

954 contributed substantially to improve the manuscript. This work has been supported by a

955 FPVI European-funded Integrated Infrastructure Initiative "SYNTHESYS“ to IM (ES-

956 TAF 023-2004), FCT, Ministério de Ciência e Tecnologia, Portugal (PD/1245/2004),

957 plus grants from the CSIC-Comunidad de Madrid, Spain, (CCG07-CSIC/AMB-1978),

958 from the Spanish Ministerio de Ciencia y Tecnología (CGL2007-66516).

959

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1253 Table 1. Characteristics of the DNA regions sequenced (outgroups included). Values between brackets in the organellar regions refer to the

1254 partial data for each of the two sequenced regions.

Organellar regions Nuclear regions Regions cpDNA mitDNA Total ITS1 5.8S ITS2 Total (ndhF, matK) (cob, atpB) 1285 2000 Aligned length (bp) 3285 255 163 247 882 (738, 547) (978, 1022) 1285 2000 Length range (bp) 3285 230-251 159-163 232-242 841-870 (738, 547) (978, 1022) No. of variable characters 144 34 178 149 40 132 346 (97, 47) (10, 24) 73 10 No. of informative characters 83 49 93 40 156 (43, 30) (9, 1)

Total number of indels 0 0 0 11 1 15 27

No. of 1-bp indels 0 0 0 3 0 8 11

Maximum length of indel (bp) 0 0 0 12 6 8 -

31.51 43.42 % of GC content 37.47 70.20 56.41 70.44 63.74 (32.55, 30.47) (41.21, 45.64) 0.01647 0.00113 Nucleotide diversity (π) 0.00713 0.10576 0.02114 0.09638 0.06559 (0.02053, 0.01101) (0.00126, 0.00099)

1255 Figure captions:

1256 Figures 1A and 1B.- Bayesian tree (50% majority-rule consensus) of organellar DNA

1257 sequences (ndhF and matK chloroplast regions, cob and atpA mitochondrial regions) from a

1258 non-redundant matrix containing 289 sequences of Narcissus and four outgroups (see text).

1259 Posterior probabilities (expressed as percentages) and parsimony bootstrap support higher

1260 than 50% are indicated above and below branches, respectively. A thicker line indicates 100%

1261 support in both analyses. Black circles: >95% support value; grey circles: 95 ≥support value

1262 ≥70; white circles: <70 support value. Color-coded branches and vertical tags follow currently

1263 accepted sections as indicated in the legend. Roman numerals denote groups discussed in the

1264 main text.

1265 Figures 2A and 2B.- Bayesian tree (50% majority-rule consensus) of nuclear ribosomal ITS

1266 from a non-redundant matrix containing 169 cloned sequences from 163 samples of Narcissus

1267 and four outgroups (see text). Posterior probabilities (expressed as percentages) and

1268 parsimony bootstrap support higher than 50% are indicated above and below branches,

1269 respectively. A thicker line indicates 100% support in both analyses. Black circles: >95%

1270 support value; grey circles: 95≥ support value ≥70; white circles: <70 support value. Color-

1271 coded branches and vertical tags follow currently accepted sections as indicated in the legend.

1272 Roman numerals denote groups discussed in the main text. Shaded names highlight the

1273 positions of different ITS copies from the same cloned individual. * indicates potential

1274 pseudogene sequences. rec indicates potential recombinant sequences. Grey inserts show

1275 differences in the ITS tree when potential pseudogenes and recombinant sequences were

1276 removed.

1277 Figure 3. Major positional and compositional incongruence between 50% majority-rule

1278 consensus BI trees from organellar DNA (left) and nrDNA ITS (right) data sets. Major clades

1279 (identified with roman numerals following Figures 1 and 2) are collapsed, dotted lines link

1280 clades (or rarely grades) of congruent composition in the two phylogenetic trees, and thick

1281 solid lines indicate sections which fall into separate clades in one or both trees. Support for

1282 branches follows Figures 1 and 2. Sections are labeled according to the infrageneric

1283 classification we followed at the beginning of this study (largely based on Fernandes, 1968).

1284 See Fig. S9 for a redrawn figure with the new sectional taxonomy here proposed.

1285

1286

Aurelia N. bulbocodium1 N. bulbocodium2 Apodanthi Fig. 1B N. bulbocodium3 N. bulbocodium4 Braxireon N. bulbocodium5 Bulbocodii N. bulbocodium6 N. bulbocodium7 Ganymedes N. bulbocodium8 Jonquillae N. bulbocodium9 N. bulbocodium10 Narcissus N. bulbocodium11 Pseudonarcissus N. tenuifolius N. bulbocodium12 Serotini N. bulbocodium13 Tazettae N. bulbocodium14 N. bulbocodium subsp. obesus10 N. bulbocodium subsp. obesus11 V N. bulbocodium var. citrinus N. ×felineri (bulbocodium × primigenius) N. ×brevitubulosus (asturiensis × bulbocodium var. nivalis N. bulbocodium var. nivalis1 N. bulbocodium15 N. bulbocodium16 N. bulbocodium var. nivalis2 N. jacquemoudii N. bulbocodium subsp. graellsii N. bulbocodium17 N. bulbocodium18 N. bulbocodium subsp. quintanilhae1 N. bulbocodium subsp. obesus12 N. jeanmonodii N. tingitanus N. bulbocodium19 N. bulbocodium20 N. bulbocodium21 N. ×barrae (bulbocodium × cantabricus) N. bulbocodium22 N. bulbocodium23 N. bulbocodium24 N. fernandesii1 N. willkommii1 N. jonquilla1 N. jonquilla2 N. jonquilla3 N. jonquilla4 N. jonquilla5 N. jonquilla6 N. jonquilla7 N. jonquilla8 subgen. Narcissus N. jonquilla var. henriquesii N. cordubensis N. fernandesii2 N. fernandesii3 N. fernandesii4 N. fernandesii × N. papyraceus N. willkommii2 N. willkommii3 N. willkommii4 N. fernandesii var. major IV N. cerrolazae N. dubius1 N. dubius2 N. dubius3 N. dubius4 N. tortifolius1 N. tortifolius2 N. tortifolius3 N. tortifolius4 N. ×pujolii (assoanus × dubius) N. viridiflorus1 N. viridiflorus2 N. viridiflorus3 N. viridiflorus4 N. viridiflorus5 N. viridiflorus6 N. ×alleniae1 (miniatus × viridiflorus) N. bulbocodium subsp. quintanilhae2 N. calcicola1 N. calcicola2 N. calcicola3 N. scaberulus1 N. scaberulus2 N. ×carringtonii (scaberulus × triandrus subsp. pallidulus) N. rupicola1 N. rupicola2 N. rupicola3 N. rupicola4 N. rupicola5 N. rupicola × N. triandrus III N. cuatrecasasii1 N. cuatrecasasii2 N. cuatrecasasii var. segimonensis N. cuatrecasasii × N. triandrus N. watieri1 N. watieri2 N. atlanticus N. albimarginatus1 N. albimarginatus2 N. marvieri1 N. marvieri2 N. ×alleniae1 (miniatus× viridiflorus) N. ×alleniae2 (miniatus× viridiflorus) N. elegans1 N. elegans2 N. elegans3 N. elegans4 N. elegans5 N. elegans6 N. miniatus1 N. miniatus2 N. miniatus3 N. miniatus4 N. ×perezlarae (cavanillesii × miniatus) N. tazetta1 II N. tazetta2 N. tazetta3 N. broussonetii1 N. broussonetii2 N. broussonetii3 N. broussonetii1 N. broussonetii2 N. broussonetii3 N. ×alentejanus (cavanillesii × serotinus) N. serotinus1 N. serotinus2 N. serotinus3 N. serotinus4 N. pannizianus1 N. pannizianus1 N. papyraceus2 N. papyraceus3 subgen. Hermione N. papyraceus4 N. papyraceus5 N. papyraceus6 N. polyanthos N. papyraceus7 I N. papyraceus8 N. pachybolbus N. papyraceus9 N. papyraceus10 N. broussonetii1 N. papyraceus11 N. broussonetii2 N. tazetta4 N. broussonetii3 Sternbergia lutea N. tazetta5 N. broussonetii1 N. broussonetii2 Leucojum aestivum N. broussonetii3 Lapiedra martinezii Nerine bowdenii 0.0030 N. asturiensis1 N. asturiensis1 Aurelia N. minor2 N. asturiensis × N. obesus Apodanthi N. asturiensis1 Braxireon N. asturiensis1 Bulbocodii N. asturiensis1 N. asturiensis1 Ganymedes N. asturiensis1 Jonquillae N. primigenius1 N. primigenius2 Narcissus N. nobilis var. leonensis1 Pseudonarcissus N. portensis Serotini N. ×somedanus (asturiensis × triandrus) N. tortuosus Tazettae N. nobilis var. leonensis2 N. cyclamineus1 N. cyclamineus2 N. minor2 N. nanus N. asturiensis1 N. cyclamineus3 N. pseudonarcissus1 N. pseudonarcissus2 N. pseudonarcissus3 N. pseudonarcissus4 N. pseudonarcissus5 N. abscissus1 N. abscissus2 N. hispanicus N. hispanicus subsp. perez-chiscanoi N. pseudonarcissus subsp. munozii-garmendiae N. ×hannibalis (hispanicus × triandrus subsp. lusitanicus) N. nobilis1 N. nobilis2 N. pallidiflorus1 N. pallidiflorus2 N. pseudonarcissus subsp. eugeniae1 N. pseudonarcissus subsp. eugeniae2 N. bicolor1 N. bicolor2 VIII N. bujei1 N. bujei2 N. jacetanus N. bujei3 N. pseudonarcissus subsp. eugeniae3 N. confusus N. bujei4 N. macrolobus N. pumilus N. triandrus subsp. pallidulus1 N. triandrus subsp. pallidulus2 N. triandrus subsp. pallidulus3 N. triandrus subsp. pallidulus4 N. triandrus subsp. pallidulus5 N. triandrus subsp. pallidulus6 N. triandrus1 N. triandrus2 N. triandrus3 N. triandrus7 N. triandrus var. loiseleurii N. triandrus4 N. ×rozeirae (bulbocodium × triandrus subsp. pallidulus) N. ×susannae (cantabricus × triandrus subsp. pallidulus) N. triandrus subsp. lusitanicus1 N. triandrus subsp. lusitanicus2 N. triandrus subsp. lusitanicus3 N. triandrus subsp. lusitanicus4 N. triandrus × N. bulbocodium N. triandrus5 N. triandrus6 N. bujei1 N. bujei2 N. bujei3 N. bujei4 N. alpestris N. moleroi N. radiiflorus N. poeticus1 N. poeticus2 N. nevadensis1

N. nevadensis2 Subgen. Narcissus N. nevadensis3 N. nevadensis4 N. nevadensis5 N. nevadensis6 N. nevadensis7 N. alcaracensis1 N. alcaracensis2 N. alcaracensis3 N. longispathus1 N. longispathus2 N. segurensis N. yepesii1 N. longispathus3 N. longispathus4 N. longispathus5 N. yepesii2 N. yepesii3 VII N. yepesii4 N. fernandesii var. rivas-martinezii N. assoanus subsp. praelongus N. gaditanus1 N. gaditanus2 N. gaditanus3 N. gaditanus4 N. assoanus1 N. assoanus2 N. assoanus3 N. assoanus4 N. baeticus N. cavanillesii1 N. cavanillesii2 N. cavanillesii3 N. cavanillesii4 N. cavanillesii5 N. cavanillesii6 N. ×alentejanus (cavanillesii × serotinus) N. ×perezlarae (cavanillesii × miniatus) N. romieuxii1 N. romieuxii2 N. romieuxii3 N. peroccidentalis N. cantabricus1 N. cantabricus2 N. cantabricus3 N. cantabricus4 N. hedraeanthus1 N. hedraeanthus2 N. ×cazorlanus (hedraeanthus × triandrus subsp. pallidulus) VI N. ×tuckeri1 (fernandesii × hedraeanthus) N. ×tuckeri2 (fernandesii × hedraeanthus) N. bulbocodium subsp. obesus1 N. bulbocodium subsp. obesus2 N. bulbocodium subsp. obesus3 N. bulbocodium subsp. obesus4 N. bulbocodium subsp. obesus5 N. bulbocodium subsp. obesus × N. calcicola N. bulbocodium subsp. obesus6 N. bulbocodium subsp. obesus7 Fig. 1A N. bulbocodium subsp. obesus8 N. bulbocodium subsp. obesus9 N. triandrus subsp. pallidulus1 N. triandrus subsp. pallidulus2 N. triandrus1 Fig. 2B N. triandrus subsp. lusitanicus1 N. triandrus subsp. lusitanicus2 Aurelia N. triandrus subsp. lusitanicus3 N. triandrus × bulbocodium N. triandrus2 Apodanthi N. triandrus2 N. triandrus3 Braxireon N. triandrus3 N. triandrus subsp. loiseleurii Bulbocodii N. triandrus subsp. loiseleurii rec Ganymedes N. ×rozeirae (bulbocodium × triandrus subsp. pallidulus) Jonquillae N. viridiflorus1 N. viridiflorus2 Narcissus N. viridiflorus3 Pseudonarcissus N. viridiflorus4 Serotini N. viridiflorus5 Tazettae N. ×alleniae1 (miniatus× viridiflorus) N. jonquilla1 N. jonquilla2 VI N. cerrolazae / N. jonquilla var. henriquesii N. willkommii1 / N. fernandesii var. major N. fernandesii1 N. fernandesii2 N. fernandesii × papyraceus N. cordubensis N. baeticus N. gaditanus1 / N. gaditanus2 N. calcicola1 N. assoanus subsp. praelongus N. fernandesii var. rivas-martinezii N. ×pujolii (assoanus × dubius) N. assoanus1 N. assoanus2 subgen. Narcissus N. cavanillesii1 N. cavanillesii2 N. cavanillesii3 N. cavanillesii4 N. cavanillesii5 N. ×alentejanus (N. cavanillesii × N. serotinus) N. ×perezlarae (N. cavanillesii × N. miniatus) N. hedraeanthus N. ×tuckeri (fernandesii × hedraeanthus) rec N. tenuifolius N. hedraeanthus N. peroccidentalis N. tenuifolius N. romieuxii1 N. peroccidentalis N. bulbocodium subsp. obesus1 N. romieuxii1 N. bulbocodium var. citrinus rec V N. bulbocodium subsp. obesus1 N. ×barrae (bulbocodium × cantabricus) rec N. ×cazorlanus (hedraeanthus × triandrus subsp. pallidulus) N. ×cazorlanus (hedraeanthus × triandrus subsp. pallidulus) N. cantabricus1 N. cantabricus1 N. ×susannae (cantabricus × triandrus subsp. pallidulus) N. ×susannae (cantabricus × triandrus subsp. pallidulus) N. cantabricus2 N. cantabricus2 N. bulbocodium subsp. quintanilhae1 rec N. bulbocodium subsp. quintanilhae2 rec N. bulbocodium1 N. bulbocodium subsp. graellsii rec N. jacquemoudii rec N. tingitanus rec / N. bulbocodium var. nivalis1 rec IV / N. ×felineri (bulbocodium × primigenius) rec rec N. ×brevitubulosus (asturiensis × bulbocodium var. nivalis) N. jeanmonodii rec N. bulbocodium1 N. bulbocodium2 N. bulbocodium2 N. bulbocodium subsp. obesus2 N. bulbocodium subsp. obesus2 N. bulbocodium subsp. obesus × N. calcicola N. bulbocodium subsp. obesus × N. calcicola N. ×rozeirae (bulbocodium × triandrus subsp. pallidulus) rec N. bulbocodium subsp. obesus1 N. bulbocodium subsp. obesus1 N. cuatrecasasii1 N. cuatrecasasii var. segimonensis N. cuatrecasasii2 N. cuatrecasasii × triandrus N. atlanticus N. albimarginatus1 N. albimarginatus2 N. rupicola1 N. rupicola2 III N. rupicola × N. triandrus N. watieri N. marvieri / N. scaberulus1 / N. ×carringtonii (scaberulus × triandrus subsp. lusitanicus) N. scaberulus2 N. calcicola2 N. calcicola3 N. tazetta1 / N. papyraceus1 N. papyraceus2 N. pachybolbus N. polyanthos / N. broussonetii1 N. broussonetii2 N. papyraceus3 N. papyraceus4 N. ×pujolii (assoanus × dubius) / N. dubius1 N. pannizianus II N. dubius2 N. ×pujolii (assoanus × dubius) N. tortifolius2 N. dubius1 N. dubius2 N. tortifolius1* N. tortifolius1* N. tortifolius2 N. tazetta2 N. tazetta3 N. tazetta4 N. tazetta5 N. serotinus1 N. serotinus2 N. serotinus3 subgen. Hermione N. serotinus4 N. miniatus1 N. ×alleniae1 (serotinus × viridiflorus) N. miniatus2 I N. miniatus3 N. miniatus4 N. elegans1 N. elegans2 N. elegans3 N. elegans4 Lapiedra martinezii Leucojum aestivum Sternbergia lutea Nerine bowdenii 0.03 Aurelia Apodanthi N. tortuosus Braxireon N. pseudonarcissus subsp. eugeniae Bulbocodii N. primigenius Ganymedes N. nobilis var. leonensis1 N. nobilis1 Jonquillae N. pumilus Narcissus N. pallidiflorus1 Pseudonarcissus N. minor N. nanus Serotini N. jacetanus Tazettae / N. asturiensis1 N. ×somedanus (asturiensis × triandrus) N. asturiensis × N. obesus N. asturiensis2 N. radiiflorus N. poeticus / N. cyclamineus1 N. segurensis VII N. yepesii1 N. yepesii2 N. longispathus1 N. alcaracensis N. segurensis N. longispathus2 / N. longispathus3 N. bujei1 N. bujei2 N. nevadensis1

N. nevadensis2 subgen. Narcissus / N. nevadensis3 N. nevadensis4 N. nevadensis5 N. pseudonarcissus subsp. perezchiscanoi N. pseudonarcissus1 N. pseudonarcissus subsp. munozii-garmendiae N. pseudonarcissus2 N. hispanicus N. ×hannibalis (hispanicus × triandrus subsp. lusitanicus) N. alpestris N. moleroi N. abscissus N. macrolobus N. portensis N. bicolor1 N. confusus Fig. 2A

1099 Supplementary Information

1100 Table S1. Comparison of recent taxonomic infrageneric classifications of Narcissus,

1101 including the one proposed in this study.

1102 Table S2. List of botanical names of Narcissus used in the study.

1103 Table S3. Taxa sampled arranged according to the traditional infrageneric classification

1104 largely based on Fernandes (1968), including populations sampled, chromosome numbers

1105 (2n) counted in this study (B- presence of B chromosome; *- new count for the taxon),

1106 number of individuals used to count chromosomes, number of individuals sampled for

1107 chloroplast and mitochondrial regions (orgDNA), number of non-redundant orgDNA

1108 sequences used in the phylogenetic analysis, number of individuals sampled for ITS, number

1109 of clones retrieved per individual, number of non-redundant clones used in the phylogenetic

1110 analyses, and GenBank accession numbers.

1111 Table S4. Results obtained from the pairwise incongruence lenght difference (ILD) tests.

1112 Numbers indicate probability of congruence between partitions. Numbers between brackets

1113 indicate probabilities without considering N. dubius and N. tortifolius sequences. Asterisks

1114 denote significant incongruence (p<0.05).

1115 Table S5. Significance values for recombinant sequences detected by several algorithms

1116 implemented in RDP.

1117 Table S6. List of populations sharing an identical cloned ITS sequence with other populations

1118 of the same taxon or with other taxa. See also Fig. 2. Underlined taxa represent reported

1119 hybrids between different sections.

1120 Table S7. Speciation (λ) and extinction (μ) rates for (a) the entire tree, (b) subg. Hermione,

1121 and (c) subg. Narcissus based on a reduced chloroplast ndhF-matK dataset.

1122

1123

1124 Figure S1. Potential methylation sites within ITS1, 5.8S and ITS2 of Narcissus.

1125 Figure S2. GC contents and estimated free energies in 5.8S, ITS1 and ITS2, among the

1126 different taxonomic groups. Group legend: BRA: Braxireon, JON: Jonquillae, APO:

1127 Apodanthi, GAN: Ganymedes, BUL: Bulbocodii, PSE: Pseudonarcissi, SER: Serotini, AUR:

1128 Aurelia and TAZ: Tazettae. The two groups of Bulbocodiii, Jonquillae and Pseudonarcissi

1129 are plotted together, and sect. Narcissus is plotted within Pseudonarcissi since no significant

1130 differences were found (p < 0.001).

1131 Figure S3. Distance-matrix rate test plot comparing nrDNA ITS and organellar rates of

1132 evolution (see Supplementary Methods). A) Global plot including all pairwise comparisons

1133 between all accessions of Narcissus. B) Pairwise comparisons between accessions of subg.

1134 Hermione. C) Pairwise comparisons between accessions of subgenera Hermione and

1135 Narcissus. D) Pairwise comparisons between accessions of subg. Narcissus.

1136 Figures S4A and 4B. Statistical parsimony haplotype networks based on 1750 Narcissus

1137 sequences concatenated from two chloroplast regions (ndhF and matK). Small circles denote

1138 single mutational steps. Color-coded shades identify the different sections as in Figs. 1 and 2.

1139 White rectangular boxes represent haplotypes, including the species where they occur

1140 (identified by a red number) and the number of sequences corresponding to that haplotype

1141 within each species.

1142 Figure S5. Bayesian molecular dating of Narcissus. Maximum clade credibility tree obtained

1143 with BEAST indicating median divergence times and 95% confidence intervals for the main

1144 lineages from concatenated plastid DNA (ndhF-matK) sequences. Mean temperature variation

1145 across the Cenozoic as inferred by Zachos et al (2008). MMCO = Middle Miocene Climatic

1146 Optimum. Clade numbers refer to calibration points (red) and lineages described in

1147 Supplementary Methods.

1148 Figure S6. Maximum clade credibility (MCC) tree from a BEAST analysis of plastid DNA

1149 sequences (ndhF-matK) of Narcissus with branches colored by speciation rate ( sp/Ma)

1150 across the tree estimated using Bayesian Analysis of Macroevolutionary Mixtures (BAMM).

1151 Branch numbers refer to clades described in Table S7.

1152 Figure S7. Bayesian tree (50% majority-rule consensus) of organellar DNA sequences (ndhF

1153 and matK chloroplast regions, cob and atpA mitochondrial regions) from a non-redundant

1154 matrix excluding the allopolyploids N. dubius, N. miniatus and N. tortifolius. Posterior

1155 probabilities (expressed as percentages) and parsimony bootstrap support higher than 50% are

1156 indicated above and below branches, respectively. A thicker line indicates 100% support in

1157 both analyses. Black circles: >95% support value; grey circles: 95 ≥support value ≥70; white

1158 circles: <70 support value. Color-coded branches and vertical tags follow currently accepted

1159 sections as indicated in the legend. Roman numerals denote groups discussed in the main text.

1160 Figure S8. Highlight of the Bayesian tree (50% majority-rule consensus) of nuclear

1161 ribosomal ITS of subg. Hermione from an analysis based on a non-redundant matrix

1162 excluding the allopolyploids N. dubius, N. miniatus and N. tortifolius (and the hybrids

1163 N. ×alleniae and N. ×pujolii). Posterior probabilities (expressed as percentages) and

1164 parsimony bootstrap support higher than 50% are indicated above and below branches,

1165 respectively. A thicker line indicates 100% support in both analyses. Black circles: >95%

1166 support value; grey circles: 95 ≥support value ≥70; white circles: <70 support value. Color-

1167 coded branches and vertical tags follow currently accepted sections as indicated in the legend.

1168 Roman numerals denote groups discussed in the main text.

1169 Figure S9. New subgeneric classification proposed here plotted on the 50% majority-rule

1170 consensus BI trees from the organellar DNA (left) and nrDNA ITS (right) data sets presented

1171 in Figure 3. Major clades (identified with roman numerals following Figures 1 and 2) are

1172 collapsed, dotted lines link clades (or rarely grades) of congruent composition in the two

1173 phylogenetic trees, and thick solid lines indicate sections which fall into separate clades in

1174 one or both trees. . Support for branches follows symbols in Figures 1 and 2.

Table S1

Table S1. Comparison of recent taxonomic infrageneric classifications of Narcissus, including the one proposed in this study. Subg: subgenus; Sect: section; Subsect: subsection.

Flora Proposed in Fernandes 1968 Webb 1978 Blanchard 1990 Mathew 2002 Zonneveld 2008 RHS 2016 Iberica this study 2016 Subg. Sect. Subsect. Sect. Subsect. Sect. Subsect. Subg. Sect. Subsect. Series Subg. Sect. Sect. Sect. Sect. Subsect.

Serotini Serotini Serotini Serotini Serotini Serotini Serotini Serotini Angustifolii Angustifoliae Angustifoliae Angustifoliae Angustifolii Hermione Tazettae Tazettae Hermione Hermione Tazettae Tazettae Hermione Tazettae Tazettae Tazettae Tazettae Hermione Albiflorae Dubii Aurelia Aurelia Aurelia Hermione Hermione Aurelia Hermione Aurelia Aurelia Apodanthi Apodanthae Apodanthi Apodanthi Apodanthi Apodanthi Apodanthi Jonquilla Jonquillae Jonquillae Jonquillae Jonquilla Jonquilla Jonquilla Jonquillae Jonquillae Jonquilla Jonquillae Juncifolii Apodanthi Chloranthi Juncifolii Juncifolii Tapeinanthus Tapeinanthus Tapeinanthus Tapeinanthus Tapeinanthus Tapeinanthus Braxireon Braxireon Ganymedes Ganymedes Ganymedes Narcissus Ganymedes Ganymedes Ganymedes Ganymedes Ganymedes Bulbocodii Bulbocodium Bulbocodium Bulbocodium Corbularia Bulbocodium Bulbocodium Bulbocodium Bulbocodii Meridionalis Pseudonarcissi Pseudonarcissus Narcissus Pseudonarcissi Pseudonarcissus Pseudonarcissi Pseudonarcissus Pseudonarcissus Pseudonarcissus Pseudonarcissi Reflexi Nevadensis Nevadensis Narcissus Narcissus Narcissus Narcissus Narcissus Narcissus Narcissus Narcissus Narcissus Narcissus Narcissus Narcissus

Chloranthi

Table S2

Table S2. List of botanical names of Narcissus followed in the study. N. abscissus (Haw.) Schult. & Schult.f., Syst. Veg. 7ii: 941 (1830). N. albimarginatus D. Müll.-Doblies & U. Müll.-Doblies, in Fl. Pl. Africa: 50(2), pl. 1986 (1989), N. alcaracensis Ríos, D.Rivera, Alcaraz & Obón, in Bot. J. Linn. Soc. 131(2):160 (1999). N. alpestris Pugsley, in J. Roy. Hort. Soc. 58(1): 79 (1933). N. assoanus Dufour ex Schult. & Schult.f., Syst. Veg. 7ii, p. 962 (1830). N. assoanus subsp. praelongus Barra & G. López, in Anales Jard. Bot. Madrid 39: 209 (1982). N. assoanus subsp.rivas-martinezii (Fern.Casas) Barra, Díez & Ureña,, in Flora Montiberica 63: 105 (2016). N. asturiensis (Jordan) Pugsley, in J. Roy. Hort. Soc. 58(1): 40 (1933). N. atlanticus Stern, in Daffodil & Tulip Year Book 16: 25 (1950). N. baeticus Fern.Casas, in Fontqueria 1: 11 (1982). N. bicolor L., Sp. Pl., ed. 2, 1: 415 (1762). N. broussonetii Lag., Gen. Sp. Pl.: 13 (1816). N. bujei Fern.Casas, in Fontqueria 2: 33 (1982). N. bulbocodium L., Gen. Sp. Pl.: 13 (1816). N. bulbocodium subsp. graellsii (Webb ex Graells) K. Richt., Pl. Eur. 1: 237 (1890). N. bulbocodium subsp. obesus (Salisb.) Maire in Jahand. & Maire, Cat. Pl. Maroc: 138 (1931) N. bulbocodium subsp. quintanilhae A.Fern., in Bol. Soc. Brot. ser. 2, 60: 305 (1987). N. bulbocodium var. citrinus Baker, in Florist Pomol.: 68 (1880). N. bulbocodium var. nivalis (Graells) Baker, Handb. Amaryll.: 3 (1888). N. calcicola Mendonça, in Compt.-Rend. Hebd. Séances Mém. Soc. Biol. 96: 1253 (1927). N. cantabricus DC. in Redouté, Liliac. 8: [126] (1816). N. cavanillesii Barra & G. López, in Anales Jard. Bot. Madrid 41: 202 (1984). N. cerrolazae Ureña, in Bot. Complut.19: 84 (1994). N. confusus Pugsley, in J. Roy. Hort. Soc. 58(1) 59 (1933). N. cordubensis Fern.Casas, in Fontqueria 1: 10 (1982). N. cuatrecasasii Fern.Casas, Laínz & Ruiz Rejón, in Cuad. Ci. Biol.2 (1): 4 (1973). N. cuatrecasasii var. segimonensis Fern.Casas, in Fontqueria 3: 27 (1983). N. cyclamineus DC. in Redouté, Liliac.8: [126] (1816). N. dubius Gouan, Ill. Observ. Bot.: 22 (1773). N. elegans (Haw.) Spach, Hist. Nat. Vég. 12: 452 (1846). N. fernandesii Pedro, in Bol. Soc. Brot. ser. 2, 21: 60 (1947). N. fernandesii var. major A.Fern., in Bol. Soc. Brot. ser. 2, 40: 239 (1966). N. gaditanus Boiss. & Reut. in Boiss, Diagn. Pl. Orient. ser. 2, 4: 96 (1859). N. hedraeanthus (Webb & Heldr.) Colmeiro, Enum. Pl. Peníns. Hispano-Lusit. 5: 80 (1889). N. hispanicus Gouan, Ill. Observ. Bot.: 23 (1773). N. hispanicus subsp. perez-chiscanoi (Fern. Casas) Fern. Casas in Fontqueria 55: 270 (2005) N. jacetanus Fern.Casas, in Fontqueria 5: 35 (1984). N. jacquemoudii Fern.Casas, in Fontqueria 10: 11 (1986). N. jeanmonodii Fern.Casas, in Fontqueria 10: 9 (1986). N. jonquilla L., Sp. Pl.: 290 (1753). N. jonquilla var. henriquesii Samp., Fl. Portugal, ed. 2: 803 (1939). N. longispathus Pugsley, in J. Roy. Hort. Soc. 58(1): 54 (1933). N. macrolobus (Jord.) Pugsley, in J. Roy. Hort. Soc. 58(1): 71 (1933). N. marvieri Jahand. & Maire, in Bull. Soc. Hist. Nat. Afrique N. 16: 70 (1925). N. miniatus Donn.-Morg., Koop. & Zonn., in Daffodil, Snowdrop & Tulip Yearb. 2005-2006: 19-25 (2005). N. minor L., Sp. Pl., ed. 2, 1: 415 (1762). N. moleroi Fern.Casas, in Fontqueria 14: 21 (1987). N. nanus (Haw.) Spach, Hist. Nat. Vég. 12: 433 (1846). N. nevadensis Pugsley, in J. Roy. Hort. Soc. 58(1): 62 (1933). N. nobilis (Haw.) Schult. & Schult.f., Syst. Veg. 7ii: 939 (1830). N. nobilis var. leonensis (Pugsley) A.Fern., in Daffodil & Tulip Year Book 33: 61 (1968). N. pachybolbus Durieu, in Rev. Bot. Recueil Mens. 2: 425 (1846). N. pallidiflorus Pugsley, in J. Roy. Hort. Soc. 58(1): 69 (1933). N. pannizianus Parl., Fl. Ital. 3: 128 (1858). N. papyraceus Ker Gawl., in Bot. Mag. 24, t. 947 (1806). N. peroccidentalis Fern.Casas, in Fontqueria 44: 256 (1996). N. poeticus L., Sp. Pl.: 289 (1753). N. polyanthos Loisel., in J. Bot. (Desvaux) 2: 277 (1809). N. portensis Pugsley, in J. Roy. Hort. Soc. 58(1): 61 (1933). N. primigenius (Fern. Suárez ex Laínz) Fern.Casas & Laínz, in Fontqueria 11: 15 (1986). N. pseudonarcissus L., Sp. Pl.: 289 (1753). N. pseudonarcissus subsp. eugeniae (Fern.Casas) Fern.Casas, in Fontqueria 4: 27 (1983). N. pseudonarcissus subsp. munozii-garmendiae (Fern.Casas) Fern.Casas, in Fontqueria 4: 27 (1983). N. pumilus Salisb., Prodr. Stirp. Chap. Allerton: 220 (1796). N. radiiflorus Salisb., Prodr. Stirp. Chap. Allerton: 225 (1796). N. romieuxii Braun-Blanq. & Maire, in Bull. Soc. Hist. Nat. Afrique N. 13: 192 (1922). N. rupicola Dufour, Syst. Veg. 7ii: 958 (1830). N. scaberulus Henriq, in Bol. Soc. Brot. ser 2, 6: 45 (1888). N. segurensis Ríos, D.Rivera, Alcaraz & Obón, in Bot. J. Linn. Soc. 131(2): 153 (1999). N. serotinus L., Sp. Pl.: 290 (1753). N. tazetta L., Sp. Pl.: 290 (1753). N. tenuifolius Salisb., Prodr. Stirp. Chap. Allerton: 222 (1796). N. tingitanus Fern.Casas, in Fontqueria 44: 262 (1996). N. tortifolius Fern.Casas, in Saussurea 8: 43 (1977). N. tortuosus (Haw.) Spach, Hist. Nat. Vég. 12: 454 (1846). N. triandrus L., Sp. Pl., ed. 2, 1: 416 (1762). N. triandrus subsp. lusitanicus (Dorda Alcaraz & Fern.Casas) Barra, in Anales Jard. Bot. Madrid, 58: 186 (2000). N. triandrus subsp. pallidulus (Graells) Rivas Goday, Veg. Fl. Guadiana: 710 (1964). N. triandrus var. loiseleurii (Rouy) A.Fern., in Bol. Soc. Brot., ser. 2, 25: 178 (1951). N. viridiflorus Schousb., Iagttag. Vextrig. Marokko: 157, tab. 2 (1800). N. watieri Maire, in Bull. Soc. Hist. Nat. Afr. Nord 12: 186 (1921). N. willkommii (Samp.) A.Fern., in Bol. Soc. Brot., 2nd series, 40: 213-215 (1966). N. yepesii Ríos, D.Rivera, Alcaraz & Obón, in Bot. J. Linn. Soc. 131(2): 161 (1999). N. ×alleniae Donnison-Morgan, in New Plantsman 7(1): 42 (2000): miniatus × viridiflorus. N. ×alentejanus Fern.Casas, in Fontqueria 55: 555 (2008): cavanillesii × serotinus. N. ×barrae Fern.Casas, in Fontqueria 14: 18 (1987): bulbocodium × cantabricus. N. ×brevitubulosus A.Fern., in Anuário Soc. Brot. 49: 31 (1983): asturiensis × bulbocodium var. nivalis. N. ×carringtonii Rozeira, in Publ. Inst. Bot. "Dr. Gonçalo Sampaio", 3rd series, 1: 712 (1962): scaberulus × triandrus subps. lusitanicus. N. ×cazorlanus Fern.Casas, in Fontqueria 2: 37 (1982): hedraeanthus × triandrus subps. pallidulus. N. ×felineri Fern.Casas, in Fontqueria 11: 16 (1986): bulbocodium × primigenius. N. ×hannibalis A.Fern., in Anuário Soc. Brot. 39: 16 (1973): hispanicus × triandrus subsp. lusitanicus. N. ×perezlarae Font Quer, in Bol. Real Soc. Esp. Hist. Nat. 27: 39 (1927): cavanillesii × miniatus. N. ×pujolii Font Quer, in Mem. Mus. Ci. Nat. Barcelona Ser. Bot. 1(2): 14 (1924): assoanus × dubius. N. ×rozeirae Pérez-Chisc. & Fernández Casas, in Fontqueria 6: 43 (1984): bulbocodium × triandrus subps. pallidulus. N. ×somedanus Fern. Casado, Nava & Suárez Pérez, in Fontqueria 48: 30 (1997 publ. 1998): asturiensis × triandrus. N. ×susannae Fern.Casas, Exsicc. Distrib. 3: 13 (1980): cantabricus × pallidulus. N. ×tuckeri Barra & G. López, in Anales Jard. Bot. Madrid 41: 203 (1984): fernandesii × hedraeanthus.

Table S3

Table S3. Taxa sampled arranged according to the traditional infrageneric classification largely based on Fernandes (1968), including populations sampled, chromosome numbers (2n) counted in this study (B- presence of B chromosome; *- new count for the taxon), number of individuals used to count chromosomes, number of individuals sampled for chloroplast and mitochondrial regions (orgDNA), number of non-redundant orgDNA sequences used in the phylogenetic analysis, number of individuals sampled for ITS, number of clones retrieved per individual, number of non-redundant clones used in the phylogenetic analyses, and GenBank accession numbers. No. of No. of No. of No. of non‐redundant No. of non‐ Genbank accession numbers individuals individuals individuals No. of ITS 2n sequences retrieved from redundant ITS ndhF matK cob atpA ITS sampled for sampled for sampled for clones retrieved orgDNA clones Taxon Population cytogenetics orgDNA ITS clones Section Serotini Narcissus serotinus1 Portugal. Ponte de Ajuda. Marques1156 & Draper 10 +B 15 8 1 1 10 1 HM371975.1 KM349545.1 HM231911.1 HM174804.1 KY971379 Narcissus serotinus2 Portugal. Granja. Marques1018 & Rosselló‐Graell 10 15 8 1 1 10 1 HM372005.1 KM349547.1 HM231941.1 HM174834.1 KY971380 Narcissus serotinus3 Portugal. Póvoa de São Miguel. Marques2002 & Draper 10 10 8 1 1 10 1 HM372065.1 KM349551.1 HM232001.1 HM174894.1 KY971381 Narcissus serotinus4 Portugal. Vidigueira. Marques1013 10 8 8 1 1 10 1 HM372110.1 KM349554.1 HM232046.1 HM174939.1 KY971382 Section Aurelia Narcissus broussonetii1 Morocco. El Jadida. Marques2080 & Draper 22 10 8 2 1 10 1 KT124406, KR872993, KT272925, KT340488, KY930351 KT124407 KR872994 KT272926 KT340489 Narcissus broussonetii2 Morocco. Essaouira. Marques2081 & Draper 22, 44 15 8 2 1 10 1 KT124408, KR872997, KT272929, KT340492, KY930352 KT124409 KR872998 KT272930 KT340493 Narcissus broussonetii3 Morocco. Safi. Marques2082 & Draper 22, 44 8 8 2 KT124410, KR872995, KT272927, KT340490, KT124411 KR872996 KT272928 KT340491 Section Tazettae Narcissus elegans1 Morocco. Ksar‐el‐Kebir. Marques1200 & Draper 20 6 6 1 1 10 1 HM372679.1 KM349596.1 HM232615.1 HM175508.1 KY971383 Narcissus elegans2 Morocco. Lalla Minouna. Marques1201 & Draper 20 6 6 1 1 10 1 HM372709.1 KM349598.1 HM232645.1 HM175538.1 KY971384 Narcissus elegans3 Morocco. Moulay‐Idriss. Marques1196 & Draper 20 6 6 1 1 10 1 HM372739.1 KM349600.1 HM232675.1 HM175568.1 KY971385 Narcissus elegans4 Morocco. Cap Martil. Marques1193 & Draper 20 6 6 1 1 10 1 HM372724.1 KM349599.1 HM232660.1 HM175553.1 KY971386 Narcissus elegans5 Italy. Sicily. S.L. Jury10252. RNG s.n. 20 4 4 1 HM372859.1 KM349608.1 HM232795.1 HM175688.1 Narcissus elegans6 Morocco. Taza. Marques1195 & Draper 20 4 4 1 HM372799.1 KM349604.1 HM232735.1 HM175628.1 Narcissus dubius1 Spain. Monegros. MIM95020043 50 4 4 1 1 10 1 KT124413 KT021577 KT316770 KT594690 KY971396 Narcissus dubius2 Spain. Coll Baix. Marques2043 & Draper 50 6 6 1 1 10 1 KT124414 KT021578 KT316771 KT594691 KY971397 Narcissus dubius3 Spain. Caravaca de la Cruz. Marques2042 ‐ 4 1 KT124412 KT021576 KT316769 KT594689 Narcissus dubius4 Spain. Grazalema. S. L. Jury10417a. RNG ‐ 4 1 KT124415 KT021579 KT316772 KT594692 Narcissus miniatus1 Spain. Medina Azahara. Marques1126 & Draper 30 15 8 1 1 10 1 HM372125.1 KM349555.1 HM232061.1 HM174954.1 KY971387 Narcissus miniatus2 Spain. Los Belones. Marques1146 & Draper 30 15 8 1 1 10 1 HM372275.1 KM349567.1 HM232211.1 HM175104.1 KY971388 Narcissus miniatus3 Spain. La Línea de la Concepcion. Marques1138 & Draper 30 15 8 1 1 10 1 HM372260.1 KM349566.1 HM232196.1 HM175089.1 KY971389 Narcissus miniatus4 Spain. Majorca. JBS s.n. 30 4 4 1 1 10 1 HM372456.1 KM349580.1 HM232392.1 HM175285.1 KY971390 Narcissus pachybolbus Spain.Taza. S.L. Jury15748. RNG. 22 4 4 1 1 10 1 KT272885 KT033056 KT808789 KT594751 KY930348 Narcissus pannizianus1 Spain. BCN890151 ‐ 411101KT272887 KT033058 KT808791 KT594753 KY930350 Narcissus pannizianus2 Spain. Puerto de Encinas Borrachas. Marques2044 22 8 8 1 KT272886 KT033057 KT808790 KT594752 Narcissus papyraceus2 Morocco. Azilah. Marques800 & Draper 22 6 6 1 1 10 1 KT272888 KT033059 KT808792 KT594754 KY930346 Narcissus papyraceus3 Morocco. Larache. S.L. Jury16231. RNG s.n. ‐ 411101KT272890 KT033061 KT808794 KT594756 KY930345 Narcissus papyraceus8 Morocco. Temara. Marques1210 & Draper 22 8 8 1 KT272895 KT033066 KT808799 KT594761 Narcissus papyraceus9 Morocco. Tanger. Marques1209 & Draper 22 6 6 1 KT272896 KT033067 KT808800 KT594762 Narcissus papyraceus4 Portugal. Fonte Benémola. Marques50, Brehm & Draper 22 8 8 1 1 10 1 KT272891 KT033062 KT808795 KT594757 KY930347 Narcissus papyraceus1 Portugal. Reguengo. Marques1070 & Draper 22 6 6 1 1 10 1 KT272893 KT033064 KT808797 KT594759 KY930344 Narcissus papyraceus5 Portugal. Ribeira de Quarteira. Marques1061 & Draper 22 +B 6 6 1 KT272892 KT033063 KT808796 KT594758 Narcissus papyraceus7 Portugal. Virtudes. Marques1065 & Draper 22 6 6 1 KT272894 KT033065 KT808798 KT594760 Narcissus papyraceus6 Spain. San Roque. Marques2045 22 4 8 1 KT272889 KT033060 KT808793 KT594755 Narcissus papyraceus10 Spain. Carcabuey. Enrique Triano s.n. ‐ 41 KT272897 KT033068 KT808801 KT594763 Narcissus polyanthos Morocco. Moulay‐Idriss. S. L. Jury13714. RNG s.n. 22 4 4 1 1 10 1 KT799134 KT033073 KT808806 KT594768 KY930349 Narcissus tazetta1 Portugal. Águas Belas. Marques1999 & Draper 20 4 6 1 1 10 1 KT799087 KT033099 KT808832 KT878438 KY930339 Narcissus tazetta2 Portugal. Alcoarche. Marques2000 & Draper ‐ 8 1 1 10 1 KT799088 KT033100 KT808833 KT878439 KY930340 Narcissus tazetta3 Japan. BCN890576 ‐ 4 1 1 10 1 KT799091 KT033103 KT808836 KT878442 KY930341 Narcissus tazetta4 Spain. Majorca. ‐ 8 1 1 10 1 KT799089 KT033101 KT808834 KT878440 KY930342 Narcissus tazetta5 Spain. Teulada. Marques1848, Soler & Draper 20 4 8 1 1 10 1 KT799090 KT033102 KT808835 KT878441 KY930343 Narcissus tortifolius1 Spain. BCN890565 ‐ 3 1 1 10 2 KT863427 KT033123 KT808856 KT878462 KY971392, KY971394 Narcissus tortifolius2 Spain. MIM s.n. 36 4 3 1 1 10 1 KT863428 KT033124 KT808857 KT878463 KY971393 Narcissus tortifolius3 Spain. Los Castaños. Juan F. Mota s.n. ‐ 4 1 KT863429 KT033125 KT808858 KT878464 Narcissus tortifolius4 Spain. Sorbas. Juan F. Mota s.n. ‐ 4 1 KT863430 KT033126 KT808859 KT878465 Narcissus xpujolii (assoanus x dubius) Spain. Coll Baix. Marques2222 & Draper 32* 4 6 1 1 10 2 KT799133 KT033070 KT808803 KT594765 KY971395, KY971422 Section Apodanthi Narcissus albimarginatus1 Morocco. Chaouen. Jbel Bouhachem. Marques801 & Draper 14* 4 6 1 1 10 1 KT210953 KR872971 KT272903 KT279959 KY971398 Narcissus albimarginatus2 Morocco. Chaouen. Jbel Kelti. Marques802 & Draper 14* 4 6 1 1 10 1 KT210954 KR872972 KT272904 KT279960 KY971399 Narcissus atlanticus Morocco. Oukaimeden. Marques803 & Draper 14* 4 6 1 1 10 1 KT863444 KT033140 KT808873 KT878479 KY971412 Narcissus calcicola1 Portugal. Serra de Aire e Candeeiros. Marques1908, Flores & Draper 14 4 4 1 1 10 1 KT340508 KT021558 KT316751 KT594671 KY971400 Narcissus calcicola2 Portugal. Rocha da Pena. Marques1907, Brehm & Draper 14 4 4 1 1 10 1 KT340509 KT021559 KT316752 KT594672 KY971401 Narcissus calcicola3 Portugal. Serra de Monte Figo. César Garcia s.n. ‐ 6 1 1 10 1 KT340510 KT021560 KT316753 KT594673 KY971402 Narcissus cuatrecasasii1 Spain. Mágina. Marques804 & Draper 14 6 8 1 1 10 1 KT340521 KT021571 KT316764 KT594684 KY971408 Narcissus cuatrecasasii2 Spain. Sierra de Cazorla. Medrano&Herrera2006‐01 ‐ 8 1 1 10 1 KT340520 KT021570 KT316763 KT594683 KY971409 Narcissus cuatrecasasii var. segimonensis Spain. Quesada. Puerto de Tiscar. Marques805 & Draper 14* 6 5 1 1 10 1 KT340519 KT021569 KT316762 KT594682 KY971410 Narcissus marvieri1 Morocco. Jbel Tazzeka. Marques806 14 6 8 1 1 10 1 KT799111 KT021617 KT619090 KT594730 KY971414 Narcissus marvieri2 Morocco. Jbel Zerhoun. Marques807 14 4 8 1 KT799112 KT021618 KT619091 KT594731 Narcissus rupicola2 Portugal. Penhas Douradas. Marques1880 & Draper 14 4 8 1 1 10 1 KT799077 KT033089 KT808822 KT878428 KY971416 Narcissus rupicola3 Portugal. Penhas Saude. Marques1881 & Draper 14 4 8 1 KT799078 KT033090 KT808823 KT878429 Narcissus rupicola1 Spain. Puerto de Canencia. Marques1982 & Draper ‐ 6 1 1 10 1 KT799076 KT033088 KT808821 KT878427 KY971415 Narcissus rupicola4 Spain. Puerto de Guadarrama. Marques1978 & Draper 14 4 6 1 KT799079 KT033091 KT808824 KT878430 Narcissus rupicola5 Spain. Sierra Segura. Marques1983 ‐ 8 1 KT799081 KT033093 KT808826 KT878432 Narcissus scaberulus1 Portugal. Ervedal da Beira. Marques1985 & Draper 14 4 8 1 1 10 1 KT799082 KT033094 KT808827 KT878433 KY971405 Narcissus scaberulus2 Portugal. Oliveira do Conde. Marques1984 & Draper 14 4 8 1 1 10 1 KT799084 KT033096 KT808829 KT878435 KY971406 Narcissus watieri1 Morocco. Marrakesh. Oukaimeden. Marques805 & Draper 14 6 8 1 1 10 1 KT863443 KT033139 KT808872 KT878478 KY971413 Narcissus watieri2 Morocco. Marrakesh. Tizi´n´Test. Marques806 & Draper 14 6 6 1 KT863442 KT033138 KT808871 KT878477 Narcissus xcarringtonii (scaberulus x triandrus subsp. pallidulus) Portugal. Ervedal da Beira. Marques799 & Draper 14 6 6 1 1 10 1 KT799083 KT033095 KT808828 KT878434 KY971407 Narcissus cuatrecasasii x N. triandrus Spain. Mágina. Marques807 & Draper 14* 4 6 1 1 10 1 KT340522 KT021572 KT316765 KT594685 KY971411 Narcissus rupicola x N. triandrus Spain. Puerto de Guadarrama. Marques808 & Draper 14* 4 6 1 1 10 1 KT799080 KT033092 KT808825 KT878431 KY971417 Section Jonquillae Narcissus assoanus1 Spain. Coll Baix. Marques2220 & Draper 14 4 8 1 1 10 1 KT210960 KR872978 KT272910 KT279966 KY971423 Narcissus assoanus2 Spain. Granadella. Marques1848, Soler & Draper 14 +B 4 8 1 1 10 1 KT210961 KR872979 KT272911 KT279967 KY971424 Narcissus assoanus3 Spain. Montgó. Marques1847, Soler & Draper 14 4 8 1 KT210962 KR872980 KT272912 KT279968 Narcissus assoanus4 Spain. Montserrat. Marques2221 & Draper 14 8 8 1 KT210963 KR872981 KT272913 KT279969 Narcissus assoanus subsp. praelongus Spain. Grazalema. Puerto de las Palomas. Enrique Triano s.n. ‐ 411101KT210964 KR872982 KT272914 KT279970 KY971425 Narcissus assoanus subsp. rivas‐martinezii Spain. Málaga. Sierra de las Nieves. Enrique Triano s.n. 14* 4 4 1 1 10 1 KT210965 KR872983 KT272915 KT279971 KY971426 Narcissus baeticus Spain. Carcabuey. Enrique Triano s.n. 14 4 8 1 1 10 1 KT326969 KR872999 KT279985 KT340494 KY971438 Narcissus cerrolazae Spain. Montecorto. Ronda. Enrique Triano s.n. 14 4 4 1 1 10 1 KT340515 KT021565 KT316758 KT594678 KY971436 Narcissus cordubensis Spain. Benamejí. Enrique Triano s.n. 28 4 4 1 1 10 1 KT340518 KT021568 KT316761 KT594681 KY971437 Narcissus fernandesii1 Portugal. Reguengo. Marques1941 & Draper 14 4 8 1 1 10 1 KT340529 KT021583 KT316776 KT594696 KY971440 Narcissus fernandesii2 Spain. Escañuela. MIM98110716 ‐ 4 1 1 10 1 KT340532 KT021586 KT316779 KT594699 KY971441 Narcissus fernandesii3 Spain. Lucena. Enrique Triano s.n. 14 4 4 1 KT340533 KT021587 KT316780 KT594700 Narcissus fernandesii4 Spain. Santuario Virgen de la Cabeza. Marques2046 ‐ 8 1 KT340531 KT021585 KT316778 KT594698 Narcissus fernandesii var. major Portugal. Reguengo. Marques1939 & Draper 28, 35 4 8 1 1 10 1 KT340516 KT021566 KT316759 KT594679 KY971442 Narcissus gaditanus1 Portugal. Benafim. Marques1946 ‐ 8 1 1 10 1 KT340534 KT021588 KT316781 KT594701 KY971403 Narcissus gaditanus2 Spain. Ojén. Juánar. Marques2073 14 6 8 1 1 10 1 KT340537 KT021591 KT316784 KT594704 KY971404 Narcissus gaditanus3 Portugal. Rocha da Pena. Marques1945 14 4 8 1 KT340535 KT021589 KT316782 KT594702 Narcissus gaditanus4 Portugal. Serra Monte Figo. César Garcia 14, 21 4 4 1 KT340536 KT021590 KT316783 KT594703 Narcissus jonquilla1 Portugal. Serra da Estrela. MIM98110716 ‐ 4 1 1 10 1 KT304268 KT021602 KT316795 KT594715 KY971433 Narcissus jonquilla3 Portugal. Ponte da Ajuda. Marques1953, Rosselló‐Graell 14 4 8 1 KT304270 KT021604 KT316797 KT594717 Narcissus jonquilla4 Portugal. Pulo do Lobo. Marques1956 14 4 8 1 KT304272 KT021606 KT316799 KT594719 Narcissus jonquilla5 Portugal. Ribeira Alcáçovas. Rosselló‐Graell1567 14 4 8 1 KT304273 KT021607 KT316800 KT594720 Narcissus jonquilla6 Portugal. Ribeira Azevelinho. Marques1952 & Draper 14 4 4 1 KT304274 KT021608 KT316801 KT594721 Narcissus jonquilla7 Portugal. Ribeira S. Cristovão. Rosselló‐Graell1568 14 4 4 1 KT304275 KT021609 KT316802 KT594722 Narcissus jonquilla8 Spain. Arroyo de Cuncos. Marques1957, & Draper ‐ 4 1 KT304267 KT021601 KT316794 KT594714 Narcissus jonquilla2 Spain. La Nava. Marques2047 ‐ 8 1 1 10 1 KT304269 KT021603 KT316796 KT594716 KY971434 Narcissus jonquilla var. henriquesii Portugal. Ponte da Ajuda. Marques1958, Rosselló‐Graell 14 4 4 1 1 10 1 KT304271 KT021605 KT316798 KT594718 KY971435 Narcissus viridiflorus1 Spain. BCN930017 ‐ 4 1 1 10 1 KT863432 KT033128 KT808861 KT878467 KY971428 Narcissus viridiflorus4 Spain. La Línea. Marques1138 & Draper 28 4 8 1 1 10 1 KT863436 KT033132 KT808865 KT878471 KY971431 Narcissus viridiflorus5 Spain. Medina‐Sidonia. Marques1172 & Draper 28 4 8 1 1 10 1 KT863437 KT033133 KT808866 KT878472 KY971432 Narcissus viridiflorus2 Morocco. Cape Spartel. Marques1208 & Draper 28 4 8 1 1 10 1 KT863433 KT033129 KT808862 KT878468 KY971429 Narcissus viridiflorus3 Morocco. Kenitra. Marques2026 & Draper 28 4 8 1 1 10 1 KT863434 KT033130 KT808863 KT878469 KY971430 Narcissus viridiflorus6 Morocco. Salé. Marques2024 & Draper 28 4 6 1 KT863435 KT033131 KT808864 KT878470 Narcissus willkommii1 Portugal. Alvor. Paiva1528 14 4 8 1 1 10 1 KT863438 KT033134 KT808867 KT878473 KY971439 Narcissus willkommii2 Portugal. COI. s. n. 14 4 6 1 KT863440 KT033136 KT808869 KT878475 Narcissus willkommii3 Portugal. Loulé. Marques2028 & Draper ‐ 8 1 KT863439 KT033135 KT808868 KT878474 Narcissus willkommii4 Portugal. Ribeira da Quarteira. Marques2027 & Draper 14 4 8 1 KT863441 KT033137 KT808870 KT878476 Narcissus xalleniae1 (serotinus x viridiflorus) Spain. Medina‐Sidonia. Marques1173 & Draper 29* 4 8 2 1 10 2 KT326957, KR872966, KT272898 KT279954, KY971427, KT326958 KR872967 KT279955 KY971391 Narcissus xalleniae2 (serotinus x viridiflorus) Spain. San José Valle. Marques1111 & Draper 29* 4 6 1 KT326959 KR872968 KT272900 KT279956 Narcissus fernandesii x N. papyraceus Portugal. Reguengo. Marques1946 & Draper 18* 4 8 1 1 10 1 KT340530 KT021584 KT316777 KT594697 KY971443 Section Bulbocodii Narcissus bulbocodium2 Portugal. Alcoarche. Marques1873 & Draper 14, 28 8 8 1 1 10 1 KT326986 KT210972 KT304225 KT594629 KY992380 Narcissus bulbocodium3 Portugal. Aldeias de Montoito. Marques1884 & Draper 28, 35 6 6 1 KT326988 KT210974 KT304227 KT594631 Narcissus bulbocodium4 Portugal. Cerdeira. Marques1878 & Draper 14, 28, 56 8 8 1 KT326989 KT210975 KT304228 KT594632 Narcissus bulbocodium5 Portugal. Cabeça de Carneiro. Azevelinho. Marques1876 & Draper 28 6 6 1 KT326990 KT210976 KT304229 KT594633 Narcissus bulbocodium6 Portugal. Cabeça Carneiro. Marques1875 & Draper 28 6 6 1 KT326991 KT210977 KT304230 KT594634 Narcissus bulbocodium7 Portugal. COI s.n. ‐ 4 1 KT326992 KT210978 KT304231 KT594635 Narcissus bulbocodium11 Portugal. Mogador. Marques1890 ‐ 8 1 KT327008 KT210994 KT304247 KT594651 Narcissus bulbocodium12 Portugal. Outeiro do Pombo. Marques1885 28, 42 6 8 1 KT326995 KT210981 KT304234 KT594638 Narcissus bulbocodium13 Portugal. Penhas Douradas. Marques2041 & Draper ‐ 8 1 KT326985 KT210971 KT304224 KT594628 Narcissus bulbocodium15 Portugal. Ponte da Ajuda. Marques1874 & Draper 28 4 4 1 KT326997 KT210983 KT304236 KT594640 Narcissus bulbocodium17 Portugal. Cape Espichel. Marques2031 & Draper 28, 42 8 8 1 KT326999 KT210985 KT304238 KT594642 Narcissus bulbocodium18 Portugal. Ribeira de Godelim. Marques1881 & Draper 42 4 6 1 KT327000 KT210986 KT304239 KT594643 Narcissus bulbocodium22 Portugal. Virtudes. Marques2040 & Draper 42, 49 4 8 1 KT327010 KT210996 KT304249 KT594653 Narcissus bulbocodium23 Portugal. JB‐MNHN s.n. ‐ 41 KT327004, KT210990, KT304243, KT594647, KT327005 KT210991 KT304244 KT594648 Narcissus bulbocodium19 Spain. Ames. Ponte Maceira. Miguel Serrano s.n. 28 4 6 1 KT327001 KT210987 KT304240 KT594644 Narcissus bulbocodium20 Spain. Santíz. Miguel Lizana s.n. ‐ 4 1 KT327002 KT210988 KT304241 KT594645 Narcissus bulbocodium21 Spain. Pico Cervero. Miguel Lizana s.n. ‐ 41 KT327003 KT210989 KT304242 KT594646 Narcissus bulbocodium14 Spain. Puerto de Peña Negra. Marques2029 ‐ 8 1 KT326996 KT210982 KT304235 KT594639 Narcissus bulbocodium16 Spain. Puerto del Tremedal. Marques2030 ‐ 8 1 KT326998 KT210984 KT304237 KT594641 Narcissus bulbocodium24 Spain. Sierra de Urbasa. Juan Carlos Moreno s.n. ‐ 4 1 KT327009 KT210995 KT304248 KT594652 Narcissus bulbocodium8 Spain. Gijón. Marques2065, H. Nava & A. Bueno ‐ 8 1 KT326993 KT210979 KT304232 KT594636 Narcissus bulbocodium9 Spain. Gijón. Marques2066, H. Nava & A. Bueno ‐ 8 1 KT326994 KT210980 KT304233 KT594637 Narcissus bulbocodium10 Spain. Sierra de Garza. Marques2072 ‐ 8 1 KT327007 KT210993 KT304246 KT594650 Narcissus bulbocodium1 Morocco. Agadir. Felipe Lopes Domínguez s.n. ‐ 4 1 1 10 1 KT326987 KT210973 KT304226 KT594630 KY992438 Narcissus bulbocodium subsp. graellsii Spain. Puerto de Guadarrama. Marques1947 & Draper 14, 28 4 6 1 1 10 1 KT326981 KT210967 KT279997 KT340506 KY992377 Narcissus bulbocodium subsp. obesus1 Portugal. Alte. Marques1901 & Draper 26 4 6 1 1 10 2 KT327013 KT210999 KT304252 KT594656 KY992365, KY992366 Narcissus bulbocodium subsp. obesus2 Portugal. Alvor. Marques1903 & Draper 26 4 6 1 1 10 1 KT327011 KT210997 KT304250 KT594654 KY992367 Narcissus bulbocodium subsp. obesus3 Portugal. Portela. C. Sergio21611 26 4 6 1 KT327012 KT210998 KT304251 KT594655 Narcissus bulbocodium subsp. obesus4 Portugal. Berlenga. Cristina Gomes s. n. ‐ 6 1 KT327024 KT211010 KT304263 KT594667 Narcissus bulbocodium subsp. obesus5 Portugal. Cape Espichel. Marques1897 & Draper 26 4 6 1 KT327014 KT211000 KT304253 KT594657 Narcissus bulbocodium subsp. obesus6 Portugal. Lagoa Comprida. Marques1898 & Draper 26 4 6 1 KT327015 KT211001 KT304254 KT594658 Narcissus bulbocodium subsp. obesus7 Portugal. Magoito. Cristina Gomes s. n. ‐ 6 1 KT327016 KT211002 KT304255 KT594659 Narcissus bulbocodium subsp. obesus9 Portugal. Arrábida. Marques1894 & Draper 26 4 6 1 KT327021 KT211007 KT304260 KT594664 Narcissus bulbocodium subsp. obesus10 Portugal. Portinho da Arrábida. Marques1896 & Draper 26 4 6 1 KT327022 KT211008 KT304261 KT594665 Narcissus bulbocodium subsp. obesus11 Portugal. Rocha da Pena. Marques1900 & Draper ‐ 8 1 KT327025 KT211011 KT304264 KT594668 Narcissus bulbocodium subsp. obesus12 Portugal. Zimbral. Marques1902 & Draper 26 4 8 1 KT327023 KT211009 KT304262 KT594666 Narcissus bulbocodium subsp. obesus8 Morocco. Oukaimeden. S. L. Jury8945. RNG ‐ 3 1 KT327018 KT211004 KT304257 KT594661 Narcissus bulbocodium subsp. quintanilhae1 Portugal. Cerdeira. Marques1903 & Draper 21, 42 6 8 1 1 10 1 KT326983 KT210969 KT304222 KT594626 KY992369 Narcissus bulbocodium subsp. quintanilhae2 Portugal. Quinta da Serra. Marques1904 & Draper 21, 42 +B 6 8 1 1 10 1 KT326984 KT210970 KT304223 KT594627 KY992370 Narcissus bulbocodium var. nivalis1 Spain. Puerto de Las Señales. Marques2068, H. Nava & A. Bueno 14 +B 6 8 1 1 10 1 KT799128 KT021634 KT619107 KT594747 KY992381 Narcissus bulbocodium var. nivalis2 Spain. Puerto de Tarna. Marques2069, H. Nava & A. Bueno 14 6 8 1 KT799129 KT021635 KT619108 KT594748 Narcissus cantabricus1 Spain. Puerto Virgen. MIM98110734 28 4 4 1 1 10 1 KT340511 KT021561 KT316754 KT594674 KY992374 Narcissus cantabricus2 Spain. Sierra de Garza. Marques2032 & Draper ‐ 8 1 1 10 1 KT340513 KT021563 KT316756 KT594676 KY992376 Narcissus cantabricus3 Spain. Mérida. Marques2033 & Draper ‐ 8 1 KT340512 KT021562 KT316755 KT594675 Narcissus cantabricus4 Spain. Granada. Sierra Nevada. El Serrallo. J. B. Hoya de Pedraza. 28 4 4 1 KT340514 KT021564 KT316757 KT594677 Narcissus bulbocodium var. citrinus Spain. Puerto de Las Señales. Marques2061 14 4 8 1 1 10 1 KT327020 KT211006 KT304259 KT594663 KY992371 Narcissus hedraeanthus1 Spain. Sierra de Segura. Collado de Gontar. Medrano&Herrera2006‐07 ‐ 6 1 1 10 1 KT340538 KT021592 KT316785 KT594705 KY992360 Narcissus hedraeanthus2 Spain. Sierra de Cazorla. La Cabrilla. Medrano&Herrera2006‐08 ‐ 6 1 KT340539 KT021593 KT316786 KT594706 Narcissus jacquemoudii Morocco. Tahnaout. Marques2078 & Draper ‐ 8 1 1 10 1 KT326982 KT210968 KT304221 KT594625 KY992378 Narcissus jeanmonodii Morocco. Oukaimeden. Marques2077 & Draper ‐ 8 1 1 10 1 KT327017 KT211003 KT304256 KT594660 KY992382 Narcissus peroccidentalis Morocco. Kénitra. Marques2075 & Draper ‐ 8 1 1 10 1 KT799132 KT033069 KT808802 KT594764 KY992363 Narcissus romieuxii1 Morocco. Chaouen. Marques2034 & Draper 28 4 6 2 1 10 1 KT799072 KT033084 KT808817 KT878423 KY992364 Narcissus romieuxii2 Morocco. Ketama. Marques2035 & Draper 28 +B 4 3 1 KT799073 KT033085 KT808818 KT878424 Narcissus tenuifolius BCN920158 ‐ 3 1 1 10 1 KT340507 KT211013 KT304266 KT594670 KY992362 Narcissus tingitanus Morocco. Tanger. Marques2076 & Draper ‐ 6 1 1 10 1 KT327019 KT211005 KT304258 KT594662 KY992379 Narcissus xbarrae (bulbocodium x cantabricus) Spain. Sierra de Garza. Marques1940 ‐ 411101KT327006 KT210992 KT304245 KT594649 KY992372 Narcissus xcazorlanus ( hedraeanthus x triandrus subsp. pallidulus) JB‐MNHN s.n. ‐ 4 1 1 10 1 KT340540 KT021594 KT316787 KT594707 KY992373 Narcissus xbrevitubulosus (asturiensis x bulbocodium) Portugal. Lagoa Comprida. Marques2083 & Draper 14 4 8 1 1 10 1 KT799130 KT021636 KT619109 KT594749 KY992384 Narcissus bulbocodium subsp. obesus x N. calcicola Portugal. Rocha da Pena. Marques1970 & Draper 26* 4 8 1 1 10 1 KT327026 KT211012 KT304265 KT594669 KY992368 Narcissus xfelineri (bulbocodium x primigenius) Spain. Puerto de Tarna. Marques2079, H. Nava & A. Bueno ‐ 8 1 1 10 1 KT799131 KT021637 KT619110 KT594750 KY992383 Narcissus xtuckeri1 (fernandesii x hedraeanthus) BCN930526 ‐ 3 1 1 10 1 KT340541 KT021595 KT316788 KT594708 KY992361 Narcissus xtuckeri2 (fernandesii x hedraeanthus) JB‐MNHN s.n. ‐ 3 1 KT340542 KT021596 KT316789 KT594709 Section Ganymedes Narcissus triandrus2 Portugal. Ervedal da Beira. Marques2036 & Draper 14 4 8 1 1 10 1 KT799096 KT033108 KT808841 KT878447 KY992386 Narcissus triandrus1 Spain. La Cabra. Enrique Triano s.n. 14 4 4 1 1 10 1 KT799092 KT033104 KT808837 KT878443 KY992385 Narcissus triandrus3 Spain. Gijon. Marques2063, H. Nava & A. Bueno 14 4 8 1 1 10 1 KT799109 KT033121 KT808854 KT878460 KY992387 Narcissus triandrus4 Spain. Gijon. Marques2064, H. Nava & A. Bueno 14 4 8 1 KT799110 KT033122 KT808855 KT878461 Narcissus triandrus5 Spain. Sierra de Mágina. Marques2037 ‐ 8 1 KT799093 KT033105 KT808838 KT878444 Narcissus triandrus6 Spain. Sierra de Segura. Navalcaballo. Medrano&Herrera2006‐05 ‐ 6 1 KT799094 KT033106 KT808839 KT878445 Narcissus triandrus7 Spain. Sierra de Cazorla. Vadillo‐Castril. Medrano&Herrera2006‐06 ‐ 6 1 KT799095 KT033107 KT808840 KT878446 Narcissus triandrus var. loiseleurii France. Glénan. CBNB s.n. 14 4 4 1 1 10 1 KT799108 KT033120 KT808853 KT878459 KY992388 Narcissus triandrus subsp. lusitanicus1 Portugal. Águas Belas. Marques1969 & Draper 14 4 8 1 1 10 1 KT799097 KT033109 KT808842 KT878448 KY992390 Narcissus triandrus subsp. lusitanicus3 Portugal. Dornes. Marques1968 & Draper 14 4 8 1 1 10 1 KT799099 KT033111 KT808844 KT878450 KY992392 Narcissus triandrus subsp. lusitanicus4 Portugal. Mogadouro. Marques2038 14 4 8 1 KT799100 KT033112 KT808845 KT878451 Narcissus triandrus subsp. lusitanicus2 Spain. BCN920167 ‐ 4 1 1 10 1 KT799098 KT033110 KT808843 KT878449 KY992391 Narcissus triandrus subsp. pallidulus2 Portugal. Miranda do Douro. Marques2014 ‐ 8 1 1 10 1 KT799107 KT033119 KT808852 KT878458 KY992395 Narcissus triandrus subsp. pallidulus3 Portugal. Penamacor. César Garcia s.n. ‐ 5 1 KT799103 KT033115 KT808848 KT878454 Narcissus triandrus subsp. pallidulus4 Portugal. Penhas saude. Marques2010 & Draper 14 4 8 1 KT799104 KT033116 KT808849 KT878455 Narcissus triandrus subsp. pallidulus5 Puerto de Guadarrama. Marques2008 & Draper 14 4 8 1 KT799102 KT033114 KT808847 KT878453 Narcissus triandrus subsp. pallidulus6 Portugal. Torre. Marques2009 & Draper 14 4 8 1 KT799105 KT033117 KT808850 KT878456 Narcissus triandrus subsp. pallidulus1 Spain. Sierra de Mágina. Marques2039 8 1 1 10 1 KT799106 KT033118 KT808851 KT878457 KY992394 Narcissus triandrus x N. bulbocodium Portugal. Mogadouro. Marques2015 & Draper 14 4 8 1 1 10 1 KT799101 KT033113 KT808846 KT878452 KY992393 Narcissus xrozeirae (bulbocodium x triandrus subsp. pallidulus) BCN920211 14 4 4 1 1 10 2 KT799075 KT033087 KT808820 KT878426 KY992389, KY992437 Narcissus xsusannae (cantabricus x triandrus subsp. pallidulus) Spain. Mérida. Marques2074 ‐ 8 1 1 10 1 KT799086 KT033098 KT808831 KT878437 KY992375 Section Braxireon Narcissus cavanillesii1 Portugal. Ajuda. Marques1155 & Draper 28 15 8 1 1 10 1 HM371429.1 KR872965 HM231365.1 HM174258.1 KY971373 Narcissus cavanillesii2 Spain. Facinas. Marques1166 & Draper 28 10 8 1 1 10 1 HM371534.1 KM349505.1 HM231470.1 HM174363.1 KY971374 Narcissus cavanillesii4 Spain. Posadas. Marques1144 & Draper 28 10 8 1 1 10 1 HM371619.1 KM349509.1 HM231555.1 HM174448.1 KY971375 Narcissus cavanillesii3 Morocco. Oued el Malah, Tetouan. Marques1191 & Draper 28 10 8 1 1 10 1 HM371729.1 KM349519.1 HM231665.1 HM174558.1 KY971376 Narcissus cavanillesii5 Morocco. Souk Khémis des Anjra. Marques1204 & Draper 28 8 8 1 1 10 1 HM371789.1 KM349523.1 HM231725.1 HM174618.1 KY971377 Narcissus cavanillesii6 Morocco. Sidi‐el‐Yamani. Marques1187 & Draper 28 8 8 1 HM371804.1 KM349524.1 HM231740.1 HM174633.1 Narcissus xalentejanus (cavanillesii x serotinus) Portugal. Ajuda. Marques1158 & Draper 29 8 8 2 1 10 1 HM371834.1 KM349526.1 HM231770.1 HM174663.1 KY971378 Narcissus xperezlarae (cavanillesii x miniatus) Spain. Oliva. Marques1174 & Draper 29 8 8 2 1 10 1 HM371924.1 KM349538.1 HM231860.1 HM174753.1 MF039090 Section Narcissus Narcissus poeticus1 Andorra. Coll de Ordino. Joan Pedrol7941 14 6 6 1 1 10 1 KT124416 KT033071 KT808804 KT594766 KY992421 Narcissus poeticus2 BCN890125 ‐ 6 1 KT124417 KT033072 KT808805 KT594767 Narcissus radiiflorus Austria. Steiermark. Enns Valley. Andreas Tribsch s.n. 14 6 6 1 1 10 1 KT124419 KT033083 KT808816 KT878422 KY992420 Section Pseudonarcissus Narcissus abscissus1 BCN920441 ‐ 411101KT210951 KR872969 KT272901 KT279957 KY992439 Narcissus abscissus2 Spain. Valle de Aran. Marques2048 ‐ 81 KT210952 KR872970 KT272902 KT279958 Narcissus alcaracensis1 Spain. Peñascosa. Laguna Fuente Raja. Medrano&Herrera2006‐03 ‐ 8 1 1 10 1 KT210955 KR872973 KT272905 KT279961 KY992401 Narcissus alcaracensis2 Spain. Peñascosa. Medrano&Herrera2005‐07 ‐ 8 1 KT210956 KR872974 KT272906 KT279962 Narcissus alcaracensis3 Spain. Peñascosa. Rio Pesebre. Medrano&Herrera2006‐04 ‐ 8 1 KT210957 KR872975 KT272907 KT279963 Narcissus alpestris Spain. San Juan de la Peña. Marques2049 ‐ 811101KT210958 KR872976 KT272908 KT279964 KY992440 Narcissus asturiensis3 Portugal. Lagoa Comprida. Marques1856 & Draper 14 +B 6 6 1 KT326961 KR872985 KT272917 KT279973 Narcissus asturiensis4 Portugal. Penhas Douradas. Marques1850 & Draper 14 6 6 1 KT326963 KR872987 KT272919 KT279975 Narcissus asturiensis5 Portugal. Penhas Longas. Marques1852 & Draper 14 8 8 1 KT326964 KR872988 KT272920 KT279976 Narcissus asturiensis7 Portugal. Lagoa Comprida. Marques1857 & Draper 14 4 8 1 KT326967 KR872991 KT272923 KT279979 Narcissus asturiensis1 Spain. Escorial. Felipe Lopes Dominguez s. n. ‐ 411101KT326960 KR872984 KT272916 KT279972 KY992423 Narcissus asturiensis2 Spain. Lago Isoba. Marques2070, H. Nava & A. Bueno ‐ 8 1 1 10 1 KT326966 KR872990 KT272922 KT279978 KY992424 Narcissus asturiensis6 Spain. Somiedo. Miguel Serrano s. n. ‐ 41 KT326965 KR872989 KT272921 KT279977 Narcissus asturiensis8 Spain. Sierra de Urbasa. Juan Carlos Moreno s.n. ‐ 31 KT326968 KR872992 KT272924 KT279980 Narcissus bicolor1 Spain. Portalet. Luis Villar s.n. ‐ 4 1 1 10 1 KT326971 KR873001 KT279987 KT340496 KY992412 Narcissus bicolor2 Spain. Valle de Aran. Marques2073 ‐ 8 1 KT326970 KR873000 KT279986 KT340495 Narcissus bujei1 Spain. Bienservida. Medrano&Herrera2006‐13 ‐ 6 1 1 10 1 KT326972 KR873002 KT279988 KT340497 KY992404 Narcissus bujei2 Spain. Charco Negro. Medrano&Herrera s.n. ‐ 6 1 1 10 1 KT326979 KR873009 KT279995 KT340504 KY992405 Narcissus bujei3 Spain. Collado Zamora. Medrano&Herrera2005‐09 ‐ 6 1 KT326973 KR873003 KT279989 KT340498 Narcissus bujei4 Spain. Sierra de Cazorla. El Chorro. Medrano&Herrera2006‐11 ‐ 6 1 KT326974 KR873004 KT279990 KT340499 Narcissus bujei5 Spain. Torredelcampo. El Megatín. Medrano&Herrera2006‐12 ‐ 6 1 KT326975 KR873005 KT279991 KT340500 Narcissus bujei6 Spain. Sierra de las Nieves. Los Quejigales. Medrano&Herrera2006‐14 ‐ 6 1 KT326976 KR873006 KT279992 KT340501 Narcissus bujei7 Spain. Sierra de Mágina. Puerto de la Mata. Medrano&Herrera2005‐08 ‐ 6 1 KT326977 KR873007 KT279993 KT340502 Narcissus bujei8 Spain. Sierra de las Nieves. Enrique Triano s. n. 14 4 4 1 KT326978 KR873008 KT279994 KT340503 Narcissus confusus Spain. Cañamero. Marques2050 ‐ 8 1 1 10 1 KT340517 KT021567 KT316760 KT594680 KY992442 Narcissus cyclamineus1 Portugal. S. Joao do Monte. Marques1753 & Draper ‐ 6 1 1 10 1 KT340523 KT021573 KT316766 KT594686 KY992432 Narcissus cyclamineus2 Spain. Santiago de Compostela. Marques1790 & Draper 14 4 6 1 KT340525 KT021575 KT316768 KT594688 Narcissus cyclamineus3 Spain. Val do Dubra. Ínsua. Miguel Serrano s. n. 14 4 4 1 KT340524 KT021574 KT316767 KT594687 Narcissus pseudonarcissus subsp. eugeniae1 Spain. Sierra de Cucalón. Fonfria. Marques2052 ‐ 8 1 1 10 1 KT340528 KT021582 KT316775 KT594695 KY992433 Narcissus pseudonarcissus subsp. eugeniae2 Spain. Sierra de Moncayo. Marques2051 ‐ 8 1 KT340526 KT021580 KT316773 KT594693 Narcissus pseudonarcissus subsp. eugeniae3 Spain. Puerto de las Señales. Marques2053, H. Nava & A. Bueno 14 +B 4 8 1 KT340527 KT021581 KT316774 KT594694 Narcissus hispanicus Portugal. Águas Belas. Marques1950 & Draper 14 4 8 1 1 10 1 KT340544 KT021598 KT316791 KT594711 KY992416 Narcissus jacetanus Spain. San Juan de la Peña. Marques2056 14 4 8 1 1 10 1 KT340546 KT021600 KT316793 KT594713 KY992426 Narcissus longispathus1 Spain. Arroyo de la Venta. Medrano&Herrera s.n. ‐ 6 1 1 10 1 KT340550 KT021613 KT619086 KT594726 KY992400 Narcissus longispathus2 Spain. Sierra de Cazorla. Fuente del Oso. Medrano&Herrera2005‐06 ‐ 4 1 1 10 1 KT340549 KT021612 KT619085 KT594725 KY992402 Narcissus longispathus3 Spain. Sierra de Cazorla. La Cabrilla. Medrano&Herrera2006‐10 ‐ 6 1 1 10 1 KT340551 KT021614 KT619087 KT594727 KY992403 Narcissus longispathus4 Spain. Sierra de Cazorla. Valdecuevas. Medrano&Herrera2006‐09 ‐ 6 1 KT340552 KT021615 KT619088 KT594728 Narcissus longispathus5 Spain. Villaverde de Guadalimar. Medrano&Herrera2006‐02 ‐ 6 1 KT340553 KT021616 KT619089 KT594729 Narcissus macrolobus BCN930825 ‐ 3 1 1 10 1 KT799113 KT021619 KT619092 KT594732 KY992411 Narcissus minor Spain. Escurial. Pico Cervero. González de Castro s.n. 14 +B 4 4 1 1 10 1 KT799114 KT021620 KT619093 KT594733 KY992427 Narcissus pseudonarcissus subsp. munozii‐garmendiae BCN920185 ‐ 3 1 1 10 1 KT799115 KT021621 KT619094 KT594734 KY992414 Narcissus moleroi Spain. Nuria. Marques2016 & Draper 14 4 8 1 1 10 1 KT210959 KR872977 KT272909 KT279965 KY992441 Narcissus nanus JB‐MNHN s.n. 14 +B 4 3 1 1 10 1 KT799116 KT021622 KT619095 KT594735 KY992428 Narcissus nevadensis1 Spain. Haza de las Piedras. J. B. Hoya de Pedraza 14 4 4 1 1 10 1 KT799122 KT021628 KT619101 KT594741 KY992406 Narcissus nevadensis2 Spain. Prados del Aire. J. B. Hoya de Pedraza 14 4 4 1 1 10 1 KT799123 KT021629 KT619102 KT594742 KY992407 Narcissus nevadensis3 Spain. Barranco del Primer Roble. J. B. Hoya de Pedraza 14 4 4 1 1 10 1 KT799124 KT021630 KT619103 KT594743 KY992408 Narcissus nevadensis4 Spain. Sierra Nevada. J. B. Hoya de Pedraza ‐ 4 1 1 10 1 KT799127 KT021633 KT619106 KT594746 KY992409 Narcissus nevadensis5 Spain. Barranco del Agudero. Medrano&Herrera s.n. ‐ 5 1 1 10 1 KT799125 KT021631 KT619104 KT594744 KY992410 Narcissus nevadensis6 Spain. Barranco de los Tejos. J. B. Hoya de Pedraza 14 4 6 1 KT799121 KT021627 KT619100 KT594740 Narcissus nevadensis7 Spain. Cortijo del Sotillo. Medrano&Herrera s.n. ‐ 6 1 KT799126 KT021632 KT619105 KT594745 Narcissus nobilis1 Spain. Puerto del Pontón. Marques2057, H. Nava & A. Bueno 28 4 8 1 1 10 1 KT799117 KT021623 KT619096 KT594736 KY992436 Narcissus nobilis2 Spain. Puerto de Tarna. Marques2058, H. Nava & A. Bueno 28 4 8 1 KT799118 KT021624 KT619097 KT594737 Narcissus nobilis var. leonensis1 Spain. Puerto de Lunada. Marques2054, H. Nava & A. Bueno 42 4 8 1 1 10 1 KT340548 KT021611 KT619084 KT594724 KY992435 Narcissus nobilis var. leonensis2 Spain. Cerneja. Marques2056, H. Nava & A. Bueno 42 4 8 1 KT340547 KT021610 KT619083 KT594723 Narcissus pallidiflorus1 Spain. Villarreal de Alava. Marques2084 ‐ 8 1 1 10 1 KT799120 KT021626 KT619099 KT594739 KY992430 Narcissus pallidiflorus2 Spain. Covadonga. RJB s.n. 14 +B 4 4 1 KT799119 KT021625 KT619098 KT594738 Narcissus portensis BCN920206 ‐ 4 1 1 10 1 KT799135 KT033074 KT808807 KT878413 KY992413 Narcissus hispanicus subsp. perez‐chiscanoi BCN920175 ‐ 4 1 1 10 1 KT799074 KT033086 KT808819 KT878425 KY992415 Narcissus primigenius1 Spain. Gijón. Marques2059, H. Nava & A. Bueno 14 4 8 1 1 20 1 KT799136 KT033075 KT808808 KT878414 KY992434 Narcissus primigenius2 Spain. Covadonga. Marques2060, H. Nava & A. Bueno 14 4 8 1 KT799137 KT033076 KT808809 KT878415 Narcissus pseudonarcissus1 Andorra. Joan Pedrol7951 14 4 4 1 1 10 1 KT799066 KT033077 KT808810 KT878416 KY992418 Narcissus pseudonarcissus2 Belgica. Sibret. Belleau. Claude Lebfrere s.n. 14 4 8 1 1 10 1 KT799067 KT033078 KT808811 KT878417 KY992419 Narcissus pseudonarcissus3 Belgium. Grand Leez. Claude Lefèbvre s.n. 14 4 8 1 KT799068 KT033079 KT808812 KT878418 Narcissus pseudonarcissus4 Belgium. Onoz. Claude Lefèbvre s.n. 14 4 4 1 KT799069 KT033080 KT808813 KT878419 Narcissus pseudonarcissus5 Spain. Soria. Felipe Lopes Domínguez s.n. 14 4 4 1 KT799070 KT033081 KT808814 KT878420 Narcissus pseudonarcissus6 Spain. Sierra de Urbasa. Juan Carlos Moreno s.n. ‐ 4 1 KT799071 KT033082 KT808815 KT878421 Narcissus pumilus Portugal. JB‐MNHN s.n. 14 +B 4 4 1 1 10 1 KT326980 KT210966 KT279996 KT340505 KY992429 Narcissus segurensis Spain. Arroyo del Tejuelo. Medrano & Herrera 2005‐01 ‐ 6 1 1 20 2 KT799085 KT033097 KT808830 KT878436 KY992396, KY992397 Narcissus ×somedanus (asturiensis x triandrus) Spain. Somiedo. Miguel Serrano s. n. 21 4 4 1 1 10 1 KT340543 KT021597 KT316790 KT594710 KY992425 Narcissus tortuosus Spain. Cabo Peñas. H. Nava s.n. 14, 28 4 4 1 1 10 1 KT863431 KT033127 KT808860 KT878466 KY992431 Narcissus yepesii1 Spain. Lower Arroyo de Navalasna. Medrano&Herrera2005‐02 ‐ 4 1 1 10 1 KT863445 KT033141 KT808874 KT878480 KY992398 Narcissus yepesii2 Spain. Upper Arroyo de Navalasna. Medrano&Herrera2005‐03 ‐ 4 1 1 10 1 KT863446 KT033142 KT808875 KT878481 KY992399 Narcissus yepesii3 Spain. Fuente de la Jordana. Medrano&Herrera2005‐04 ‐ 6 1 KT863447 KT033143 KT808876 KT878482 Narcissus yepesii4 Spain. Navalcaballo. Medrano&Herrera2005‐05 ‐ 6 1 KT863448 KT033144 KT808877 KT878483 Narcissus asturiensis x N. obesus Portugal. Lagoa Comprida. Marques1858 & Draper 14* 4 8 1 1 10 1 KT326962 KR872986 KT272918 KT279974 KY992422 Narcissus xhannibalis (hispanicus x triandrus subsp. lusitanicus) Portugal. Águas Belas. Marques1949 & Draper 14 4 8 1 1 10 1 KT340545 KT021599 KT316792 KT594712 KY992417 Outgroups Lapiedra martinezii Spain. Gata de Gorgos. Marques2071 & Draper 22 4 5 1 1 10 1 KM359734 KM359730 KT279981 KT279950 KY971418 Leucojum aestivum RJB s.n. ‐ 5 1 1 10 1 KM359736 KM359732 KT279983 KT279952 KY971419 Sternbergia lutea RJB s.n. ‐ 5 1 1 10 1 KM359735 KM359731 KT279982 KT279951 KY971420 Nerine bowdenii RJB s.n. ‐ 5 1 1 10 1 KM359737 KM359733 KT279984 KT279953 KY971421 Nothoscordum dialystemon Genbank; used only to date the phylogeny AF508406.1 JQ435522.1 Asparagus cochinchinensis Genbank; used only to date the phylogeny JX903371.1 AB029804.1 TOTAL 913 1770 289 163 1650 169 Table S4

Table S4. Results obtained from the pairwise incongruence lenght difference (ILD) tests. Numbers indicate probability of congruence between partitions. Numbers between brackets indicate probabilities without considering N. dubius and N. tortifolius sequences. Asterisks denote significant incongruence (p<0.05).

ndhF matK cob atpB ITS - ndhF

0.90 matK - (0.85) 0.65 0.70 cob - (0.67) (0.78) 0.74 0.81 0.91 atpB - (0.74) (0.83) (0.92) <0.01* <0.01* <0.01* <0.01* ITS - (<0.01*) (<0.01*) (<0.01*) (<0.01*)

Appendix

Table S5. Significance values for recombinant sequences detected by several algorithms implemented in RDP.

Recombinant sequences MaxChi Chimaera Siscan N. bulbocodium subsp. graellsii 1.33 x 10-5 1.34 x 10-5 3.08 x 10-7 N. bulbocodium subsp. quintanilhae1 1.86 x 10-4 1.85 x 10-4 1.02 x 10-6 N. bulbocodium subsp. quintanilhae2 1.92 x 10-4 1.93 x 10-4 1.02 x 10-6 N. citrinus 4.60 x 10-6 3.85 x 10-7 4.45 x 10-8 N. jacquemoudii 1.34 x 10-6 1.33 x 10-5 3.08 x 10-7 N. jeanmonodii 2.45 x 10-6 2.46 x 10-6 2.58 x 10-8 N. nivalis 2.40 x 10-6 2.41 x 10-6 5.37 x 10-7 N. tingitanus 2.35 x 10-5 2.35 x 10-5 8.12 x 10-8 N. ×barrae 4.78 x 10-5 4.44 x 10-7 4.50 x 10-8 N. ×brevitubulosus 2.67 x 10-6 2.41 x 10-6 5.36 x 10-7 N. ×felineri 2.41 x 10-6 2.51 x 10-6 5.37 x 10-7 N. ×tuckeri 3.46 x 10-8 3.22 x 10-7 3.34 x 10-8 N. ×rozeirae2 9.05 x 10-7 9.06 x 10-7 5.26 x 10-8

Table S6

Table S6. List of populations sharing an identical cloned ITS sequence with other

populations of the same taxon or with other taxa. See also Fig. 2. Underlined taxa

represent reported hybrids between different sections.

Group I

 N. serotinus1 - N. serotinus2 - N. serotinus3 - N. serotinus4

 N. miniatus3 - N. miniatus4

 N. elegans1 - N. elegans2 - N. elegans3 - N. elegans4

Group II

 N. dubius1 - N. dubius2 - N. ×pujolli (assoanus × dubius)

 N. tazetta1 - N. papyraceus1 - N. papyraceus2

Group III

 N. scaberulus1 - N. ×carringtonii (scaberulus × triandrus subsp. lusitanicus)

 N. rupicola1 - N. rupicola2 - N. rupicola × triandrus

 N. cuatrecasasii2 - N. cuatrecasasii × triandrus

 N. rupicola1 - N. rupicola2 - N. rupicola × triandrus

Group IV

 N. bulbocodium var. nivalis - N. ×brevitubulosus - N. jeanmonodii

 N. bulbocodium subsp. graellsii - N. jacquemoudii

 N. bulbocodium2 - N. bulbocodium subsp. obesus × N.calcicola

Group V

 N. hedraeanthus - N. ×tuckeri (fernandesii × hedraenthus)

 N. ×barrae - N. ×cazorlanus - N. ×susannae

Group VI

 N. triandrus subsp. pallidulus1 - N. triandrus subsp. pallidulus2

 N. triandrus1 - N. triandrus subsp. lusitanicus1 - N. triandrus subsp.

lusitanicus3 - N. triandrus subsp. lusitanicus3 - N. triandrus ×bulbocodium

 N. triandrus2 - N. triandrus3 - N.×rozeirae (bulbocodium × triandrus subsp.

pallidulus)

 N. viridiflorus1 - N. viridiflorus2 - N. viridiflorus3 - N. viridiflorus4 - N.

viridiflorus5 - N. ×alleniae1

 N. jonquilla1 - N. jonquilla2 - N. willkommii1

 N. fernandesii1 - N. fernandesii2

 N. fernandesii var. major - N. fernandesii ×payraceus

 N. assoanus1 - N. ×pujolii (assoanus × dubius)

 N. cavanillesii1 - N. cavanillesii2 - N. cavanillesii3 - N. cavanillesii4 - N.

cavanillesii5 - N. ×perezlarae (cavanillesii × miniatus)

Group VII

 N. longispathus1 - N. segurensis - N. yepesii1 - N. yepesii2

 N. minor - N. nanus

 N. longispathus2 - N. longispathus3

 N. nevadensis1 - N. nevadensis2

 N. nevadensis3 - N. nevadensis4  N. pseudonarcissus1 - N. hispanicus subsp. perez-chiscanoi

 N. pseudonarcissus2 - N. pseudonarcissus subsp. munozii-garmendiae

 N. hispanicus - N. ×hannibalis (hispanicus × triandrus subsp. lusitanicus)

Table S7

Table S7. The speciation (λ) and extinction (μ) rates for (a) the entire tree, (b) subgenus

Hermione, and (c) subgenus Narcissus.

(a) Speciation rate (λ) 1.62 (90 % HPD: 1.19- 2.16) Extinction rate (μ) 1.41 (90 % HPD: 0.91– 2.02)

(b) Speciation rate (λ) for the subgenus Hermione 1.58 (90 % HPD: 1.14- 2.11) Extinction rate (μ) for the subgenus Hermione 1.41 (90 % HPD: 0.90– 2.02)

(c) Speciation rate (λ) for the subgenus Narcissus 1.63 (90 % HPD: 1.20- 2.17) Extinction rate (μ) for the subgenus Narcissus 1.41 (90 % HPD: 0.90– 2.02)

Figure S1

ITS1 5.8S ITS2

69 126 227 273 378 400 563 591 601 642 644 N. serotinus 1-4 69 126 227 233 400 563 591 601 644 N. miniatus 1 69 126 227 400 563 591 601 644 N. miniatus 2 14 69 111 233 417 578 632 659 N. miniatus 3-4

14 69 233 578 659 Sub-genus N. elegans 1-4 Hermione 75 400 508 588 N. tazetta 1-2 24 400 588 N. tazetta 3 14 30 41 158 467 588 N. tazetta 4 111 142 233 289 314 563 578 601 N. tortifolius1 246 273 316 427 632 N. tortifolius 2 316 N. alcaracensis 126 227 563 591 601 632 644 N. ×alleniae1 400 N. ×alleniae2 233 N. assoanus 2 632 Sect. Jonquillae + Sect. Ganymedes 138 528 Sect. Apodanthae 528 578 632 Sub-genus Sect. Ganymedes 578 Narcissus N. bulbocodium 1-3 + N. graellsii + N. jacquemoudii + N. tingitanus + N. nivalis 417 578 N. jeanmonodii + N. ×felineri + N. brevitubulosus 126 578 N. bulbocodium subsp. quintanilhae 1 75 126 N. bulbocodium subsp. quintanilhae 2 62 2nd group Sect. Bulbocodium

Figure S1. Potential methylation sites within ITS1, 5.8S and ITS2 in the sequences of Narcissus. Figure S2

60 GC contents (%)

40 BRA JON APO GAN BUL PSE SER AUR TAZ -20

-40

-60

-80

-100

Free energy (Kcal/mol) -120 5.8S -140 ITS1

-160 ITS2

-180 BRA JON APO GAN BUL PSE SER AUR TAZ

Figure S2. GC contents and estimated free energies in 5.8S, ITS1 and ITS2 regions, among the di erent taxonomic groups. Group legend: BRA: Braxireon, JON: Jonquillae, APO: Apodanthi, GAN: Ganymedes, BUL: Bulbocodii, PSE: Pseudonarcissi, SER: Serotini, AUR: Aurelia and TAZ: Tazettae. The two groups of Bulbocodiii, Jonquillae and Pseudonarcissus are plotted together, as is section Narcissus within Pseudonarcissus since no signicant di erences were found (p < 0.001). Figure S3

.18 A .18 B N. dubius N. tortifolius .16 .16

.14 .14

.12 .12

.10 .10

.08 . .08

.06 .06

.04 .04

.02 .02

0.00 0.00 0.000 .002 .004 .006 .008 .010 .012 .014 .016 0.000 .002 .004 .006 .008 .010 .012 .014 .016 nr DNA ITS .18 N. dubius C .18 D N. tortifolius .16 .16

.14 .14

.12 .12 Hybrids .10 .10

.08 .08

.06 .06

.04 .04

.02 .02

0.00 0.00 0.000 .002 .004 .006 .008 .010 .012 .014 .016 0.000 .002 .004 .006 .008 .010 .012 .014 .016

orgDNA

Figure S3. Distance-matrix rate test plot comparing nrDNA ITS and organellar rates of evolution (see Supplementary Methods). A) Global plot including all pairwise comparisons between all accessions of Narcissus. B) Pairwise comparisons between accessions from subgenus Hermione. C) Pairwise comparisons between accessions from subgenera Hermione and Narcissus. D) Pairwise comparisons between accessions from subgenus Narcissus. Figure S4A

subgen. Hermione subgen. Narcissus

sect. Jonquillae D6 11. N. serotinus C1 12. N. ×alentejanus C1 D1 22-6 13. N. cerrolazae D3 14-4 14. N. cordubensis D1 C1 11-32,12-2 sect. Serotini 15. N. dubius D4 16. N. fernandesii D2 D5 17. N. fernandesii var. major D3 8-4, 22-36 D2 18. N. fernandesii × papyraceus D2 16-24, 18- 8 10. N. broussonetii B1, B2 19. N. jonquilla D2 19-48, 20-4 20. N. jonquilla var. henriquesii D2 23-30 21. N. tortifolius D4 sect. Aurelia 22. N. viridi orus D5, D6 D3 23. N. willkommii D2 B1 8. N. ×alleniae D5 10-15 13-4,17-8 24. N. ×pujolii D4

B2 10-9 D4 15-18 21-20, 24-6

A9 7-8

A8 A10 A11 7-8 1-32,2-28 8-10 9-2 E1 E2 84-6 84-12

A7 7-6 E5 E4 E3

84-4 84-4 84-24

E1 1 E12 1. N. elegans A10 E6 E7 2. N. miniatus A10 86-16 88-8 3. N. pachybolbus A3 84-10 84-18 4. N. pannizianus A2, A4 5. N. papyraceus A4 6. N. polyanthos A1 E8 E13 7. N. tazetta A5-A9 8. N. ×alleniae A 1 1 84-16 84-4 9. N. ×perezlarae A10 E10 E14 92-3 84-10

A6 E9 7-4 sect. Tazettae 84-8

E15 E16 84-4 84-6

84 N. bulbocodium E1-E9, E13-E18, E23 85. N. bulbocodium subsp. graellsii E21 E17 86. N. bulbocodium subsp. obesus E11, E20, G1, G2 84-4, 94-4 87. N. bulbocodium subsp. quintanilhae E19, M1 A5 88. N. bulbocodium var. citrinus E12 7-8 89. N. bulbocodium var. nivalis E22 90. N. jacquemoudii E21 91. N. jeanmonodii E20 92. N. tenuifolius E10 93. N. tingitanus E20 94. N. ×barrae E17 A2 A4 A3 95. N. ×brevitubulosus E22 E18 4-4 4-4, 5-62 3-4 96. N. ×felineri E22 84-8

A1 6-4 E21 E19 E20 86-6,91-8 85-6, 90-8 87-8 93-6

E23 E22

sect. Bulbocodii 84-16 89-16,95-8 96-8 Figure S4B

subgen. Narcissus

sect. Braxireon

I1 30. N. cavanillesii I1 9- 6, 11- 6 9. N. ×perezlarae I1 30 -48 11. N. ×alentejanus I1

50. N. alcaracensis J4 51. N. longispathus J2, J5 52. N. nevadensis J6 53. N. segurensis J2, J3 54. N. yepesii J1, J2 25. N. assoanus H2 26. N. assoanus subsp. praelongus H4 sect. Pseudonarcissus 31. N. triandrus L1, L3 27. N. baeticus H1 32. N. triandrus var. loiseleurii L4 J3 J4 28. N. assoanus subsp. rivas-martinezii H3 33. N. triandrus subsp. lusitanicus L1,L3 29. N. gaditanus H3 53-6 50 -24 34. N. triandrus subsp. pallidulus L2,L3 35. N. triandrus × bulbocodium L1 36. N. ×rozeirae L3 J5 37. N. ×susannae L3 sect. Jonquillae H4 J2 51- 14 26-4 51-14, 53-6 54-4 sect.

H1 Ganymedes J1 J6 87-8 27-8 32-4 sect. Apodanthi 54-16 52-33 M1 L4 H3 F2 38-12 31-28, 33-20 38. N. albimarginatus F2 28-4, 29-28 L1 L2 34-24 L3 31-20, 33-8,34-5 39. N. atlanticus F3 F1 F3 35-8 36-4,37-8 40. N. calcicola F7 41. N. cuatrecasasii F4 44-16 39-6, 48-14 42. N. cuatrecasasii H2 var. segimonensis F4 F4 F5 K7 K16 K19 43. N. cuatrecasasii 25-32 K9 × triandrus F4 41-16, 42-5 45-36, 46-6 sect. K3 55-12 67-4, 69-3 74-4 44. N. marvieri F1 43 -6 76-32 45. N. rupicola F5 Pseudonarcissi 64-4 F6 K10 46. N. rupicola K1 K8 K18 F5 × triandrus 47-16 68-8,73-12 63-8 47. N. scaberulus F6 66-3 81-4, 82-4 80-6 78-3, 83-8 48. N. watieri F3 49. N. ×carringtonii F8 F7 F8 K2 K4 K11 K17 K20 60-46, 61-4 40-14 49-6 62-16 71-16 75-16 56-8 77 -24

K5 K12 K15 K22 79-4 65-12 57-8 57-32

K6 K13 K14 K23

59-12 70-16 72-12 57-7,58-8

86-12 55. N. abscissus K9 sect. Narcissus 56. N. alpestris K2 G1 sect. Bulbocodii 57. N. asturiensis K15, K22, K23 58. N. asturiensis × bulbocodium subsp. obesus K23 G7 59. N. bicolor K6 60. N. bujei K4 99-12 102-4 61. N. confusus K4 62. N. cyclamineus K11 63. N. hispanicus K8 64. N. hispanicus subsp. perezchiscanoi K3 G2 65. N. jacetanus K12 G4 66. N. macrolobus K10 67. N. minor K16 86-39, 97-8 101-9 68. N. moleroi K1 69. N. nanus K16 G3 70. N. nobilis K13 71. N. nobilis var. leonensis K17 100 -8 72. N. pallidi orus K14 73. N. poeticus K1 74. N. portensis K19 75. N. primigenius K20 76. N. pseudonarcissus K7 77. N. pseudonarcissus subsp. eugeniae K4 78. N. pseudonarcissus subsp. munozii-garmendiae K8 79. N. pumilus K5 N. bulbocodium subsp. obesus 86. E11, E20, G1, G2 80. N. radii orus K1 N. bulbocodium subsp. obesus x calcicola G2 97. 81. N. tortuosus K18 N. cantabricus G5 G5 98. 82. N. ×somedanus K18 99. N. hedraeanthus G7 98-24 83. N. ×hannibalis K8 100. N. peroccidentalis G3 101. N. romieuxii G4 102. N. ×cazorlanus G7 G6 103. N. ×tuckeri G6 103-6

Figures S4A and 4B. Statistical parsimony haplotype networks based on 1750 Narcissus sequences concatenated from two chloroplast regions (ndhF and matK). Small circles denote single mutational steps. Color-coded shades identify the di erent sections as in Fig. 1 and 2. White rectangular boxes represent haplotypes, including the species where they occur (identied by a red number) and the number of sequences corresponding to that haplotype within each species. FigureAppendix S5

+ 4

MMCO 0 Global mean temperature - 4

- 8 7 0 6 0 5 0 4 0 3 0 2 0 1 0 0

Asparagus cochinchinensis Nothoscordum dialystemon Sternbergia lutea

0.11 N. dubius 8x 0.4 N. tortifolius 6x N. ×pujolii 2.89 0.2 N. fernandesii var. major 4x,5x N. cerrolazae 2x 0.88 7 N. cordubensis 4x 4.31 0.18 N. jonquilla 2x 0.39 N. willkommii 2x 0.09 N. fernandesii 2x N. viridiflorus 4x 5.56 1.88 N. tenuifolius N. bulbocodium 2x, 4x, 5x, 6x, 7x

0.08 N. bulbocodium var. nivalis 2x 0.25 N. ×felineri

3.43 0.8 N. ×brevitubulosus N. jacquemodii 0.09 14 1.19 N. bulbocodium subsp. graellsii

0.12 N. jeanmonodii 6 1 1.99 N. tingitanus 0.38 N. bulbocodium subsp. obesus 4x

0.16 N. bulbocodium 2x, 4x, 6x N. ×barrae N. triandrus 2x N. ×rozeirae 11 1.05 0.26 N. triandrus subsp. pallidulus 2x 0.08 N. ×susannae 0.17 0.43 N. triandrus subsp. lusitanus 2x N. triandrus var. loiseleurii 2x 0.18 N. confusus 0.6 N. pseudonarcissus subsp. eugeniae 2x N. pumilus 2x

1.7 N. abscissus 16.05 0.56 N. hispanicus 2x 0.09 N. ×hannibalis 2x 1.44 0.34 10 3.29 N. pseudonarcissus subsp. munozii-garmendiae 0.09 N. pseudonarcissus 2x N. bicolor

2.24 N. macrolobus N. asturiensis 2x 1.19 0.41 1 54,05 N. cyclamineus 2x 5 9.99 0.8 N. nanus 2x 0.1 1.11 N. minor 2x N. tortuosus 2x, 4x 0.09 1.57 N. ×somedanus 3x 0.26 4.94 N. portensis 2x 0.72

2.8 N. primigenius 2x 0.52 N. nobilis var. leonensis 8x 0.38 N. jacetanus 2x N. nobilis 4x 0.76 N. pallidiflorus 2x 0.14 N. moleroi 2x 6.22 0.43 N. radiiflorus 2x 1.43 N. poeticus 2x x=7 0.59 N. alpestris N. bujei 2x N. bulbocodium subsp. quintanilhae 3x, 6x

0.08 N. cavanillesii 4x N. ×alentejanus 0.04 N. ×perezlarae 4.93 1.03 N. assoanus 2x N. baeticus 2x 2.84 0.83 N. assoanus subsp. praelongus 8.08 N. gaditanus 2x, 3x 12 0.19 2.31 N. assoanus subsp. rivas-martinezii 2x N. yepesii 0.59 18.1 N. nevadensis 2x 3 1.01 N. alcaracensis 13.8 0.18 N. longispathus 0.34 N. segurensis

0.63 N. calcicola 2x N. scaberulus 2x 0.32 8.76 6 2.64 N. ×carringtonii N. rupicola 2x 0.52 N. cuatrecasasii 2x 1.3 N. marvieri 2x 0.31 N. albimarginatus 2x 0.62 N. atlanticus 2x 0.09 N. watieri 2x 1.01 N. bulbocodium subsp. obesus 4x x=5 N. bulbocodium subsp. obesus 4x

135.37 0.13 N. ×cazorlanus 1.89 N. hedraeanthus N. ×tuckeri 2 37,08 3.37 0.7 N. peroccidentalis N. romieuxii 4x 1.67 0.24 N. romieuxii 4x N. cantabricus 4x N. pachybolbus 4x 8 1.29 N. pannizianus 4x 0.57 N. polyanthos 4x 0.29 N. papyraceus 4x 4 5.98 N. tazetta 4x 1.35 N. elegans 4x 0.39 N. miniatus 6x 3.6 0.2 N. ×perezlarae N. ×alleniae N. broussonetii 4x,8x 9 2.57 0.14 N. serotinus 2x N. ×alentejanus

12.68 Leucojum aestivum Lapiedra martinezi Nerine bowdeni

7 0 6 0 5 0 4 0 3 0 2 0 1 0 0

Cretaceous Paleocene Eocene Oligocene Miocene Pliocene Holocene Pleistocene

Figure S5. Figure S6

subgenus Hermione 9 4 8

13 λ 6 1.7

subgenus Narcissus 5 1.5 10

14 1.2 7

Figure S6. Maximum clade credibility (MCC) tree from a BEAST analysis of plastid DNA sequences (ndhF-matK) of Narcissus with branches colored by speciation rate (λ, sp/Ma) across the tree estimated using Bayesian Analysis of Macroevolutionary Mixtures (BAMM). Branch numbers refer to clades described in Table S7. Figure S7A

Aurelia Apodanthi Braxireon Bulbocodii N. bulbocodium1 Ganymedes N. bulbocodium2 Fig. 1B N. bulbocodium3 Jonquillae N. bulbocodium4 Narcissus N. bulbocodium5 N. bulbocodium6 Pseudonarcissi N. bulbocodium7 Serotini N. bulbocodium8 N. bulbocodium9 Tazettae N. bulbocodium10 N. bulbocodium11 N. tenuifolius N. bulbocodium12 N. bulbocodium13 N. bulbocodium14 N. bulbocodium subsp. obesus10 N. bulbocodium subsp. obesus11 N. bulbocodium var. citrinus N. ×felineri (bulbocodium × primigenius) V N. ×brevitubulosus (asturiensis × bulbocodium var. nivalis N. bulbocodium var. nivalis1 N. bulbocodium15 N. bulbocodium16 N. bulbocodium var. nivalis2 N. jacquemoudii N. bulbocodium subsp. graellsii N. bulbocodium17 N. bulbocodium18 N. bulbocodium subsp. quintanilhae1 N. bulbocodium subsp. obesus12 N. jeanmonodii N. tingitanus N. bulbocodium19 N. bulbocodium20 N. bulbocodium21 N. ×barrae (bulbocodium × cantabricus) N. bulbocodium22 N. bulbocodium23 N. bulbocodium24 N. fernandesii1 N. willkommii1 N. jonquilla1 N. jonquilla2 subgen. Narcissus N. jonquilla3 N. jonquilla4 N. jonquilla5 N. jonquilla6 N. jonquilla7 N. jonquilla8 N. jonquilla var. henriquesii N. cordubensis N. fernandesii2 N. fernandesii3 N. fernandesii4 N. fernandesii × N. papyraceus N. willkommii2 N. willkommii3 N. willkommii4 IV N. fernandesii var. major N. cerrolazae N. viridiflorus1 N. viridiflorus2 N. viridiflorus3 N. viridiflorus4 N. viridiflorus5 N. viridiflorus6 N. ×alleniae1 (miniatus × viridiflorus) N. bulbocodium subsp. quintanilhae2

N. calcicola1 N. calcicola2 N. calcicola3 N. scaberulus1 N. scaberulus2 N. ×carringtonii (scaberulus × triandrus subsp. pallidulus) N. rupicola1 N. rupicola2 N. rupicola3 N. rupicola4 N. rupicola5 N. rupicola × N. triandrus III N. cuatrecasasii1 N. cuatrecasasii2 N. cuatrecasasii var. segimonensis N. cuatrecasasii × N. triandrus N. watieri1 N. watieri2 N. atlanticus N. albimarginatus1 N. albimarginatus2 N. marvieri1 N. marvieri2

N. ×alleniae1 (miniatus × viridiflorus) N. ×alleniae2 (miniatus × viridiflorus) N. elegans1 N. elegans2 N. elegans3 N. elegans4 N. elegans5 N. elegans6 N. ×perezlarae (cavanillesii × miniatus) N. tazetta3 II N. tazetta1 N. tazetta2 N. broussonetii1 N. broussonetii2 N. broussonetii3 N. broussonetii1 N. broussonetii2 N. broussonetii3 N. ×alentejanus (cavanillesii × serotinus) N. serotinus1 N. serotinus2 N. serotinus3 N. serotinus4 N. pannizianus1 N. pannizianus2 N. papyraceus2 N. papyraceus3 subgen. Hermione N. papyraceus4 N. papyraceus5 N. papyraceus6 N. polyanthos N. papyraceus7 I N. papyraceus8 N. pachybolbus N. papyraceus9 N. papyraceus10 N. papyraceus1 N. tazetta4 Sternbergia lutea N. tazetta5 Leucojum aestivum Lapiedra martinezii Nerine bowdenii 0.0030 Figure S7B

N. asturiensis1 N. asturiensis2 Aurelia N. pseudonarcissus8 N. asturiensis × N. obesus Apodanthi N. asturiensis3 Braxireon N. asturiensis4 Bulbocodii N. asturiensis5 N. asturiensis6 Ganymedes N. asturiensis7 Jonquillae N. primigenius1 N. primigenius2 Narcissus N. nobilis var. leonensis1 Pseudonarcissi N. portensis Serotini N. ×somedanus (asturiensis × triandrus) N. tortuosus Tazettae N. nobilis var. leonensis2 N. cyclamineus1 N. cyclamineus2 N. minor1 N. nanus N. asturiensis8 N. cyclamineus3 N. pseudonarcissus1 N. pseudonarcissus2 N. pseudonarcissus3 N. pseudonarcissus4 N. pseudonarcissus5 N. abscissus1 N. abscissus2 N. hispanicus N. hispanicus subsp. perez-chiscanoi N. pseudonarcissus subsp. munozii-garmendiae N. ×hannibalis (hispanicus × triandrus subsp. lusitanicus) N. nobilis1 N. nobilis2 N. pallidiflorus1 N. pallidiflorus2 N. pseudonarcissus subsp. eugeniae1 N. pseudonarcissus subsp. eugeniae2 N. bicolor1 N. bicolor2 VIII N. bujei1 N. bujei2 N. jacetanus N. bujei3 N. pseudonarcissus subsp. eugeniae3 N. confusus N. bujei4 N. macrolobus N. pumilus N. triandrus subsp. pallidulus1 N. triandrus subsp. pallidulus2 N. triandrus subsp. pallidulus3 N. triandrus subsp. pallidulus4 N. triandrus subsp. pallidulus5 Figure S7. Bayesian tree (50% majority-rule N. triandrus subsp. pallidulus6 N. triandrus1 consensus) of organellar DNA sequences (ndhF N. triandrus2 N. triandrus3 and matK chloroplast regions, cob and atpA N. triandrus7 N. triandrus var. loiseleurii mitochondrial regions) from a non-redundant N. triandrus4 N. ×rozeirae (bulbocodium × triandrus subsp. pallidulus) matrix excluding the allopolyploids N. dubius, N. ×susannae (cantabricus × triandrus subsp. pallidulus) N. miniatus and N. tortifolius. Posterior probabilities N. triandrus subsp. lusitanicus1 N. triandrus subsp. lusitanicus2 (expressed as percentages) and parsimony N. triandrus subsp. lusitanicus3 N. triandrus subsp. lusitanicus4 bootstrap supports greater than 50% are indicated N. triandrus × N. bulbocodium N. triandrus5 above and below the branches, respectively. N. triandrus6 N. bujei1 A thicker line indicates 100% support in both N. bujei2 N. bujei3 analyses. Black circles: >95% support value; N. bujei4 N. alpestris grey circles: 95 ≥support value ≥70; N. moleroi N. radiiflorus white circles: <70 support value. N. poeticus1 N. poeticus2 Color-coded branches and vertical tags follow N. nevadensis1

N. nevadensis2 Subgen. Narcissus currently accepted sections as indicated in the N. nevadensis3 N. nevadensis4 legend. Roman numerals denote groups N. nevadensis5 N. nevadensis6 discussed in the main text. N. nevadensis7 N. alcaracensis1 N. alcaracensis2 N. alcaracensis3 N. longispathus1 N. longispathus2 N. segurensis N. yepesii1 N. longispathus3 N. longispathus4 N. longispathus5 N. yepesii2 N. yepesii3 VII N. yepesii4 N. assoanus subsp. rivas-martinezii N. assoanus subsp. praelongus N. gaditanus1 N. gaditanus2 N. gaditanus3 N. gaditanus4 N. assoanus1 N. assoanus2 N. assoanus3 N. assoanus4 N. baeticus N. cavanillesii1 N. cavanillesii2 N. cavanillesii3 N. cavanillesii4 N. cavanillesii5 N. cavanillesii6 N. ×alentejanus (cavanillesii × serotinus) N. ×perezlarae (cavanillesii × miniatus) N. romieuxii1 N. romieuxii2 N. romieuxii1 N. peroccidentalis N. cantabricus1 N. cantabricus2 N. cantabricus3 N. cantabricus4 N. hedraeanthus1 N. hedraeanthus2 N. ×cazorlanus (hedraeanthus × triandrus subsp. pallidulus) VI N. ×tuckeri1 (fernandesii × hedraeanthus) N. ×tuckeri2 (fernandesii × hedraeanthus) N. bulbocodium subsp. obesus1 N. bulbocodium subsp. obesus2 N. bulbocodium subsp. obesus3 N. bulbocodium subsp. obesus4 N. bulbocodium subsp. obesus5 N. bulbocodium subsp. obesus × N. calcicola N. bulbocodium subsp. obesus6 N. bulbocodium subsp. obesus7 Fig. 1A N. bulbocodium subsp. obesus8 N. bulbocodium subsp. obesus9 Figure S8

N. pannizianus N. papyraceus1 N. papyraceus2 N. pachybolbus N. polyanthos N. broussonetii1 N. broussonetii2 II N. papyraceus3 N. papyraceus4 N. tazetta2 N. tazetta3 N. tazetta4 N. tazetta5 N. tazetta1 N. serotinus1 N. serotinus2

N. serotinus3 subgen. Hermione N. serotinus4 I N. elegans1 N. elegans2 N. elegans3 N. elegans4

0.03 Figure S8. Highlight of the Bayesian tree (50% majority-rule consensus) of nuclear ribosomal ITS for subgenus Hermione from an analyses based on a non-redundant matrix excluding the allopolyploids N. dubius, N. miniatus and N. tortifolius (and the hybrids N. ×alleniae and N. ×pujolii). Posterior probabilities (expressed as percentages) and parsimony bootstrap supports greater than 50% are indicated above and below the branches, respectively. A thicker line indicates 100% support in both analyses. Black circles: >95% support value; grey circles: 95 ≥support value ≥70; white circles: <70 support value. Color-coded branches and vertical tags follow currently accepted sections as indicated in the legend. Roman numerals denote groups discussed in the main text. Figure S9

orgDNA nrDNA

Narcissus subsect. Pseudonarcissi VII Narcissus

Nevadensis Ganymedes

VIII Narcissus subsect. Pseudonarcissi Narcissus subsect. Narcissus

Nevadensis Ganymedes VII Juncifolii Jonquillae

Braxireon VI / VI Juncifolii Meridionalis Braxireon

V V Meridionalis Bulbocodii

IV Bulbocodii

Jonquillae

IV III Dubii Apodanthi Jonquillae Bulbocodii III Apodanthi Aurelia II Tazettae Angustifolii Dubii Tazettae II outgroups Aurelia outgroups Serotini Serotini I I Tazettae Angustifolii

Figure S9. New subgeneric classi cation here proposed accommodated to the 50% majority-rule consensus BI trees from organellar DNA (left) and nrDNA ITS (right) data sets presented in Figure 3. Major clades (identi ed with roman numerals following Figures 1 and 2) are collapsed, dotted lines link clades (or rarely grades) whose composition coincides in both phylogenetic trees, and thick solid lines indicate sections whose composition is split into separate clades in one or both trees. Support for branches follows symbols in Figures 1 and 2. Manuscript

A three-genome five-gene comprehensive phylogeny of the bulbous genus Narcissus (Amaryllidaceae) challenges 1 current taxonomic classifications and reveals multiple hybridization events Isabel Marques, Javier Fuertes Aguilar, Maria Amélia Martins-Louçao, Farideh Moharrek, Gonzalo Nieto Feliner

SUPPLEMENTARY MATERIAL

INCONGRUENCE TESTS

Material and Methods—In order to measure the homogeneity among the three

matrices, a series of incongruence length difference (ILD) tests implemented as the

partition homogeneity test in PAUP* (Swofford, 1999) were performed. ILD test has

been shown to be sensible to factors such as between-partition differences in

evolutionary rates (Darlu & Lecointre, 2002), and size of the data partitions (Dowton &

Austin, 2002), which increase the probability of type I errors, i.e., incorrectly rejecting

the correct hypothesis of congruence. However, it is less susceptible to type II errors

when sufficient numbers of informative sites are available (Darlu & Lecointre, 2002).

We have use it here in a conservative way, following the arguments that criticisms of

the ILD test do not support its categorical rejection, that there is no completely

satisfactory test for data incongruence (Hipp & al., 2004), and that it can be particularly

useful in large data sets as ours. Pairwise analyses of all the partitions involved 104

replicates, saving 10 trees per replicate by stepwise addition that were subjected to TBR

branch swapping. Although the generally accepted significance value is p = 0.05,

Cunningham (1997) suggested that such threshold may be too conservative and thus we

have used p = 0.01.

Results —Since no phylogenetic incongruence was found between any of the

organellar regions (Table S4), they were combined in a single matrix. In contrast, the

significant non-homogeneity detected between the ITS and the plastid regions (p < 0.01;

Table S4), argued against combining these data sets.

A three-genome five-gene comprehensive phylogeny of the bulbous genus Narcissus (Amaryllidaceae) challenges 2 current taxonomic classifications and reveals multiple hybridization events Isabel Marques, Javier Fuertes Aguilar, Maria Amélia Martins-Louçao, Farideh Moharrek, Gonzalo Nieto Feliner

HAPLOTYPE NETWORK OF ORGANELLAR SEQUENCES.

Material and Methods——Haplotype networks based on statistical parsimony were constructed using the algorithm described in Templeton & al., (1992). This method is appropriate for reticulated data (i.e., tokogenetic) and allows a better visualization of frequency of haplotypes, the haplotype distribution across species, and the underlying taxonomic information. Networks of the chloroplast, and mitochondrial dataset were separately constructed using TCS 1.21 (Clement & al., 2000) with gaps coded as missing data.

Results — The statistical parsimony analyses of the combined ndhF-matK plastid matrix collapsed the sequences into 96 haplotypes (Fig. S4). TCS results identified two unconnected networks, in agreement with the chloroplast tree. The statistical parsimony analysis of the mitochondrial sequences detected two networks with poor resolution, which are congruent with the two major networks found for the chloroplast data (data not shown).

The network corresponding to subgenus Hermione connected 14 haplotypes

(Fig. 1A). Haplotypes are clustered into three major groups corresponding to sections

Aurelia, Serotini and Tazettae. Nine haplotypes are found within section Tazettae, which required thirteen missing haplotypes to be all connected along the network. The highest haplotype diversity was found in N. tazetta, which presented five different haplotypes (A5-A9). Narcissus elegans and N. miniatus and N. ×perezlarae share the same haplotype (A10), as do N. serotinus and N. ×alentejanus (C1) in section Serotini.

The network corresponding to subgenus Narcissus arranged the remaining 82 haplotypes into 10 groups (Fig. S4A-B). More than half (45) of those haplotypes are found in the first group of Bulbocodii and the first group of Pseudonarcissus. Several A three-genome five-gene comprehensive phylogeny of the bulbous genus Narcissus (Amaryllidaceae) challenges 3 current taxonomic classifications and reveals multiple hybridization events Isabel Marques, Javier Fuertes Aguilar, Maria Amélia Martins-Louçao, Farideh Moharrek, Gonzalo Nieto Feliner species share the same haplotype and, conversely, some species have two or even three haplotypes but the extreme case was represented by N. bulbocodium that presented 16 haplotypes. Three groups of haplotypes match the taxonomic sections Apodanthi,

Ganymedes and Braxireon, respectively. The other three sections, Pseudonarcissus,

Jonquillae and Bulbocodii appear each in two different groups separated by several mutational steps, which corroborate the results depicted in the phylogenetic trees. Other unexpected results with respect to taxonomy are (i) the placement of the haplotypes of

N. dubius and N. tortifolius (section Tazettae) within one of the two groups of section

Jonquillae; (ii) that of N. poeticus and N. radiiflorus (section Narcissus) with species of section Pseudonarcissus, sharing the same haplotype with N. moleroi Fern.Casas (K1); and (iii) the disparate position of the haplotype (M1) of one population of N. bulbocodium subsp. quintanilhae (which should be related to those of section

Ganymedes) and one of the groups of section Pseudonarcissus.

RECOMBINATION ANALYSES.

Material and Methods—Overlooking potential recombined sequences may distort phylogenetic inferences (Schierup & Hein, 2000a,b; Posada & Crandall, 2002). This is particularly important in nuclear genes subject to concerted evolution like ITS. Since the accuracy of detecting recombination events depends on the amount of recombination, genetic diversity and rate variation among sites (Maynard Smith, 1999;

Posada & Crandall, 2001; Posada & Crandall, 2002), it is advisable to use several methods (Martin & al., 2005; Poke & al., 2006). Following Posada & Crandall, 2001 and Wiuf & al., 2001, six recombination tests were applied to the non-redundant matrices of the organellar and ITS data: RDP (Martin & Rybicki, 2000), GENECONV

(Padidam & al., 1999), BOOTSCAN (Salminien & al., 1995), MAXCHI (Maynard A three-genome five-gene comprehensive phylogeny of the bulbous genus Narcissus (Amaryllidaceae) challenges 4 current taxonomic classifications and reveals multiple hybridization events Isabel Marques, Javier Fuertes Aguilar, Maria Amélia Martins-Louçao, Farideh Moharrek, Gonzalo Nieto Feliner

Smith, 1992), CHIMAERA (Posada & Crandall, 2001) and SISCAN (Gibbs & al.,

2000). The tests were run using RDP beta24 (Martin & al., 2005) with the default settings except in the RDP method, where the internal sequence of N. cavanillesii1 was used as reference due to the large size of our data set. For all methods, the highest p- value, i.e., the probability that sequences share high identities in a potentially recombinant region due to chance alone was set to 0.01. The significance of χ2 peaks in

MAXCHI and CHIMAERA was determined with 1000 permutations. Sequential

Bonferroni corrections were applied to compensate for multiple tests (Rice, 1989).

Potential recombinant copies were identified whenever more than one method detected them as such.

Results — Detection of recombination by RDP yielded significantly supported events only for 13 accessions (Table S5). Of the six methods used, MaxChi, Chimaera and

Siscan always gave a strong support for two-breakage recombination points in the ITS regions, specifically in the ITS2 between positions 627 bp and 779 bp.

The recombinant sequences detected were likely the result of hybridization between N. bulbocodium subsp. obsesus3 and a second unknown parent, not identified in our dataset.

Some of these accessions exhibit a high chromosome number (e.g., some specimens of N. bulbocodium subsp. quintanilhae, 2n = 42) while others coincide in being reported to be of hybrid origin either intersectional (N. ×brevitubulosus A.Fern.,

N. ×felineri Fern.Casas, N. ×tuckeri Barra & G. López, N. ×rozeirae) or intrasectional

(N. ×barrae Fern.Casas, N. bulbocodium var. citrinus Baker). No recombination events were detected in the plastid or mitochondrial data.

A three-genome five-gene comprehensive phylogeny of the bulbous genus Narcissus (Amaryllidaceae) challenges 5 current taxonomic classifications and reveals multiple hybridization events Isabel Marques, Javier Fuertes Aguilar, Maria Amélia Martins-Louçao, Farideh Moharrek, Gonzalo Nieto Feliner

DETECTION OF PSEUDOGENES IN ITS.

Material and Methods— —DNA ribosomal loci sometimes contain non- functional ITS copies (pseudogenes) besides functional ones (e.g., Buckler & al., 1997;

Hartmann & al., 2001; Hughes & al., 2002; Bailey & al., 2003), which may lead to incorrect phylogenetic inferences (Mayol & Rosselló, 2001). Several approaches alone or combined can be used to detect the presence of pseudogenes (Razafimandimbison & al., 2004). For each sequence that was incongruent with the organellar trees, we have checked the following features in ITS1, 5.8S and ITS2 regions: (1) substitutions at potential methylation sites and length variation since pseudogenes are often characterized by an accumulation of cytosine mutations at CpG or CpNpG sites

(Buckler & al., 1997; Li, 1997); (2) Nucleotide composition and free energy since non- functional copies often have a lower GC content (Li, 1997) and a lower stability in

RNA structure frequently associated to the presence of indels in conserved regions of the ITS (Baldwin, 1992; Mayol & Rosselló, 2001; Bailey & al., 2003); and (3)

Mutations in nucleotide sequence of structural motifs in ITS1 and ITS2 regions, namely the 21 bp conserved motif GGCRY-(N)4-7- GYGYCAAGGAA found in ITS1 across

Spermatophytes (Liu & Schardl, 1994), as well as in the 5.8S gene. In ITS2, we searched for the presence and length of the six conserved and six variable motifs described in Hershkovitz & Zimmer (1996), the number of helices present, the universally conserved pyrimidine mismatch in helix II and the GCU triplet on the 5’ apex of helix III (Mai & Coleman, 1997). We used PAUP*4.0 (Swofford, 1999) to compute the %GC content for each sequence. RNA secondary structures were predicted using Quikfold implemented in the DINAMelt server (Markham & Zuker, 2005; http://www.bioinfo.rpi.edu/applications/hybrid/), which was also used to estimate the free energy (G at 37°C) for each sequence. All the above features were compared A three-genome five-gene comprehensive phylogeny of the bulbous genus Narcissus (Amaryllidaceae) challenges 6 current taxonomic classifications and reveals multiple hybridization events Isabel Marques, Javier Fuertes Aguilar, Maria Amélia Martins-Louçao, Farideh Moharrek, Gonzalo Nieto Feliner between ITS1, 5.8S and ITS2 within each sequence, between lineages in Narcissus and between putative hybrid and non-hybrid taxa.

Results — We found that 68 of the 169 cloned ITS sequences (47%) presented substitutions at potential methylation sites. Distribution of the number of deamination- like substitutions followed the pattern ITS1>ITS2>5.8S. Three cloned sequences of N. tortifolius1 showed a deletion in the 5.8S region between positions 137 and 140, while the remaining sequences were 163 bp long, similar to other angiosperms. The same three sequences of N. tortifolius1 also showed a 10 bp deletion in the ITS1 region.

Sequences from subgenus Hermione always had higher GC content averages than those from subgenus Narcissus both in ITS1 (73.25 % vs. 69.43%) and ITS2

(72.89% vs. 69.82%) (Fig. S2). The highest variability in GC contents occurred in sections Bulbocodii (x = 1.53 ± 2.13) and Tazettae (x = 73.92 ± 4.28). In the whole data set, significant differences in GC content were found between 5.8S and ITS1 (Χ2 =

349.146, p = 0.0001) as well as between 5.8S and ITS 2 region (Χ2 = 241.000, p =

0.0001) but not between ITS1 and ITS2 (Χ2 = 78.970, p = 0.667).

Secondary structure models from sequences of subgenus Hermione also had a higher mean free energy values than those of subgenus Narcissus both in ITS1 (-129.57 vs. -132.92) and ITS2 (-106.29 vs. -113.64). The eighteen reported putative hybrids reached higher values than the remaining species in ITS1 (-130.98 vs. -133.90) and in

ITS2 (-107.80 vs -116.55). In general, sequences with less stable secondary structures

(high free energies) and those with low GC content clustered together. As expected, a significant negative correlation between free energies and GC content was found in the

5.8S gene (rs = -0.850, p = 0.0001), in ITS1 (rs = -0.747, p = 0.0001) and ITS2 (rs = -

0.906, p = 0.0001). A three-genome five-gene comprehensive phylogeny of the bulbous genus Narcissus (Amaryllidaceae) challenges 7 current taxonomic classifications and reveals multiple hybridization events Isabel Marques, Javier Fuertes Aguilar, Maria Amélia Martins-Louçao, Farideh Moharrek, Gonzalo Nieto Feliner

The conserved 21 bp-region motif in ITS1 was found in all but three sequences of N. tortifolius1, where a deletion of 10 bp occurred. Like in other plants (Liu &

Schardl, 1994), the 3’ portion (AAGGAA) of the 15 bp-paired stem of that motif was always found outside the stem structure, except in the same three sequences of N. tortifolius1. In the sequences of N. serotinus and N. miniatus, several substitutions were found in the 21 bp-region. All twelve conserved and variable motifs were present in

ITS2 and their length fell within that reported in the literature for functional ITS

(Hershkovitz & Zimmer, 1996). A 5’-ATCG was found in all sequences at the first conserved motif. The predicted secondary structure of the ITS2 generally presented the common four-helix structure found in algal and plant vascular species (Baldwin & al.,

1995; Mai & Coleman, 1997). In some sequences, two helices were found in the position occupied by helix IV as reported also in Oryza (Mai & Coleman, 1997) but no association between the presence of helix V and either taxonomy or ploidy level was found. The conserved pyrimidine mismatch in helix II as well as the GCU triplet on the

5’ apex of helix III were absent in the sequences of N. miniatus, N. tortifolius, N. albimarginatus D.Müll.-Doblies & U.Müll.-Doblies, N. rupicola Dufour, N. moleroi

Fern.Casas and N. rupicola × N. triandrus L.

All together these results suggest that only three N. tortifolius1 sequences can be identified as potential pseudogenes.

DISTANCE-MATRIX RATE TEST.

Material and Methods——We have used the distance-matrix test approach to graphically compare the evolution of one gene against another from the same group of organisms without previous knowledge of time of divergence between species or their phylogenetic relationship (Syvanen, 2002; Hao & Golding, 2006). We first computed A three-genome five-gene comprehensive phylogeny of the bulbous genus Narcissus (Amaryllidaceae) challenges 8 current taxonomic classifications and reveals multiple hybridization events Isabel Marques, Javier Fuertes Aguilar, Maria Amélia Martins-Louçao, Farideh Moharrek, Gonzalo Nieto Feliner pairwise genetic distances between all the sequences independently for the nuclear and organellar matrices using the GTR model implemented in the beta 10 version of

PAUP*4.0 (Swofford, 1999). The resulting pairwise distances within ITS and organellar sequences were represented in a XY scatter plot for all accessions. For each pair of accessions the genetic distance between their organellar sequences was represented in the x axis and that between their ITS sequences was represented in the y axis. Due to the existence of cloned ITS sequences, a single pair of accessions could be represented by several dots in the plot, all with the same value for the x axis and thus parallel to the y axis. This pattern corresponds to a single organellar distance between the two accessions but several distances between the ITS sequences. The relationship between organellar distances and nuclear distances was compared between and within phylogenetic groups (Hermione and Narcissus lineages) using SPSS 11.0 (SPSS, Inc.,

Chicago, Illinois, USA) to better discriminate the differences among groups.

Results — The pairwise distances between sequences of Narcissus showed a complex pattern, not consistent with a random model. The main source of complexity is the mutation rates in ITS although the inclusion of cloned sequences that results in several pairwise distances between the same two individuals, i.e., vertical lines, also obscure the picture. The mutation rate in ITS differs from that of the organellar sequences, which was particularly evident when comparing sequences of subgenus

Hermione between themselves or against sequences from subgenus Narcissus (Fig.

S4A).

Pairwise distance in ITS between subgenus Hermione sequences (y = 12.230x; p

= 0.0001) or between sequences from both subgenera (y = 10.390x; p = 0.0001) are higher than between subgenus Narcissus sequences (y = 7.0781x; p = 0.0001). A three-genome five-gene comprehensive phylogeny of the bulbous genus Narcissus (Amaryllidaceae) challenges 9 current taxonomic classifications and reveals multiple hybridization events Isabel Marques, Javier Fuertes Aguilar, Maria Amélia Martins-Louçao, Farideh Moharrek, Gonzalo Nieto Feliner

Considering only the pairwise distances values between sequences from subgenus Hermione, the highest levels of divergence are caused by the organellar distances between N. tortifolius or N. dubius and the remaining taxa (Χ2 = 241.105, p =

0.0001; Fig. S4B). This is due to the fact that unlike the ITS tree where they fall within the Hermione lineage, these two species fall in the other main lineage in the organellar tree. Thus the pairwise distances with the other species of subgenus Hermione are markedly greater and they appear in a separate cloud (Fig. S4B,C). When comparing only pairwise distances between subgenus Narcissus sequences, the highest distances were found in hybrid sequences (Fig. S4D).

DIVERSIFICATION RATE ANALYSIS.

Material and Methods— —To detect and quantify heterogeneity in evolutionary rate, we used BAMM version 2.2.0 (Bayesian Analysis of

Macroevolutionary Mixtures; Rabosky & al., 2014a). We based our analysis on the reduced chloroplast sequence dataset, from which the time-calibrated phylogeny in

Newick format resulting from the divergence time inference using BEAST was pruned in R (R Development Core Team, 2014) to have just ingroup taxa and thus facilitate unambiguous assignment of species richness. Priors for the reversible-jump Markov

Chain Monte Carlo model in BAMM were estimated using BAMMtools (Rabosky & al., 2014b). BAMM was run for 2 × 106 generations of reverse jump MCMC.

Convergence of BAMM runs was assessed by computing ESS of log-likelihoods and numbers of shifts using the CODA library for R: both parameters had effective sample sizes > 1000. The first 10 % of samples was discarded as burn-in. Post-run analysis and visualisation was performed using the R package BAMMtools.

A three-genome five-gene comprehensive phylogeny of the bulbous genus Narcissus (Amaryllidaceae) challenges 10 current taxonomic classifications and reveals multiple hybridization events Isabel Marques, Javier Fuertes Aguilar, Maria Amélia Martins-Louçao, Farideh Moharrek, Gonzalo Nieto Feliner

Results — BAMM did not detect any significant rate-shift configuration, and the best fit model to the phylogeny was one involving a homogeneous process of near constant per-lineage diversification rates. Mean speciation (λ) (Fig. S6) and extinction rates (μ) within the subgenera Hermione and Narcissus are similar to those of the entire tree (Table S7).

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