1 Title: The genetic diversity and structure of the Ferula communis L. complex (Apiaceae) in
2 the Tyrrhenian area
3
4 Authors:
5 Caterina Angela Dettori a,*, Maria Cecilia Loi a, Salvatore Brullo b, Pere Fraga i Arguimbau c,
6 Elena Tamburini d, Gianluigi Bacchetta a.
7 1 Centro Conservazione Biodiversità (CCB), Sezione di Botanica ed Orto Botanico,
8 Dipartimento di Scienze della Vita e dell’Ambiente - Università degli Studi di Cagliari. Viale
9 S. Ignazio da Laconi, 11-13 - I-09123 Cagliari, Italy.
10 b Dipartimento di Scienze Biologiche, Geologiche e Ambientali - Università degli Studi di
11 Catania. Via A. Longo 19 - I-95125 Catania, Italy.
12 c Secció de Ciències Naturals, Institut Menorquí d'Estudis (IME). Camí des Castell 28 - E-
13 07702 Maó, Menorca, Illes Balears, Spain.
14 d Sezione di Microbiologia e Virologia, Dipartimento di Scienze Biomediche - Università
15 degli Studi di Cagliari. Sesta strada Ovest, Z.I. Macchiareddu - I-09010 Uta, Italy.
16
17 *Corresponding author:
18 Caterina Angela Dettori
19 Viale S. Ignazio da Laconi, 11-13 - I-09123 Cagliari, Italy.
20 Tel: +390706753681
21 Fax: +390706753509
22 E-mail: [email protected]
23
24 Abstract
25 The giant fennel Ferula communis L. is a circum-Mediterranean complex characterized by a
26 great morphological variability and comprising several species and subspecies. In this work, 1
27 we used AFLP markers to investigate the pattern of genetic variation of the F. communis
28 complex in the Tyrrhenian area and to compare the levels of genetic diversity between the
29 widespread F. communis and the Corso-Sardinian endemic congener F. arrigonii.
30 Our study indicates fairly high levels of genetic diversity for all populations (Fragpoly = 58.2-
31 88%; Hj = 0.186-0.313), with no significant differences between F. arrigonii and F.
32 communis. The genetic structure is only partially coherent with the geographic provenance of
33 the populations: while individuals of F. arrigonii constituted a separate genetic group, the
34 individuals of F. communis were partitioned into three main genetic clusters. These
35 corresponded respectively to F. communis cf. subsp. glauca, to populations from Tunisia (F.
36 cf. vesceritensis) and from Gozo Island, and to all populations from the rest of the
37 investigated areas; this last cluster was characterized by a marked substructure.
38
39 Keywords: AFLP, Ferulinae, genetic differentiation, genetic diversity, giant fennel,
40 Tyrrhenian Islands.
41
2
42 1. Introduction
43 The genus Ferula L. (Apiaceae) is represented by 172 perennial herbaceous species
44 occurring from Central Asia, where it has its main centre of endemism, westward throughout
45 the Mediterranean region to Northern Africa and the Macaronesian Region (Mabberley,
46 2008). According to Kurzyna- Młynik et al. (2008), Ferula forms a monophyletic genus
47 within the Ferulinae subtribe as part of the Scandiceae tribe. The same authors confirmed that
48 the Mediterranean group originated from Asian ancestors, this hypothesis being also
49 supported by the general theory of the westward colonization by Asian steppe plants
50 (Frantzke et al., 2004). Pérez-Collazos et al. (2009) estimated that the split of the Ferulinae
51 into a Central Asian lineage and an Asian-Mediterranean lineage occurred 12.4 ± 3.7 Mya,
52 whereas, within the latter group, the divergence of an Asian clade and its Mediterranean sister
53 clade would have taken place 10.7 ± 3.5 Mya. The Mediterranean Ferula lineages would have
54 originated in the Late Miocene (6.7 ± 3 Mya), concurrent with the Messinian salinity crisis,
55 with subsequent species divergence in the Pliocene and the Early Pleistocene.
56 The focus of this work is on the putative taxa inhabiting the main Tyrrhenian Islands
57 and coasts, more specifically on: i) Ferula communis L.. subsp. communis and F. c. subsp.
58 glauca (L.) Rouy & Camus, two widespread taxa with a long history of taxonomical
59 uncertainties, up to the point that even today they are alternatively considered species (e.g.,
60 Conti et al., 2005; Jeanmonod and Gamisans, 2013) or subspecies (e.g., Bolòs and Vigo,
61 1984; Pignatti, 1982) by different authors; ii) F. c. subsp. cardonae Sánchez-Cux. & M.
62 Bernal, endemic to the island of Minorca; iii) F. vesceritensis Coss. & Dur., endemic to
63 Algeria and Tunisia; and iv) Ferula arrigonii Bocchieri, endemic to Sardinia and Corsica,
64 described relatively recently as a distinct species with respect to F. communis, from which it
65 differs mainly in phenological and morphological traits (Bocchieri, 1988; Dettori et al.,
66 2014a).
3
67 A large amount of literature is available regarding the phytochemistry of both F. arrigonii
68 (e.g. Delair et al., 1994; Filippini et al., 2000) and F. c. subsp. communis. In particular, two
69 main chemotypes, one poisonous to animals (with different degrees of toxicity, Sacchetti et
70 al., 2003) and the other one non-poisonous, have been distinguished and studied within this
71 latter subspecies in Sardinia (Appendino, 1997; Arnoldi et al., 2004; Rubiolo et al., 2006;
72 Sacchetti et al., 2003). These two chemotypes have been reported to be indistinguishable from
73 a morphological and a karyological point of view (Appendino, 1997; Rubiolo et al., 2006;
74 Sacchetti et al., 2003); however, the analyses carried out by Marchi et al. (2003) using
75 allozyme electrophoresis revealed that they are genetically distinct. The genetic diversity and
76 spatial genetic structure of F. arrigonii in Sardinia and Corsica were investigated by Dettori et
77 al. (2014b) by means of AFLP markers, revealing that, in spite of its endemicity and
78 fragmented distribution, this species is characterized by high levels of genetic polymorphism
79 and genetic diversity, as well as by relatively low differentiation among populations. Apart
80 from these studies on Sardinian populations, the only molecular data produced up to date on
81 Western Mediterranean taxa regard the close Iberian endemic F. loscosii (Lange) Willk,
82 which was investigated by means of both allozymes (Pérez-Collazos and Catalán, 2008) and
83 AFLP markers (Pérez-Collazos et al., 2009). Currently, there are no available comparative
84 studies comprising plants from the whole Tyrrhenian area. In this context, the use of
85 molecular markers to study this species complex may be particularly useful to evaluate the
86 species’ genetic diversity and their inter-specific relationships, to shed light into the genetic
87 structuring of widespread taxa such as F. communis, to complement previous studies which
88 did not make use of molecular data and to delve into different biological questions. A
89 particularly interesting issue when investigating species complexes such as F. communis is
90 the comparison of the levels of genetic diversity between endemic and widespread species. In
91 these cases, comparisons against a common congener provide a useful standard against which
92 rare species can be evaluated (Ellis et al., 2006) and contribute to our understanding of the 4
93 relationship between levels of genetic diversity and geographic range size in clades containing
94 rare and widespread plant species (Edwards et al., 2014). In this context, the F. communis
95 complex represent an ideal study case, since the endemic F. arrigonii and the widely
96 distributed F. c. subsp. communis are closely related congeners which share similar life
97 history traits; therefore their geographic range is the factor that might most likely explain
98 differing levels of genetic diversity. In general, the expectation is that endemic species, and
99 particularly island endemics (Frankham, 1997), exhibit lower levels of genetic diversity than
100 widespread ones (Cole, 2003; Hamrick and Godt 1996, 1989; Karron, 1987). This is thought
101 to be due to genetic drift, the founder effect and directional selection operating in some
102 environments and leading to genetic uniformity (Babbel and Selander, 1974; Franklin, 1980;
103 Nei et al., 1975; Van Valen, 1965). Kruckeberg and Rabinowitz (1985) reviewed the
104 characteristics of endemic taxa and concluded that more "comparative studies to contrast the
105 biology of rare taxa with those of related common ones would be particularly valuable". In the
106 last decades, many researchers have included widespread congeners when examining the
107 genetic variation of species with a narrow distribution. In some cases, endemic species have
108 displayed equivalent or higher levels of genetic diversity compared to their more widely
109 distributed congeners (Dodd and Helenurm, 2002; Ellis et al., 2006; Gitzendanner and Soltis,
110 2000; Karron, 1988; Purdy and Bayer, 1996; Turchetto et al., 2016). Furthermore, in some
111 genera both narrow-distribution and widespread species showed either very low or very high,
112 but similar, levels of polymorphism (e.g., Whittall et al., 2010; Young and Brown, 1996), thus
113 suggesting that the classic view that narrow-distribution species have less genetic variability
114 than more widespread ones may be an overgeneralization (Mateu-Andrés, 2004).
115 In this study, Amplified Fragment Length Polymorphism (AFLP) markers were used
116 to address the following issues: i) what is the genetic diversity of F. communis s.l. and of its
117 subspecies in the Tyrrhenian area? ii) do the endemic F. arrigonii and the widespread F. c.
5
118 subsp. communis differ in their levels of genetic diversity?, and iii) are the patterns of
119 molecular variation structured across the investigated area?
120
121
122 2. Materials and methods
123 2.1. Sampling sites and plant material
124 Leaf material was collected from a total of 16 populations (Table 1, Fig. 1). Sampling
125 of F. c. subsp. communis (hereafter F. communis) included one population from Minorca (Es
126 Grau, GRA), two from Corsica (Restonica, RES and Agheri, AGH), three from Sardinia
127 (Monte Albo, ALB; Bindua, BIN and Monte Crasta, CRA), three from Sicily (Monte Pizzuta,
128 PIZ; Caltagirone, CAL and Rometta, ROM), one from the Aeolian Islands (Filicudi Island,
129 FIL), two from Gozo Island (Xlendi, XLE and Ta Cenc, TAC), and one from the Italian
130 Peninsula (Latina, LAT). F. c. subsp. cardonae (hereafter F. cardonae) was sampled in
131 Minorca in its locus classicus, Cala en Blanes (BLA), while F. c. subsp. glauca (hereafter F.
132 cf. glauca to distinguish it from the typical subsp. glauca, which was originally described for
133 Southern France; Rouy and Camus, 1901) samples were collected from a population located
134 in Central Italy (Monte Calvi, CLV). Sampling of F. cf. vesceritensis included one population
135 from Northern Tunisia (Hammamet, HAM).
136 Leaf material was immediately dried with silica gel and stored in a dry room at 15%
137 R.H. (Relative Humidity) and 15 °C. Sampling of plants was done throughout the populations
138 in order to cover the whole occupied area and to minimize sampling of related individuals;
139 10-15 individuals per population were analyzed. As regards F. arrigonii, data regarding three
140 populations (one from Corsica and two from Sardinia) were retrieved from Dettori et al.
141 (2014b) and used as a comparison in the present study.
142
6
143
144 2.2. AFLP analyses
145 Genomic DNA was extracted from 20 mg of silica gel-dried leaf tissue using the
146 DNeasy Plant Mini Kit (Qiagen, Italy) following the manufacturer’s protocol; quality and
147 quantity was checked by both agarose gel electrophoresis and spectrophotometry
148 (BioPhotometer, Eppendorf srl, Milan, Italy). The AFLP (Amplified Fragment Length
149 Polymorphism) technique was chosen to carry out the study because of its high
150 reproducibility and no previous requirements of knowledge on DNA sequences. The primer
151 combinations EcoRI + ACC with MseI + CAC and EcoRI + AAT with MseI + CAG were
152 chosen after a preliminary screening on the basis of the clarity of fragment profiles and the
153 level of information provided. The original protocol by Vos et al. (1995) was followed with
154 slight modifications, as described in Dettori et al. (2014b).
155 To assess the reproducibility of the analysis the whole procedure (i.e. from DNA extraction to
156 capillary electrophoresis) was repeated for 20 samples. The error rate was calculated as the
157 number of phenotypic differences related to the total number of phenotypic comparisons
158 (Bonin et al., 2004).
159
160
161 2.3. Data analyses
162 In order to avoid excessive fragment size homoplasy (Vekemans et al., 2002) only
163 fragments between 150 and 500 bp were scored by means of GeneMarker v. 2.4.0
164 (Softgenetics, State College, PA, USA) to produce a binary matrix. Input files for subsequent
165 analysis were either obtained by using Transformer-4 (Caujapé-Castells et al., 2013) or edited
166 manually.
7
167 Several parameters were computed to estimate the genetic diversity at the population
168 level. Number and proportion of polymorphic loci (Fragpoly at the 95% level, corresponding to
169 P95 in most publications) were computed using AFLP-SURV v. 1.0 (Vekemans, 2002). The
170 same software was used to calculate Hj (Nei’s gene diversity, analogous to H or He in most
171 publications; Nei, 1973; Vekemans, 2002), Ht (the total gene diversity, i.e. expected
172 heterozygosity or gene diversity in the overall sample), Hw (the average gene diversity within
173 populations). Allele frequencies were generated using the default Bayesian method with non-
174 uniform prior distribution and Hardy-Weinberg genotypic proportions (Zhivotovsky, 1999).
175 The number of private fragments was examined by means of FAMD v. 1.30 (Schlüter and
176 Harris, 2006). The frequency and distribution of rare bands, i.e. those present in less than
177 twenty individuals on the whole data set, was calculated following Stehlik et al. (2001) and
178 considering ten individuals from every population (being n = 10 the minimum number of
179 individuals genotyped per population). POPGENE v. 1.32 (Yeh et al., 2000) was used to
180 compute the effective allele number (ne) and Shannon’s information index (I; Lewontin,
181 1972) at the population level. ANalysis Of VAriance (ANOVA) and Sheffé’s post hoc test
182 were used to examine the significance of differences in genetic diversity and fragment rarity
183 parameters between taxa and genetically homogeneous groups using the R software package
184 (R Development Core Team, 2011).
185 To explore the global genetic structure, both a principal co-ordinate analysis (PCoA)
186 and a neighbour-joining tree were computed based on a matrix of Nei & Li distances
187 (following Nei and Li, 1979) among individuals using FAMD 1.30 (Schlüter and Harris,
188 2006). The tree was graphically edited using SplitsTree v. 4.13 software (Huson and Bryant,
189 2006), and support was assessed by means of 500 bootstrap replicates generated by FAMD
190 1.30. To quantify the amount of genetic differentiation attributable to geographic and
191 population subdivision, both hierarchical and non-hierarchical analysis of molecular variance
192 (AMOVA; Excoffier et al., 2005) using Arlequin v.3.5, significance was assessed by means 8
193 of 1023 permutations as set by default by the software. The corresponding F-statistics were
194 also estimated: Fst (general fixation index), Fct (F-statistic among regions) and Fsc (F-statistic
195 among populations within regions). To further investigate the population structure, a Bayesian
196 model-based approach was used, as proposed by Pritchard et al. (2000) and implemented in
197 the software Structure v. 2.3 (Pritchard et al., 2000; Falush et al., 2007), to assign the
198 genotypes into genetically structured groups. Twenty independent runs for each K (from one
199 to 19) were performed using 50,000 burn-in periods and 100,000 MCMC (Markov Chain
200 Monte Carlo) repetitions, using no prior population information and assuming independent
201 allele frequencies and admixture. In order to further investigate the genetic structure within
202 the F. communis complex, the same analysis was carried out using two partial datasets, the
203 first one comprising all populations of F. communis s.l. and assuming K=1-16 (n = 220), the
204 second one excluding the population of F. cf. glauca and assuming K=1-15 (n = 205). For
205 each of the analyses the most accurate value of K was evaluated following the method
206 proposed by Evanno et al. (2005) and implemented in the software STRUCTURE
207 HARVESTER (Earl and vonHoldt, 2012). Based on these previous analyses and in order to
208 gain insight into the genetic substructure of F. communis, STRUCTURE analyses were
209 carried out with the same parameters after excluding XLE, TAC and HAM populations and
210 assuming K=1-12 (n = 165). Based on the results of this latter analysis, the dataset was split
211 and the analyses run separately for populations from Corsica and Sardinia (assuming K = 1-5;
212 n = 70), and for populations from Sicily, Minorca, Filicudi Island and the Italian Peninsula (K
213 = 1-7; n = 95). The program CLUMPP v. 1.1.2 (Jakobsson and Rosenberg, 2007) was used to
214 determine the optimum alignment of clusters across individual runs for each K; outcomes
215 from CLUMPP were imported into Distruct v. 1.1 (Rosenberg, 2004) for viewing the
216 individuals’ assignment probabilities.
217
218 9
219 3. Results
220 3.1. Genetic diversity
221 The information obtained by the analysis of the profiles is summarized in Table 1. The
222 error rate based on phenotypic comparisons among replicated individuals amounted to 3.2%.
223 The final dataset consisted of 280 individuals from 19 populations, generating 251 fragments
224 in the range of 150-500 bp, of which 245 (97.6%) were polymorphic. The degree of
225 polymorphism was fairly high for all populations, ranging from 58.2% (TAC) to 88.0%
226 (BLA). TAC also showed the lowest values all genetic diversity parameters except Fragrare
227 (the lowest values for this parameter were recorded for CRA and FIL), while the highest were
228 found in BIN (ne and I), BLA (Hj) and CLV (Fragrare). The average gene diversity within
229 populations (Hw) was 0.263 and the total gene diversity (Ht) was 0.317. No private allele to
230 any single population was detected with the exception of CLV (F. cf. glauca, one private
231 allele). However, when grouping populations into its current taxonomic treatments, nine
232 private alleles were detected in F. arrigonii and four in F. communis. Grouping populations
233 according to the genetic clusters detected by STRUCTURE analyses resulted in three alleles
234 being private to XLE, TAC and HAM, while grouping them according to their geographic
235 provenance resulted in one allele being private to Gozo Island. None of the alleles identified
236 as being private was fixed. A slightly significant difference was found between F. arrigonii
237 and all other populations of F. communis s.l. for the Fragrare parameter (P < 0.05 by One-Way
238 ANOVA followed by the Sheffé’s post hoc test), which was on average higher in the first
239 than in the second group; while grouping populations into the genetic clusters identified by
240 STRUCTURE resulted in the group XLE+TAC+HAM (Gozo Island and Tunisia) having
241 significantly lower genetic diversity values. than the other groups.
242
243 3.2. Genetic structure
10
244 The PCoA based on the matrix of Nei & Li distances failed to group individuals
245 belonging to the same populations. However, four major groups of individuals could be
246 identified: a first group was formed by individuals of F. cf. glauca (CLV population), which
247 constituted a separate cluster, as did those of F. arrigonii (BON, CAV, SMA) and individuals
248 of F. communis from Gozo Island and F. cf. vesceritensis from Tunisia (XLE, TAC, HAM).
249 The remaining individuals of F. communis and those of F. cardonae formed a single group;
250 however, individuals tended to cluster into two different subgroups, the first one comprising
251 the populations from Corsica and Sardinia, the second one constituted by populations from
252 Sicily, Minorca, Filicudi Island and the Italian Peninsula. The first axis explained 11.9% of
253 the variation, the second one 8.1%, the third one 5.3% (Fig. 2).
254 The same pattern was evident in the neighbour-joining analysis carried out at the
255 individual level (Fig. S1). The only populations whose individuals clustered together were
256 LAT, CLV, FIL and TAC; among these, only CLV and TAC were supported by high
257 bootstrap values (99% and 70%, respectively). The overall pattern highlighted the presence of
258 one highly supported group comprising individuals from CLV (F. cf. glauca from the Italian
259 Peninsula) and two poorly supported groups (bootstrap values <50%), one comprising
260 individuals from XLE, TAC and HAM (F. communis from Gozo Island and F. cf.
261 vesceritensis from Tunisia) and one comprising individuals of F. arrigonii. All other
262 individuals (attributable to F. communis and F. cardonae) generally clustered according to
263 two major geographic regions, with only a few intermingled individuals; the first one
264 comprised most individuals from Corsica and Sardinia, while the second included most
265 individuals from Minorca, Sicily, Filicudi Island and the Italian Peninsula (Fig. S1).
266 The results of AMOVA analysis are presented in Table 2. When no grouping was
267 applied, the analysis returned Fst = 0.285, meaning that 28.47% of the total genetic variation
268 of the 19 investigated populations was attributable to differences among populations and
269 71.53% to differences within populations. The percentage of variation among regions was 11
270 maximised when grouping the populations of F. arrigonii (BON, CAV, SMA) vs. all other
271 populations of F. communis s.l. (Table 2). Further subdivisions according to taxonomic
272 treatments and/or geographic provenance also rendered highly significant fixation indexes but
273 did not remarkably alter the partitioning of the genetic variation.
274 The STRUCTURE analyses were coherent with previous results. As regards the
275 analysis carried out on the total datasets, a sharp signal was found at K = 2, the two clusters
276 corresponded to individuals of F. arrigonii (BON, CAV and SMA) and F. communis s.l. (all
277 other populations), respectively (Fig. 3A). RES, AGH, CRA, BIN and ALB populations (F.
278 communis from Corsica and Sardinia) showed a slight degree of admixture with the cluster
279 formed by BON, CAV and SMA (F. arrigonii). When individuals of F. communis s.l. were
280 considered alone the best K was 3, the first cluster corresponded to individuals of F. cf.
281 glauca (CLV), the second one comprised individuals from XLE, TAC (Gozo Island) and
282 HAM (Tunisia), the third one comprised all other individuals (Fig. 3B). When excluding CLV
283 population the best K was again 2, and the structuring did not differ substantially from that
284 obtained in the previous analysis (results not shown). Exclusion of XLE, TAC and HAM
285 resulted again in K = 2, the cluster corresponding respectively to populations from Corsica
286 and Sardinia and to populations from Sicily, Filicudi Island, Minorca and the Italian Peninsula
287 (Fig. 3C). When Bayesian clustering analysis was carried out on these two groups separately,
288 individuals of the former were partitioned into three genetic groups approximately
289 corresponding to RES and AGH (F. communis from Corsica), ALB and CRA (F. communis
290 from Northern Sardinia) and BIN (F. communis from Southern Sardinia), respectively (Fig.
291 3D). The analysis on the second group of individuals resulted in K = 5 with some populations,
292 namely PIZ, FIL and GRA, showing a considerable degree of admixture. One cluster
293 corresponded to LAT population (F. communis from the Italian Peninsula), a second one
294 comprised CAL, ROM (F. communis from Sicily) and, to a lesser extent, FIL (F. communis
295 from Filicudi Island) and BLA (F. cardonae from Minorca); a third one was mainly attributed 12
296 to BLA but was also present in PIZ (F. communis from Sicily); a fourth one characterized
297 GRA (F. communis from Minorca) but was predominant also in PIZ; a fifth one characterized
298 FIL and PIZ.
299
300
301
302 4. Discussion
303 4.1. Genetic diversity
304 The present study using AFLP analysis provided information on the magnitude and
305 pattern of genetic variation existing in 19 natural populations of the F. communis complex in
306 the Tyrrhenian area.
307 Overall, the obtained results revealed high levels of genetic diversity and moderate
308 levels of differentiation among populations. Some Sardinian populations of F. communis were
309 also investigated by means of allozymes (Marchi et al., 2003), showing a similar
310 differentiation among populations (overall Fst = 0.223) and a lower expected heterozygosity
311 (ranging from 0.097 to 0.165). Both the levels of heterozygosity and the differentiation among
312 populations were consistent with the trends of genotypic variation revealed through AFLP
313 data in previous studies, e.g., in the review carried out by Nybom et al. (2004), where they
314 reported a mean within-population genetic diversity of 0.23 and a mean among-populations
315 differentiation of 0.35 (ɸst) or 0.21 (Gst).
316 Many biological factors can influence both the species genetic diversity and its
317 distribution among populations. Among these, the geographic distribution has been
318 acknowledged to be one of the most important (Hamrick and Godt, 1989). Interestingly, when
319 Nybom et al. (2004) reviewed the genetic diversity and differentiation values based on
320 RAPD-derived data and sorting them by different life history traits, the geographical range
13
321 seemed not have any particular influence: the mean within-population heterozigosity for
322 widespread species was 0.22, it was 0.20 for endemics but 0.28 for taxa with a narrow
323 distribution. The same pattern was observed in our study case: the genetic diversity values are
324 similar and not significantly different between F. arrigonii and F. c. subsp. communis, and are
325 even higher in the former than in the latter species for the Fragrare parameter. Conversely, in
326 Nybom et al. (2004) review, life form and breeding system seemed to a have significant
327 influence on the genetic parameters. Both F. arrigonii and F. communis are perennial species
328 and, although there are no available detailed studies on their breeding system, their genetic
329 diversity values fall within the range reported by Nybom et al. (2004) for outcrossing or for
330 species with a mixed breeding system. Moreover, according to the AMOVA most of the
331 genetic variability is retained within populations, which is also in accordance with the
332 common expectations for long-lived, outcrossing species as reported by the same authors.
333 Preliminary germination studies also backup our results: Sanna et al. (2009) reported that the
334 germination ability of F. arrigonii is not significantly different from F. communis and final
335 germination values are even higher in the endemic taxon; both species have high germination
336 percentages (>80%), with an optimal germination temperature of 10-15° C and no pre-
337 treatments, thus suggesting the existence of a successful outcrossing reproductive system in
338 both taxa.
339 Our results are not in accordance with the theoretical expectations that endemic species
340 (including island endemics; Frankham, 1997) should exhibit lower levels of genetic diversity
341 than widespread ones (Cole 2003; Hamrick and Godt 1996, 1989; Karron, 1987). Mateu-
342 Andrés (2004) argued that there are actually many studies reporting either low (e.g. Gemmill
343 et al., 1998; Mateu-Andrés, 2004; Segarra-Moragues and Catalán, 2002) or high (e.g. Lewis
344 and Crawford, 1995; Ranker, 1994; Young and Brown, 1996) levels of genetic variation for
345 narrow-distribution plant species. Also, many other studies have reported either lower (e.g.
346 Edwars et al., 2014; Franceschinelli et al., 2006; Furches et al., 2013; Maki et al., 2002; 14
347 Moreira da Silva et al., 2007; Purdy and Bayer, 1995; Talve et al., 2012), similar or even
348 higher (e.g. Dodd and Helenurm, 2002; Ellis et al., 2006; Karron et al., 1988; Purdy and
349 Bayer, 1996) levels of genetic diversity for endemic and narrow-distribution species with
350 respect to their widespread congeners. Moderate to high levels of genetic diversity have been
351 reported also for several plants living in the Tyrrhenian territories, e.g. the Sardinian endemic
352 Rhamnus persicifolia (HS = 0.1105 and Ht = 0.2066 through ISSRs; Bacchetta et al., 2011),
353 the Sicilian Brassica rupestris (HS = 0.212 and HT = 0.307 using ISSRs; Raimondo et al.,
354 2012), the Western Mediterranean Ambrosina bassii (He = 0.208 - 0.395 through allozymes;
355 Troia et al., 2012), or Cyclamen repandum and C. hederifolium in Corsica (He = 0.157 and
356 0.221 respectively through allozymes; Affre and Thompson, 1997). In summary, there seem
357 to be no universal pattern; our results are rather more in accordance with the view proposed
358 by Gitzendanner and Soltis (2000), who concluded that each rare species should be
359 considered a separate case and that there is no more such thing as a reference mean for rare
360 and widespread species. Rather, they argued that the comparison of genetic diversity values
361 with those of widespread congeners is much more informative than comparison against means
362 reported by other authors based on studies on species that have different biological
363 characteristics. In this sense, our comparison has also a conservation value, as it confirms that
364 the endemic F. arrigonii is not genetically depauperated (Dettori et al., 2014b).
365
366 4.1. Genetic structure
367 The analyses of the structuring of the genetic variation were highly coherent with one
368 another. All of them indicated that three groups of populations, namely BON, CAV, SMA
369 populations (F. arrigonii), CLV (F. cf. glauca), and XLE, TAC (F. communis from Gozo
370 Island), HAM (F. cf. vesceritensis from Tunisia) are well differentiated with respect to the
371 other investigated populations of F. communis s.l. This pattern was confirmed by the presence
15
372 of nine private fragments in F. arrigonii, three in XLE, TAC and HAM and one in F. cf.
373 glauca. Moreover, both F. arrigonii and F. cf. glauca had a significantly higher number of
374 rare fragments. As regards F. cf. glauca, our results suggest the distinctiveness of this taxon;
375 however, it might be worth analysing more populations across a wider distributional range.
376 Furthermore, data suggest that the two populations from Gozo Island are attributable to F. cf.
377 vesceritensis, which is reported to be endemic to Tunisia and Algeria. Further evidence, both
378 at the molecular and at the morphological level, would be helpful in order to assess the status
379 of these populations. As regards the remaining populations of F. communis s.l. from the rest
380 of the investigated territories, a certain amount of substructuring was detected by the
381 STRUCTURE, the PCoA and the neighbor-joining analyses, showing that the Sardinian and
382 Corsican populations are more closely related to each other than they are to all other
383 investigated populations. On the one hand, this confirms the peculiarity and distinctiveness of
384 the Corso-Sardinian floristic elements in the Mediterranean context, as revealed also by recent
385 phylogeographical and population genetic studies. For example, in a study employing AFLPs
386 on Erodium maritimum L., a species distributed along the coasts of the European Atlantic and
387 the Central and Western Mediterranean basin, the populations from Corsica and Sardinia were
388 the only ones which were not admixed with populations from the remaining territories, the
389 Sardinian population also had a considerable number of private fragments; therefore
390 suggesting its ancestral origin (Alarcón et al., 2013). Further examples, each of them linked to
391 different evolutionary and biological processes, are the Genista ephedroides DC. species
392 complex (De Castro et al., 2015), Astragalus L. sect. Tragacantha DC. (Hardion et al., 2016)
393 and Lamyropsis microcephala (Moris) Dittrich et Greuter (Gentili et al., 2015). On the other
394 hand, this work confirms the findings of Sánchez-Cuxart and Bernal (1998), who found great
395 similarities between plants from Minorca and Sicily based on morphological, phenological
396 and karyological characteristics and they thus hypothesized they could belong to the same
397 taxonomical entity. This pattern was further confirmed by the STRUCTURE analyses carried 16
398 out on the two subclusters comprising populations from Corsica and Sardinia and from
399 Minorca, Sicily, Filicudi and the Italian Peninsula, which revealed the presence of a highly
400 hierarchical substructure among the populations. Bayesian subclustering on the Corsican and
401 Sardinian populations of F. communis might reflect the geographic provenance of the
402 individuals: RES and AGH (Corsica) formed a separate cluster, while the Sardinian
403 individuals were partitioned into two groups corresponding respectively to populations from
404 Northern (ALB and CRA) and Southern (BIN) Sardinia. This subdivision might also reflect
405 differences in chemical properties. Sacchetti et al. (2003) identified two chemotypes, a
406 poisonous and a non-poisonous one, based on UV microscopy examinations of the vittae
407 fluorescence and TLC analyses on individuals sampled throughout the Sardinian territory.
408 According to their results and to the distribution map of the two chemotypes provided in
409 Marchi et al. (2003), both CRA and ALB would fall within the distributional range of the
410 non-poisonous chemotype, whereas BIN would fall within the range of the poisonous one.
411 Our results therefore support the distinctiveness of these two chemotypes and are in
412 accordance with the genetic structure of the species reported by Marchi et al. (2003), who
413 found a similar pattern using allozymes. The remaining populations of F. communis and F.
414 cardonae clustered into five genetically distinct groups. Among them, the one constituted by
415 individuals belonging to LAT (Italian Peninsula) clearly reflect their geographic provenance.
416 The closeness between Filicudi Island and Sicily might also explain the genetic structure of
417 individuals from FIL, which showed a high degree of admixture with PIZ, and, to a slighter
418 degree, with CAL and ROM. The Minorcan (BLA and GRA) and the Sicilian populations
419 showed a considerable degree of admixture with one another. In particular, individuals from
420 BLA (Minorca), locus classicus of F. cardonae, did not cluster with individuals from GRA,
421 but showed a slight degree of admixture with individuals from the Sicilian populations CAL
422 and ROM, therefore suggesting that further evidence is needed to clarify the status of this
17
423 taxon, as well as to gain insight into the relationships among the Minorcan and the Sicilian
424 populations.
425 Overall, all of the analyses, as well as the absence of fragments exclusive to any
426 population or island (with the exception of Gozo Island) suggest that all investigated
427 populations of F. communis are closely related. This finding is somewhat surprising
428 considering both the distances that separate the populations and the fact that they are found in
429 different islands, as well as their current taxonomic treatments. However, the marked genetic
430 substructure at a more local level backups the distinctiveness among populations from
431 different areas based on previous morphological, karyological and genetic evidence.
432 Furthermore, our results suggest, on the one hand, that this species complex is currently
433 undergoing a process of differentiation. On the other hand, the results also highlight the
434 importance of considering multiple geographic levels of investigation when studying species
435 and species complexes that are widely distributed.
436
437
438 Acknowledgements
439 We thank Prof. Pilar Catalán, Prof. Frédéric Médail, Dr. José Luis Garrido Sánchez and two
440 anonymous reviewers for their comments on early versions of the manuscript. Dr. Martino
441 Orrù and Dr. Andrea Santo helped with field work; Dr. Valerio Lazzeri provided material
442 from Tuscany and Prof. Pietro Minissale from Filicudi Island. This work was funded by the
443 Autonomous Region of Sardinia, Promozione della ricerca scientifica e dell’innovazione
444 tecnologica in Sardegna (L.R. 7/2007).
445
446
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27
Population Mean (code, geographic elevation (m Fragpoly location) Taxon Coordinates a.s.l.) Ni (%) ne Hj Fragrare I Bonifacio (BON, 200 1.38 0.274 Corsica) F. arrigonii 41° 23' N - 9° 09' E 25 20 (79.7) (±0.39) (±0.0112) 1.40 0.325 Isola dei Cavoli (CAV, 196 1.37 0.262 Sardinia) F. arrigonii 39° 05' N - 9° 31' E 15 20 (78.1) (±0.38) (±0.0112) 1.30 0.318 Capo San Marco (SMA, 195 1.34 0.254 Sardinia) F. arrigonii 39° 52' N - 8° 26' E 25 20 (77.7) (±0.35) (±0.0107) 1.20 0.309 Monte Albo (ALB, F. c. subsp. 40° 27’ N - 9° 31’ 206 1.39 0.294 Sardinia) communis E 1050 12 (82.1) (±0.38) (±0.0110) 0.40 0.335 Monte Crasta (CRA, F. c. subsp. 214 1.40 0.298 Sardinia) communis 40° 21' N - 8° 40' E 540 14 (85.3) (±0.39) (±0.0108) 0.30 0.346 F. c. subsp. 199 1.43 0.306 Bindua (BIN, Sardinia) communis 39° 17' N - 8° 29' E 110 15 (79.3) (±0.39) (±0.0110) 1.00 0.370 F. c. subsp. 42° 15’ N - 9° 05’ 196 1.37 0.280 Restonica (RES, Corsica) communis E 830 14 (78.1) (±0.39) (±0.0107) 0.80 0.319 F. c. subsp. 42° 08’ N - 9° 17’ 196 1.36 0.273 Agheri (AGH, Corsica) communis E 520 15 (78.1) (±0.37) (±0.0106) 0.40 0.318 Monte Pizzuta (PIZ, F. c. subsp. 181 1.29 0.236 Sicily) communis 37° 59' N - 13° 15' E 1065 14 (72.1) (±0.36) (±0.0108) 0.40 0.262 Caltagirone (CAL, F. c. subsp. 192 1.34 0.270 Sicily) communis 37° 15' N - 14° 30' E 480 14 (76.5) (±0.38) (±0.0106) 0.40 0.303 F. c. subsp. 216 1.35 0.291 Rometta (ROM, Sicily) communis 38° 09' N - 15° 24' E 450 12 (86.1) (±0.36) (±0.0099) 0.40 0.312 F. c. subsp. 162 1.31 0.231 Xlendi (XLE, Gozo) communis 36° 02' N - 14° 12' E 22 15 (64.5) (±0.36) (±0.0116) 0.40 0.279 F. c. subsp. 146 1.23 0.186 Ta Cenc (TAC, Gozo) communis 36° 01' N - 14° 13' E 107 15 (58.2) (±0.33) (±0.0110) 0.70 0.209 Pecorini a Mare (FIL, F. c. subsp. 193 1.29 0.238 Filicudi) communis 38° 33' N - 14° 33' E 98 12 (76.9) (±0.37) (±0.0111) 0.30 0.251
28
Carretera des Grau F. c. subsp. 189 1.36 0.271 (GRA, Minorca) communis 39° 54' N - 4° 14' E 20 15 (75.3) (±0.37) (±0.0109) 0.40 0.321 Cala en Blanes (BLA, F. c. subsp. 221 1.42 0.313 Minorca) cardonae 40° 00' N - 3° 49' E 30 13 (88.0) (±0.39) (±0.0104) 0.70 0.354 Latina (LAT, Italian F. c. subsp. 177 1.32 0.250 Peninsula) communis 41° 45' N - 12° 26' E 70 15 (70.5) (±0.38) (±0.0111) 1.20 0.284 Monte Calvi (CLV, F. c. cf. subsp. 183 1.29 0.232 Italian Peninsula) glauca 43° 05' N - 10° 36' E 230 15 (72.9) (±0.34) (±0.0106) 2.10 0.268 Hammamet (HAM, 176 1.29 0.239 Tunisia) F. cf. tunetana 36° 25' N - 10° 33' E 50 10 (70.1) (±0.36) (±0.0114) 0.60 0.255 1
2 Table 1. Populations under study, their characteristics and estimates of genetic diversity and fragment rarity. Ni = number of individuals analysed;
3 Fragpoly = number and proportion of polymorphic fragments at the 5% level; ne = effective number of alleles ± standard deviation; Hj = Nei’s
4 heterozygosity ± standard error; Fragrare = mean number of rare fragments per individual; I = Shannon’s information index.
5
6
29
1
2
3
30
1
Variance Grouping N Source of variation d.f. SS (%) Fixation index
No groups 19 Among populations 18 3228 28.47 Fst = 0.285 *** Within populations 261 6829 71.53
[CAV-BON-SMA] 2 Among groups 1 715 14.04 Fst = 0.349 ***
[XLE-TAC-HAM-FIL-CRA-ALB-BIN- Among populations within groups 17 2512 20.89 Fct = 0.140 **
RES-AGH-GRA-BLA-PIZ-CAL-ROM-LAT] Within populations 261 6829 65.06 Fsc = 0.243 ***
[CAV-BON-SMA] 4 Among groups 3 1547 18.48 Fst = 0.335 ***
[XLE-TAC-HAM] [FIL-CRA-ALB-BIN- Among populations within groups 15 1680 15.05 Fct = 0.185*** RES-AGH-GRA-BLA-PIZ-CAL-ROM-LAT] [CLV] Within populations 261 6829 66.47 Fsc = 0.185 ***
[CAV-BON-SMA] [XLE-TAC-HAM] 7 Among groups 6 2101 17.74 Fst = 0.301 ***
[CRA-ALB-BIN-RES-AGH] [FIL-PIZ-CAL-ROM] Among populations within groups 12 1127 12.40 Fct = 0.177***
[GRA-BLA] [LAT] [CLV] Within populations 261 6829 69.86 Fsc = 0.151 *** 2
3 Table 2. Results of three analyses of molecular variance (AMOVA). d.f. = degrees of freedom; SS = mean sum of squares; general fixation index
4 (Fst), fixation index for the region (Fct), and population within region (Fsc) level are shown.
31
1 Figure legends:
2
3 Fig. 1. Geographic location of the populations under study. Dots = F. communis; diamond =
4 F. cf. glauca; triangle up = F. cardonae; square = F. cf. vesceritensis; triangles down = F.
5 arrigonii.
6
7
8 Fig. 2. PCoA based on Nei & Li pairwise distances. Symbols and colours were assigned
9 based on the results of the Structure analysis.
10
11 Fig. 3. Results of the STRUCTURE analysis. Each vertical bar represents an individual; black
12 lines delimit sites. The diagram was redrawn from STRUCTURE (see materials and methods
13 for further details). A = full dataset; B = partial dataset with individuals of F. communis s.l.; C
14 = partial dataset after excluding F. cf. glauca (CLV), F. communis from Gozo Island (XLE
15 and TAC) and F. cf. vesceritensis from Tunisia (HAM); D = partial dataset with individuals
16 of F. communis from Corsica and Sardinia; E = partial dataset with individuals of F.
17 communis and F. cardonae from Minorca, Sicily, Filicudi Island and the Italian Peninsula.
18
19 Supplementary Fig. S1. Unrooted NJ tree based on Nei & Li pairwise distances. Bootstrap
20 values above 50% are indicated based on 500 replicates. Green = F. arrigonii; red = F.
21 communis from Corsica and Sardinia; yellow = F. communis from Minorca, Sicily, Filicudi
22 Island and the Italian Peninsula, F. cardonae from Minorca; blue = F. communis from Gozo
23 Island and F. cf. vesceritensis from Tunisia.
24
32