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).