Molecular phylogeny and genetic diversity of Tunisian Quercus species using chloroplast DNA CAPS markers

Biochemical Systematics and Ecology 60 (2015) 258e265

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Molecular phylogeny and genetic diversity of Tunisian Quercus species using chloroplast DNA CAPS markers Hela Sakka a, b, Ghada Baraket a, Abdesslem Abdessemad a, c, Kamel Tounsi d, Mustapha Ksontini c, Amel Salhi-Hannachi a, * Facult
e des Sciences de Tunis, Laboratoire de G
en
etique Mol
eculaire, Immunologie et Biotechnologie, Universit
e Tunis El Manar, 2092 El Manar. Tunis, Tunisia b Facult
e des Sciences de Bizerte, 7021 Zarzouna, Tunisia c Institut National de Recherche en G
enie Rural, Eaux et For^ ets, BP 2, 2080 Ariana, Tunisia d Institut Sylvo-Pastoral de Tabarka, Tunisia a

a r t i c l e i n f o

a b s t r a c t

Article history: Received 2 April 2014 Accepted 20 September 2014 Available online 28 May 2015

Chloroplast DNA variation was studied in five evergreen Quercus species from the Western part of Tunisia using Cleaved Amplified Polymorphic Sequence (CAPS) technique. Five primer pair/endonuclease combinations have been used. Chlorotypes of Quercus species have been identified. The enzyme HinfI was more efficient in detecting polymorphism in oak species than TaqI. The phenogram showed five groups defining the five studied oak species: suber group, afares group, coccifera group, canariensis group and ilex group. The topology of phenogram showed that the classification depends only on species and independently of their geographic origin. The principal component analysis (ACP) corroborated the results of the tree branching and confirmed the existence of five species groups. Our results showed a genetic proximity between Quercus afares and Quercus coccifera species that may be due to temperature tolerance or the demographic history of these species. Nevertheless, a high value of GST calculated (GST ¼ 1), suggesting that the maximum of variation is maintained among oak species. This result was confirmed by the low value of the genetic diversity within species (hS ¼ 0), the value obtained of the total genetic diversity (hT ¼ 0.378) and the absence of gene flow between species (Nm ¼ 0). A high genetic proximity has been registered between Q. afares, Quercus suber and Quercus canariensis. Moreover, Q. afares shared the chlorotype of Q. suber and Q. canariensis which suggests its hybrid origin. © 2015 Elsevier Ltd. All rights reserved.

Keywords: Quercus suber Quercus canariensis Quercus afares Quercus coccifera Quercus ilex cp DNA Chlorotypes Phylogenetic relationships Tunisia

1. Introduction Evergreen oaks (2n ¼ 24) are diploid and they dominate most forest habitats in the Mediterranean basin (Takhtajan, 1986). Five species are particularly important in this region: holm oak (Quercus ilex L., 1753), the cork oak (Quercus suber L., 1753), canary oak (Quercus canariensis, Willd, 1809), Pomel oak (Quercus afares, Maire, 1933) and kermes or holly oak (Quercus coccifera L., 1753). Although their distributions and habitats are overlapping but ecological differences exist among them. Q. suber is an evergreen tree which has a great importance in the North-Western of Tunisia, for both economic and ecological values of cork (Passarinho et al., 2006) and plays a key role in maintaining biodiversity (Hidalgo et al., 2008). The distribution of Q. suber is limited to the Northern part of Tunisia and represents 10% of the total forest area in the North-West of Tunisia * Corresponding author. Tel.: þ216 71 87 26 00; fax: þ216 70 86 04 32. E-mail address: [email protected] (A. Salhi-Hannachi). http://dx.doi.org/10.1016/j.bse.2014.09.025 0305-1978/© 2015 Elsevier Ltd. All rights reserved.

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and 4.3% of the world's cork oak forest area. Although, the conservation of this forest genetic patrimony is very important. However, Q. Ilex dominates the forests of all kinds of substrates. The Holm oak acorns represent a major component in the feeding systems of many Mediterranean wild and livestock species. Moreover, it is the basic feed ingredient for domestically meat pigs (Ruperez, 1957). Q. coccifera and Q. suber are more thermophilous than Q. ilex and appear to have contrasting environmental requirements. Kermes oak, is more frequent in arid and disturbance-prone environments, predominantly on limestone substrates (Tutin et al., 1993), while the cork oak has strict humidity and soil requirements being only found on acidic or decarbonated soils (Martínez-Ferri et al., 2004). These species are sympatric in many areas, but some differences in their ecological requirements produce distinct responses to environmental conditions and hence different evolutionary histories. Previous studies have shown differences in their genetic variation patterns in both nuclear and cytoplasmic levels (Belahbib et al., 2001; Lumaret and Jabbour-Zahab, 2009). Inter-specific gene flow has already been detected in the crosses Q. cocciferaeQ. ilex and Q. subereQ. ilex (Belahbib et al., 2001). However, Q. suber and Q. canariensis are sympatric with Q. afares over most of their geographical distribution. Most studies on genetic variation of forest trees have been carried out using nuclear markers (Belahbib et al., 2004e2005; Shiran et al., 2011). The chloroplast DNA, is a slow mutating, non-recombining, clonally inherited genome (Wolfe et al., 1987) which is maternally inherited in oaks (Dumolin et al., 1995) and typically shows low intra-specific variation. Although in the last decade, the development of new markers has allowed the detection of enough variation to assess phylogeographic patterns. More recently, chloroplast microsatellites (cp SSR) have been developed in French oak trees for haplotype discrimination of oak material (Deguilloux et al., 2004). Moreover, studies in Quercus species have been reported using PCRRFLP or CAPS technique (Petit et al., 2002) to elucidate the postglacial recolonization history in Europe (Petit et al., 2003) and America (Grivet et al., 2006). This approach has been shown to be a powerful tool for phylogenetic reconstruction at both inter- and intra-specific levels and for identifying populations that have expanded from refugia during the last glacial period (Abbott et al., 2000). Palynological data suggest a southern origin for the European oaks (Huntley and Birks, 1983). Phylogue et al., 1997a) carried out using chloroplast DNA markers have demonstrated the exgeographic studies (Dumolin-Lape istence of three glacial refugial areas localized in the three peninsulas: Iberia, Italy and the Balkans. Studies based on cp DNARFLP of cork oak (Lumaret et al., 2005) showed that this technique cannot differentiate between very small fragment-size changes (insertions/deletions) and are easily detected by PCR-RFLP. This technique was used successfully to analyze the variation of cpDNA fragments in Q. suber, Q. ilex and Q. coccifera populations sampled predominantly in Iberia and Morocco
pez-de-Heredia et al., 2007). In the present work, cp PCR-RFLP analysis of five Tunisian Quercus (Lumaret et al., 2005; Lo species (Q. canariensis, Q. suber, Q, ilex, Q. afares and Quercus cocciffera) using several DNA fragment/endonuclease, was performed to identify phylogenetically informative characters based on small fragment changes, to identify oak chlorotypes, to establish the genetic relationships between them and to make a comparative study of the genetic structure of the three Tunisian oaks (Q. canariensis, Q. suber and Q. afares) in order to verify if Q. afares acts as species apart or as a hybrid species. 2. Material and methods 2.1. Plant material Five oak species were used in this study: Q. suber (cork oak), Q. coccifera (kermes oak), Q. canariensis (Canry oak) Q. afares (Pomel oak) and Q. ilex (holm oak) and are collected from the North-Western region of Tunisia (Table 1). Distribution range and local species presence are governed by climatic and edaphic factors (Rameau et al., 1989). A total of 210 individuals from Tunisia of Q. suber (85 samples consisted of 17 populations), Q. ilex (25 samples represented by 5 populations), Q. coccifera (3 populations composed of 15 samples) Q. canariensis (14 populations consisted of 70 samples) and Q. afares (3 populations represented by 15 samples) covering the Western area of Tunisia were sampled. Moreover, samples of Q. canariensis, Q. suber, and Q. ilex from Morocco (5 individuals, for each species) have been used as controls for each species. In each stand, adult leaves were collected from 5 nonadjacent trees per species. 2.2. Extraction of DNA and amplification The DNA was extracted using a modified protocol of Doyle and Doyle (1990) modified by Dumolin et al. (1995). The polymerase chain reaction (PCR) method was detailed in Demesure et al. (1995). Five pairs of primers were used to amplify the following chloroplast fragments: trnC[tRNA-Cys(GCA)]- trnD[tRNA-Asp(GUC)] (CD), trnT[tRNA-Thr(GUG)]- trnF[tRNAPhe(UGU)] (TF), trnD[tRNA-Asp(GUC)]- trnT[tRNA-Thr(GGU)] (DT), psaA[PSI (P700 apoproteine A1)]-trnS[tRNA-Ser(GGA)] gue et al., (AS) and trnS[tRNA-Ser(GCU)]- trnR[tRNA-Arg(UCU)] (SR) (Taberlet et al., 1991; Demesure et al., 1995; Dumolin-Lape 1997b; Grivet et al., 2001). 2.3. Enzymatic digestion The amplified fragments were digested with five units of either HinfI or TaqI enzymes. Digestion products were separated by electrophoresis in polyacrylamide gels as described in El Mousadik and Petit (1996). Fragments were revealed with ethidium bromide method in order to check polymorphisms. Fragment sizes were estimated using a 1 kb ladder (Gibco BRL).

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Table 1 Geographical locations, sample code numbers and forest name of oak species (Q. coccifera, Q. ilex, Q. suber, Q. canariensis and Q. afares) studied in North Western of Tunisia. Geographic origin

Coordinates

Tabarka

Touristic zone Ras Rajel

36 570 N; 8 450 E 36 560 N; 8 530 E

7 70

Houamdia

36 560 N; 8 520 E

150

Bizerte

Sejnane

36 480 N; 8 290 E

130

Fernana

Fernana

36 370 N; 8 370 E

400

Nefza Aïn Drahem

El Feija

Siliana



0



Altitude (m)

0

Bellief Aïn Baccouche

37 00 N; 9 04 E 36 470 N; 8 460 E

160 720

Aïn Snoussi

36 480 N; 8 460 E

715

Hammam Borguiba Hotel Chenes

36 440 N; 8 350 E

570

36 47 N; 8 41 E

730

Souiniet

36 460 N; 8 460 E

500

Tbainia

36 440 N; 8 450 E

620





0

0





0

0

Bni Mtir

36 44 N; 8 44 E

450

Aïn Zena

36 470 N; 8 440 E

850

Douar

36 390 N; 8 390 E

750

Rhaim

36 460 N; 8 400 E

700

Poste forestier El Feija Plateau Kesra Sidi Aouidet Bou Abdellah Bni Hazem Poste Forestier Kessra

36 300 N; 8 150 E

800

35 480 N; 35 500 N; 35 510 N; 35 510 N; 35 480 N;

730 720 700 725 820

9 220 E 9 240 E 9 250 E 9 230 E 9 210 E

Oak species

Sample code numbers

Forest name

Q. Q. Q. Q. Q. Q. Q. Q. Q. Q. Q. Q. Q. Q. Q. Q. Q. Q. Q. Q. Q. Q. Q. Q. Q. Q. Q. Q.

coccifera coccifera suber suber canariensis coccifera suber canariensis suber canariensis suber suber canariensis suber canariensis suber canariensis suber canariensis suber canariensis suber canariensis suber canariensis suber canariensis afares

Forest of north of Tabarka

Q. Q. Q. Q. Q. Q. Q. Q. Q. Q. Q.

suber canariensis suber canariensis suber canariensis ilex ilex ilex ilex ilex

Coc1, Coc2, Coc3, Coc4, Coc5 Co6, Coc7, Coc8, Coc9, Coc10 Sub1, Sub2, Sub3, Sub4, Sub5 Sub11, Sub12, Sub13, Sub14, Sub15 Can1, Can2, Can3, Can4, Can5 Coc11, Coc12, Coc13, Co14, coc15 Sub16, Sub17, Sub18, Sub19, Sub20 Can6, Can7, Can8, Can9, Can10 Sub21, Sub22, Sub23, Sub24, Sub25 Can11, Can12, Can13, Can14, Can15 Sub26, Sub27, Sub28, Sub29, sub30 Sub31, Sub32, Sub33, Sub34, Sub35 Can16, Can17, Can18, Can19, Can20 Sub36, Sub37, Sub38, Sub39, Sub40 Can21, Can22, Can23, Can24, Can25 Sub41, Sub42, Sub43,Sub44, Sub45 Can26, Can27, Can28, Can29, Can30 Sub46, Sub47, Sub48, Sub49, Sub50 Can31, Can32, Can33, Can34, Can35 Sub51, Sub52, Sub53, Sub54, Sub55 Can36, Can37, Can38, Can39, can40 Sub56, Sub57, Sub58, Sub59, Sub60 Can41, Can42, Can43, Can44, Can45 Sub61, Sub62, Sub63, Sub64, Sub65 Can46,Can47, Can48, Can49, Can50 Sub66, Sub67, Sub68, Sub69, Sub70 Can51, Can52, Can53, Can54, Can55 Afa1, Afa2, Afa3, Afa4, Afa5, Afa6, Afa7, Afa8, Afa9, Afa10, Afa11, Afa12, Afa13, Afa14, Afa15 Sub71, Sub72, Sub73, Sub74, Sub75 Can56, Can57, Can58, Can59, Can60 Sub76, Sub77, Sub78, Sub79, Sub80 Can61, Can62, Can63, Can64, Can65 Sub81, Sub82, Sub83, Sub84, Sub85 Can66, Can67, Can68, Can69, Can70 Ile1, Ile2, Ile3, Ile4, Ile5 Ile6, Ile7, Ile8, Ile9, Ile10 Ile11, Ile12, Ile13, Ile14, Ile15 Ile16, Ile17, Ile18, Ile19, Ile20 Ile21, Ile 22, Ile23, Ile24, Ile25

Forest of Sejnane

Forest of Fernana Foest of Bellief Forest of Aïn Drahem

Forest of the southern edge of Aïn Drahem

Forest of El Feija

Forest of Kessra plateau

Forest of Kessra

2.4. Identification of cp DNA mutations The cp DNA restriction endonuclease patterns of individual trees were scored for fragment-length differences. Changes in gue the cp DNA were identified as length or site mutation. Data were scored as multistate characters as in Dumolin-Lape et al. (1997b): each polymorphic restriction fragment is considered as a character and the states are the different sizes of this fragment. The length variants were noted from 1 to 6, 9 and 0 being reserved for restriction site mutations (9 means the appearance of a new restriction point and, consequently, two new bands instead of the expected one; and 0 the disappearance of a restriction point implying a new band with size equal to the sum of two missing bands). Numbers increase from the highest to the lowest molecular weight fragments to ease the notation, but this does not imply any mutational sequence. 2.5. Phylogenetic analyses The banding profiles of the different chlorotypes generated by PCR-RFLP were compiled into a binary data matrix. The presence of each polymorphic band was scored 1 and its absence as 0. Data were then analyzed with the Gendist program (version 3.69c) to produce a genetic distance matrix using the formula of Nei and Li (1979), which assesses the similarity between both populations on the basis of the number of bands. The neighbor program was then used to produce a dendrogram using the unweighted pair group method, genetic with the arithmetic averaging (UPGMA) algorithm (Sneath and Sokal, 1973). The TreeView program was used to draw phylogenetic diagrams from the resulting dendrogram. Appropriate programs in PHYLIP (Phylogeny interference package, version 3.69c) and Page's Tree View (Win32, version 1.5.2) were

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used to carry out all these analyses (Felsenstein, 1993; Page, 1998). In addition, principal component analysis (PCA) was performed by computing the data matrix with appropriate programs of the XLSTAT-Pro (Version 7.0). 2.6. Genetic diversity analyses Chloroplast DNA haplotypes frequencies were computed for each population. The genetic diversity statistics, GST is defined as the proportion of genetic diversity that resides among populations (Nei, 1973). The average intra-population diversity (hS), the total diversity within species (hT), the coefficient of differentiation (GST ¼ 1hS/hT) and their standard deviations were estimated. The gene flow (Nm) has been calculated for each species and between species with the software genepop version 4.2.1 (Rousset, 2008). 3. Results and discussion Using the reported trnC-trnD primers, the designed PCR has permitted to generate banding profiles using templates of total cellular DNA. A single DNA band of approximately 3000 bp was amplified from each oak species. The primers trnD-trnT, trnT-trnF, trnS-trnR and psaA-trnS generate a single band of approximately 1200 bp, 1750 bp, 1800 bp and 3700 bp respectively for each oak species. The sizes of these amplified fragments are similar to those obtained for Nicotiana tabacum, (1213 bp, 1557 bp, 1814 bp 3167 bp and 3681 bp for DT, TF, SR, CD and AS, respectively) (Demesure et al., 1995) and for Quercus robur gue et al., 1997b). (Dumolin-Lape 3.1. Identification of chlorotypes in Quercus species 3.1.1. Identification of chlorotypes in Quercus suber (S1, S2 and S3) Only the combination SR-HinfI permitted to discriminate between cork oaks from Tunisia and Morocco. However, the combinations DT-TaqI, TF-HinfI, CD-TaqI and AS-HinfI showed monomorphic patterns. The combination SR-HinfI provided additional information and permitted to identify the chlorotype S3 in the analyzed Tunisian cork oak. Nevertheless, the use of chloroplast CAPS approach permitted to identify the chlorotype of Tunisian Q. suber. The chlorotype S3 was also identified in Algeria, Sardinia, Corsica and Provence suggesting that in Q. suber, a North African refuge has contributed to the recolonization of this part of Europe after the last glaciation (Lumaret et al., 2005). Moreover, a rare chlorotype S7 has been described in Tunisia which was identified also in populations of Sardinia and Corsica (Lumaret et al., 2005). The studied Cork oak trees from Morocco showed two chlorotypes S1 and S2. The Tunisian chlorotype S3 differs from the Moroccan chlorotype S1 by the mutation of SR-HinfI-5 (Table 2). Indeed, the study of Belahbib et al. (2001) showed the existence of these two chlorotypes (S1 and S2) in Q. suber populations of Morocco which differed from each other by only one mutation. 3.1.2. Identification of chlorotypes in Quercus ilex (I1 and I2) Among the five combinations primer/enzymes (DT-TaqI, TF-HinfI, CD-TaqI, AS-HinfI and SR-HinfI) tested, only the combination SR-HinfI was polymorphic and permitted the identification of two major chlorotypes I1 and I2 in Tunisian Holm oak with the pre-dominance of the former in this region. Monomorphic patterns were observed with the combinations DT-TaqI, TF-HinfI, CD-TaqI and AS-HinfI. The haplotype I2 is present in the Bou Abdallah region in Siliana. Whereas, the chlorotype I1 is described in Beni Hazem, Sidi Aouidet, Plateau kessra, and the poste Forestier of Kessra regions. The populations of Q. ilex from Morocco showed four chlorotypes I1, I2, I3 and I4. Characteristics of these chlorotypes are given in Table 2. The presence of two chlorotypes of Q. ilex in Kessra (Siliana) can be explained by Two hypotheses: (i) these chlorotypes are different for each other and constituted two different taxonomic genera such as Quercus rotundifolia and Q. ilex species; (ii) One taxonomic genus diverged and gave these two different chlorotypes of Q. ilex which are represented by two infra-specific taxonomic ranks. The number of chlorotypes described in Tunisia is lower than that registered in Italy (Fineschi et al., 2005). The combination SR-HinfI was very efficient and permitted the identification of three chlorotypes in Tunisian cork oak and holm oak. The chlorotypes S3 and I1 are the most frequent ones. Chlorotype I2 is present in the center of the “plateau Kessra”. In Morocco, the chlorotypes S1 and I2 are the most frequent ones (Belahbib et al., 2001). Chlorotype S2 is present in the North of the Central Plateau and I4 is found in the “Rif” populations (Belahbib et al., 2001). Moreover, the chlorotype I2 is the most frequent in Q. ilex, but it is also found in Q. suber when both species grow together or in close proximity (Belahbib et al., 2001) suggesting cytoplasmic introgression of Q. suber by Q. ilex in Morocco (Belahbib et al., 2001). In our study, pure populations of Q. ilex have been used, and no introgression has been detected between Q. suber and Q. ilex. 3.1.3. Identification of chlorotypes in Quercus coccifera The three combinations primer-endonuclease (DT-TaqI, TF-HinfI and SR-HinfI) showed monomorphic patterns and permitted to determinate only one coccifera chlorotype in Tunisian kermes oaks (Table 2). Studies of Toumi and Lumaret (2010), based on allozymes, showed morphotype variation in the whole holly oak range. Our study based on cp CAPS didn't show an introgression between Q. coccifera and Q. ilex oaks because these species are collected from two separate regions in Tunisia. Indeed, Q. coccifera was sampled from littoral region and Q. ilex from “Kessra plateau”. In contrast, morphological studies (Natividade Viera, 1937) and genetic analyses based on allozyme and cytoplasmic markers (Toumi and Lumaret, 2001) showed frequent hybridization and introgression between Q. ilex (holm oak) and Q.

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Table 2 Description of the nine chlorotypes identified in Q. suber (S1, S2 and S3), Q. ilex (I1, I2, I3 and I4), Q. coccifera (C1) and Q. afares (A1). Chlorotype

DT-Taq-3

TF-Hinf-1

TF-Hinf-3

SR-Hinf-2

SR-Hinf-4a

SR-Hinf-4b

SR-Hinf-5

S1 S2 S3 I1 I2 I3 I4 C1 A1

1 1 1 2 2 2 2 1 1

2 2 1 1 1 1 1 1 e

1 1 1 2 2 2 2 2 e

2 2 2 2 2 2 1 1 2

9 9 9 1 1 1 1 0 9

1 2 1 e e e e 0 1

3 3 5 1 2 4 2 5 3

coccifera (holly oak). However, the nuclear introgression of holly oak by other oak species was estimated to be low and insufficient to modify significantly the genetic characteristics of the species (Lumaret and Jabbour-Zahab, 2009; Mir et al., 2009). 3.1.4. Identification of chlorotypes in Q. afares Q. afares is an endemic North African deciduous species which occurs in one population in Tunisia in Aïn Zena region (36 430 N, 8 510 E, 950 m elevation). About 750 trees remained in a mixed coppice (Mhamdi et al., 2013) with Q. canariensis and Q. suber (Hasnaoui, 1992). Closer examination indicates that Q. afares combines morphological, physiological and ecological traits of the semideciduous Q. canariensis Willd. and Q. suber species (Tutin et al., 1993), it could result from Q. subereQ. canariensis hybridization. In order to clarify the genetic relationships between the three species, a genetic analysis based on cpDNA PCR-RFLP has been conducted. Three populations of Q. afares from Aïn Zena are used, as well as several populations of both Q. suber and Q. canariensis which are sampled in the same area. The four combinations primer-endonuclease (DT-TaqI, TF-HinfI, SR-HinfI and CD-TaqI) showed monomorphic patterns and only one Q. afares chlorotype is identified. The combinations SR-HinfI and DT-TaqI showed that Q. afares predominantly possesses a similar pattern than Q. suber (Table 2). However, the combination TF-HinfI showed that Q. afares shared the same pattern as Q. canariensis. The species Q. afares exclusively presented the same bands as Q. suber and Q. canariensis for each combination primer/endonuclease which suggests that Q. afares originates from a Q. subereQ. canariensis hybridization. This is consistent with the fact that the range of both Q. suber and Q. canariensis includes that of Q. afares. Our results are in agreement with those of Mir et al. (2006) using allozymes and chloroplast markers. 3.1.5. Identification of chlorotypes in Quercus canariensis Among the four used combinations primer-endonuclease (DT-TaqI, TF-HinfI, AS-HinfI and CD-TaqI), only the combination TF-HinfI permitted to identify the chlorotype of Tunisian Q. canariensis (chlorotype 17cd). The chlorotype 25 was described in Q. canariensis from Morocco (Petit et al., 2002). Characteristic of Q. canariensis chlorotypes are given in Table 3. 3.2. Molecular polymorphism in Quercus species The five combinations primer/endonuclease analyzed in Q. suber showed a total number of 34 sites and only 2 polymorphic sites. The level of polymorphism obtained was very low (5.88%) (Table 4). Among the analyzed combinations, only the combination SR-HinfI showed a relatively high level of polymorphism (25%). Indeed, two polymorphic sites are detected in this combination. Among a total number of 33 sites, 3 sites are polymorphic. The lower level of polymorphism was registered (9%) for the studied combinations primer/endonuclease in Q. ilex species. Moreover, the combination SR-HinfI showed a total number of eight sites, three of them were polymorphic. The polymorphism level obtained in Q. ilex was 37.5%. According to these results, the combination SR-HinfI appears informing for both species Q. suber and Q. ilex (Table 4) and showed more polymorphism in Q. ilex than in Q. suber. The three combinations primer/endonuclease tested in Q. coccifera showed a total of 23 monomorphic restriction sites (Table 4). The total number of sites scored in Q. canariensis for the overall combination primer/endonuclease analyzed is 26 and 9 sites are polymorphic. The level of polymorphism observed (34.6%) was relatively low for all datasets (Table 4). The polymorphism's level registered for each combination primer/endonuclease was 20%, 33.3%, 36.36% and 50% for CD-TaqI, AS-HinfI, TF-HinfI and DT-TaqI, respectively.

Table 3 Description of chlorotypes identified in Q. canariensis. Chlorotype

TF-Hinf-1

TF-Hinf-2

CD-Taq-1

CD-Taq-3

CD-Taq-4

AS-Hinf-1

AS-Hinf-2

AS-Hinf-5

Q. canariensis from Tunisia Q. canariensis from Morocco

0 1

1 2

1 1

2 2

3 3

1 1

3 3

3 2

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Table 4 Efficiency of endonuclease/enzyme combinations used in this study.

Q. suber Number of total sites Number of polymorphic % Polymorphism Q. ilex Number of total sites Number of polymorphic % Polymorphism Q. coccifera Number of total sites Number of polymorphic % Polymorphism Q. afares Number of total sites Number of polymorphic % Polymorphism Q. canariensis Number of total sites Number of polymorphic % Polymorphism

sites

SR-HinfI

TF-HinfI

DT-TaqI

CD-TaqI

8 2 25%

10 0 0

4 0 0

5 0 0

6 0 0

4 0 0%

6 0 0%

33 3 9%

e e

23 0 0%

e e e

27 0 0%

6 2 33.33%

26 9 34.61%

sites

8 3 37.5%

11 0 0%

4 0 0%

sites

8 0 0%

11 0 0%

4 0 0%

sites

8 0 0%

11 0 0%

4 0 0%

4 0 0%

4 2 50%

5 1 20%

sites

e e e

11 4 36.36%

e e

AS-HinfI

Total 34 2 5.88%

The combinations SR-HinfI, DT-TaqI, TF-HinfI and CD-TaqI showed 27 monomorphic restriction sites in Q. afares (Table 4). Many bands in Q. afares are specific to either Q. suber or Q. canariensis and weren't observed in Q. ilex or Q. coccifera. The size of bands in Q. suber ranged from 630 bp to 180 bp for DT-TaqI and from 540 bp to 85 bp for SR-HinfI combination. However, the size of bands in Q. canariensis varied from 450 bp to 110 bp for TF-HinfI combination. Indeed, five bands (630 bp; 400 bp; 375 bp; 220 bp and180 bp) are specific to Q. suber and Q. afares, for DT-TaqI combination and eight bands (540 bp; 310 bp; 285 bp; 225 bp; 210 bp; 115 bp; 105 bp and 85 bp) are shared between the two latter species for SR-HinfI combination.

Fig. 1. UPGMA Dendrogram of the studied Quercus species constructed from Nei and Li's genetic distances matrix estimated from cp PCR-RFLP data.

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However, six bands (450 bp; 270 bp; 230 bp; 150 bp; 125 bp and 110 bp) are specific to Q. afares and Q. canariensis, for TF-HinfI combination. Our results demonstrate that the enzyme HinfI was more efficient in detecting polymorphism than TaqI for the analyzed oak species. The low levels of polymorphism registered could be due to the low intra-specific variation within the five oak species. In contrast, high levels of polymorphism have been registered in the common beech (Fagus sylvatica L.) (Demesure gue et al., 1997b). et al., 1995) and the European oaks (Dumolin-Lape The total genetic diversity (hT), the mean genetic diversity within species (hS) and the genetic differentiation among species (GST) were estimated. A high value of the GST was obtained (GST ¼ 1), suggesting that the maximum of variation is maintained between oak species. This result was confirmed by the low value of the genetic diversity within species (hS ¼ 0) and the value obtained of the total genetic diversity (hT ¼ 0.378). Additionally, this result shows the absence of gene flow between species (Nm ¼ 0). Similarly, the level of genetic differentiation Gst among the Moroccan Q. suber populations was higher (0.84) than the value found in Q. ilex (0.33) (Belahbib et al., 2001). 3.3. Genetic relationships The genetic distance, based on Nei and Li (1979), ranged from 0.045 to 1.167 with a mean of 0.606. Thus, it may be assumed that Quercus chlorotypes are characterized by a high degree of genetic diversity. The UPGMA phenogram (Fig. 1) showed the genetic divergence observed between overall species analyzed and supported significantly the chlorotypes clustering. Moreover, the tree branching showed five groups that represent the five studied oak species. Indeed, each species defined a group: the suber group consisted of S1, S2 and S3 chlorotypes, the afares group, the coccifera group, the canariensis group composed by Q. canariensis from Tunisia and Morocco, and finally, the ilex group which is composed by I1, I2, I3 and I4 chlorotypes. The topology of the phenogram is independent of the geographic distribution of oak species and depends only on the species. Similar results, based on cp PCR-RFLP, have been reported in Belahbib et al. (2001). In addition, principal correspondence analysis was used to refine details of the genetic diversity. The distribution of oak species in plan 1e2 (68.43% of the global genetic diversity) corroborated our result cited below and showed a high genetic structure between the studied oak species. Each species make an independent and separated group. Nevertheless, genetic proximity has been observed between Q. afares and Q. coccifera species. This result is corroborated by the presence of a low genetic distance (0.195) between these species. The close relationship between the two latter species could be due to temperature tolerance or the demographic history of these species. Nevertheless, other analyses using chloroplast and or nuclear markers would be required to determine the evolutionary history of these species. Low genetic distance has been registered between Q. suber (S3) and Q. afares (0.169) suggesting a high genetic proximity between these two species. Moreover, the genetic distance between Q. afares and Quercus canariensis was 0.474. This result, evidenced the hybrid origin of Q. afares between the two latter species. Similarly, studies based on morphological (Mhamdi et al., 2013) and genetic analyses (Welter et al., 2012) showed that Q. afares Pomel is considered to be a fixed hybrid between Q. suber and Q. canariensis species. The current study proved that PCRRFLP approach is efficient not only for examining the genetic diversity structure in the genus Quercus but for inferring the phylogenetic relationships between Quercus species. Acknowledgments re de l'Enseignement Supe
rieur, de Recherche This work was partially supported by grants from the Tunisian Ministe Scientifique et de la Technologie. The authors would like to thank Dr Petit J.R. and Dr Kremer A. (INRA, Bordeaux, France) for their help and comments. Special thanks to Pr Hasnaoui Ibrahim and El Mokni Ridha (Institut Sylvo-Pastoral de Tabarka) for their fruitful comments. References Abbott, R.J., Smith, L.C., Milne, R.I., Crawford, R.M.M., Wolff, K., Balfour, J., 2000. Molecular analysis of plant migration and refugia in the Arctic. Science 289, 1343e1346. Belahbib, N., Pemonge, M.H., Ouassou, A., Sbay, H., Kremer, A., Petit, R.J., 2001. Frequent cytoplasmic exchanges between oak species that are not closely related: Quercus suber and Q. ilex in Morocco. Mol. Ecol. 10, 2003e2012.  l'e
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