African Journal of Biotechnology Vol. 11(36), pp. 8810-8819, 3 May, 2012 Available online at http://www.academicjournals.org/AJB DOI: 10.5897/AJB11.1584 ISSN 1684–5315 © 2011 Academic Journals Full Length Research Paper Inter-specific relationships among two Tunisian Thymus taxa: Thymus capitatus Hoffm. et Link. and Thymus algeriensis Boiss. et Reut. using molecular markers Imen BEN EL HADJ ALI*, Arbi GUETAT and Mohamed BOUSSAID National Institute of Applied Sciences and Technology, Laboratory of Plant Biotechnology B. P. 676, 1080 Tunis Cedex, Tunisia. Accepted 28 October, 2011 Genetic relationships between two sympatric species Thymus capitatus Hoffm. et Link. and Thymus algeriensis Boiss. et Reut. ( Thymus hirtus Willd. subsp. algeriensis Boiss. et Reut.) were assessed using random amplified polymorphic DNA (RAPD) markers. Eighteen natural populations from different geographical and bioclimatic zones were evaluated. The seven selected primers generated 121 RAPD markers for T. capitatus (103 polymorphic; P = 85.12%) and 154 for T. algeriensis (141 polymorphic; P = 91.56%). The genetic diversity within T. capitatus and T. algeriensis populations based on Shannon’s index was high (H’ pop = 0.303 and 0.307, respectively). A high genetic differentiation was revealed (GST = 0.359 and ΦST = 0.284 for T. capitatus , GST = 0.335 and ΦST = 0.296 for T. algeriensis ). The large proportions of the genetic variation were observed within populations for the two studied taxa. A high genetic population’s structure was also estimated by a principal component analysis (PCA). Unweighted pair-group method using arithmetic average (UPGMA) cluster analysis based on Nei and Li's coefficient among populations identified that T. algeriensis and T. capitatus populations were clustered into two distinct groups. The high genetic divergence between the two species corroborates their taxonomic status. Key words: Genetic diversity, inter-specific relationships, molecular markers, Thymus capitatus , Thymus algeriensis . INTRODUCTION Thymus is a large genus of Lamiaceae family comprising four species. Among them, T. capitatus and T. about 215 species and particularly prevalent in the algeriensis, which can be sympatric in a wide part of their Mediterranean area (Jalas, 1971). The systematics of distribution area, are well represented (Pottier-Alapetite, species remains difficult because of the interspecific 1981; Ben ElHadj Ali et al., 2010). Almost all species of hybridization, polyploidy and morphological similarities this genus are currently used in popular medicine for among species (Morales, 1996; Tzakow and stimulant, aromatic, antispasmodic, sedative, antioxidant, Constantinidis, 2005). The genus Thymus is known in antibacterial and antiaflatoxigenic, and also in the several countries as a spice and food preservative, as treatment of diarrhoea, digestive and respiratory system well as a protective and curative remedy for many disorders (Dob et al., 2006; Matta et al., 2007; Hazzit et ailments. In Tunisian flora, the genus is represented by al., 2009; Razzaghi-Abyaneh et al., 2009; Safaei-Ghomi et al., 2009). In Tunisia, T. capitatus is known under the vernacular name of ‘‘Zaater’’. This shrub is about 20 to 60 cm height, *Corresponding author. E-mail: [email protected]. Tel: the leaves (6 to 12 mm) are opposite and (+216) 71703829 (929). Fax: (+216) 71704329. linear/lanceolate, the flowers are hermaphrodite large (7 Ben El Hadj Ali et al. 8811 to 10 mm) and grouped in dense terminal heads with an bioclimates (Table 1 and Figure 1). The random sample size in uneven calyx (5 mm), with the upper three lobed and the each population was ten individuals. Fresh leaves were sampled lower cleft, and the corolla (5 to 8 mm) is deeper and pink from individuals, at least 20 m apart, to reduce the chance of consanguinity. or purple or white. The flowering period begins in June and continues until August. The species is a diploid (2n = 2x = 30) (Morales, 1996) and predominantly bee- Genomic DNA extraction and PCR conditions pollinated outcrossing shrub, exhibiting both vegetative and sexual propagations (Petanidou and Vokou, 1993). Total genomic DNA was isolated from about 0.5 g young leaves using a modified CTAB protocol, as outlined by Lodhi et al. (1994). Natural regeneration of genets from seeds is extremely DNA quantity was estimated spectrophotometrically (absorbance at rare because of the difficulty of seedling survival, mainly 260 nm). An agarose gel (0.8%) stained with ethidium bromide was consequent to the low habitat quality (climatic factors, soil used to verify the quality of DNA. degradation, etc) (Eriksson, 1998; Pérez-Garcia et al., PCR was performed in a 25 µL reaction volume containing 50 ng 2003). In Tunisia, T. capitatus populations are located in DNA templates, 5 µL of 5 X reaction buffer, 40 pmoles of primer, different bioclimatic zones (extending from the sub-humid 200 µM of each dNTP, 2.5 mM MgCl 2 and 1.5 U Taq polymerase (Promega). The mixture was overlaid with one drop of mineral oil. to the upper arid) on sandy and often on rocky soils, Reactions were performed in Stuart Thermal Cycler (Maxi-Gene) under a rainfall varying from 300 to 1000 mm/year and at programmed for an initial denaturation step of 94°C for 2 min, altitudes ranging from 150 to 500 m (Nabli, 1995). followed by 45 cycles of 30 s at 94°C, 1 min at 36°C (annealing T. algeriensis is an endemic plant of semi arid and arid step), and 2 min at 72°C (elongation step). An additional 10 min areas of Tunisia and Algeria (Pottier-Alapetite, 1981; Le period for elongation at 72°C followed this cycle. To test Floc’h and Boulos, 2008). It is a short lived, diploid (2n = reproducibility between and within runs, DNA from same two plants was included in every PCR run. A negative control without DNA 2x = 30) and gynodioecious shrub (Morales, 1996). It was also used in every PCR run. Eight primers were tested and reproduces by seeds and can reach 20 to 50 cm in only seven out of them were selected (OPJ-06, OPJ-08, OPJ-10, height. Leaves are opposite and linear/lanceolate (6 to 12 OPJ-12, OPJ-13, OPJ-14 and OPJ-16). Amplification products were mm). Flowers with ovate bracts and pink purplish or separated by electrophoresis in 1.5% agarose gels in TAE buffer whitish purple corolla are small (5 to 7 mm). (pH 8), stained with ethidium bromide, and visualized under UV light using a DOC PRINT Photo Documentation System. Molecular Hermaphrodite (male fertile) and female (male sterile) weights were estimated using a 200 bp DNA Promega ladder. plants can occur in the same populations. Flowering takes place between April and June. The species is believed to be an outcrosser as most thymes and often Data analysis pollinated by bees (Tarayre and Thompson, 2002; Orellana et al., 2005). However, self pollination may RAPD bands were scored as presence (1) or absence (0) of bands and transformed into a binary matrix. Each marker band was occur in hermaphrodite plants. In Tunisia, T. algeriensis assumed as being a single locus. For each primer, the percentage populations are distributed from the sub-humid to the of polymorphic bands (P%) was estimated for the studied lower arid bio-climates at altitudes ranging from 120 to populations. The genetic diversity within population was estimated 1100 m. The species grows on poor fertile calcareous using P% and Shannon’s index (H’). Shannon’s index was also soils and occurs in scattered small populations showing used to estimate the average diversity (H pop ) over all populations different level of destruction, mainly due to (Hpop = −1/ n Σ H’; where n is the number of populations), and the diversity (H sp ) at species level (Hsp = −Σps log2 ps ; where ps is the overharvesting and overgrazing. frequency of presence or absence of the RAPD in the whole In the present study, we used RAPD approaches to sample). The proportion of diversity within populations was characterize two Tunisian species of Thymus and estimated as H pop /H sp . Population differentiation was analyzed for evaluate their genetic relationships. This method is polymorphism between populations by the gene differentiation broadly used in plant population genetic and coefficient (G ST ) [GST = (Hsp - Hpop)/Hsp]. The different indices were calculated by the POPGENE computer package (Yeh et al., differentiation studies (Monteleone et al., 2006; Solouki et 1999). The comparison among Shannon’s diversity indices at the al., 2008; Zheng et al., 2008; Trindade et al., 2009). population levels was performed using a variance analysis (ANOVA Generally, RAPD has allowed the resolution of complex procedure, SAS, 1990) and Duncan’s test (Dagnelie, 1975). taxonomic relationships (Wolff and Richards, 1999; The partitioning of the genetic variation within and among Casiva et al., 2002; Ruana et al., 2004) and phlogenetic populations, besides H pop /H sp and G ST estimates, was evaluated by studies (Dababneh, 2007; Mariette et al., 2007; Paolini et AMOVA using WINAMOVA program, version 1.55 (Excoffier et al., 1992). Φ-statistics: Φ (differentiation among populations), Φ al., 2009). ST CT (differentiation among ecological groups) and ΦSC (differentiation among populations within groups) were calculated. The significance of variance components and that of Φ-statistics were estimated MATERIALS AND METHODS using permutation procedures (NTSYS-pc, version 2.0, Rohlf, 1998). The genetic similarity between individuals was estimated Surveyed populations and sampling using the Nei and Li’s (1979) similarity coefficient S xy [S xy = 2m xy / (m x + m y), where, mxy is the number of bands shared by samples x The plant materials used in this study were collected from nine and y, and m x and m y are the number of bands in samples x and y, populations across 6 bioclimatic zones according to Emberger’s respectively]. The genetic distance (D xy ) between individuals was (1966) pluviothermic coefficient (Q 2): sub-humid, upper semi-arid, estimated using the complementary value Sxy [D xy =1-Sxy ].