Nuclear DNA C-values measurements as rapid tool for biodiversity screening: Case of the Lebanese flora

MAGDA BOU DAGHER-KHARRAT*, NOUR ABDEL-SAMAD, BOUCHRA C. DOUAIHY, MICKAEL BOURGE, SPENCER C. BROWN, SONJA SILJAK-YAKOVLEV. *Laboratoire “Caractérisation Génomique des Plantes”, Faculté des sciences, Université Saint-Joseph, Beirut, [email protected]

General context: One of the ten hotspots in the Mediterranean basin is the -Lebanon-/Palestine area (Médail et al. 1997). Lebanon is a hinge between three continents (fig. 1), Europe, Asia and Africa and thus its flora is influenced by the four bio-geographical adjacent regions. Mosaic habitat variation in Lebanon : shrubs, woodlands, limestone cliffs, scree slopes, open cultivated and abandoned fields, Thus Lebanon is home for more than 3000 . diversity in Lebanon is based on the coexistence, within a single flora, of plants of diverse biogeographically origins and a large number of narrow endemic species. Genome size evaluation was used in this study to screen rapidly differences between taxonomically related plants. Genome size is indeed, an important biodiversity character of fundamental significance (Bennett, et al. 2000) and it varies considerably between angiosperm species (Bennett and

Smith 1976, Bennett and Smith 1991). The amount of DNA per chromosome set is known to be a fairly constant characteristic of The Lebanese territory is divided species. Its interspecific variation could be of a great help to address taxonomic questions. Moreover, this work presents a between mountainous slopes, fertile contribution to enlarge the plant DNA-C value database by adding 250 values of Lebanese native taxa. The availability of these data valleys which separate two could be profitably used in biodiversity evaluation, systematic and evolution studies. mountainous chains parallel with the sea and of the steppe areas in North Material & Methods: Table 1: Representation in our East of the country. The mountainous study of C-value data relief is characterized by deep Plant material (leaves, seedling and ) were collected in wild populations. The 250 canyons and a great number of rivers investigated taxa belong to 56 families and 135 genera (table 1). C-values were estimated by flow cytometry using the propidium iodide as intercalary fluorochrome stain. Each sample comprises at least five individuals and was measured twice. When possible, several populations were measured for each species. Mount - Lebanon 3000 m Anti - Lebanon 2000 m Results and Discussion 1500 m 1000 m New C values are reported for 193 taxa including 132 endemic to the Eastern Mediterranean region. Our data reported Bekaa SYR the first count for 48 . Genome size of studied taxa ranged from 1C=0,28 pg in Hypericum thymifolium to 54.69 pg Mediterranean sea valley IA in Fritillaria alfredae (Fig. 1) Where intraspecific C-value variation appears most significant for is as an indicator of taxonomic heterogeneity, pointing to the need for a re-evaluation of the delimitation of the species in question (Murray 2005).

Figure 1. Cumulative frequency of 1C-value for investigated taxa When we analyzed genome size distribution according to the Life Growth form (Raunkier 1934) we found that intermediate, large 1C (pg) versus Cumulative Frequency and very large genome sizes categories are dominated by geophytes (fig. 2). Moreover, low nuclear DNA content was clearly 1.0 associated with short life cycles (Fig. 3). This is a general finding corroborated by (Arumuganathan and Earle 1991). In fact it 0.9 very large >35 seems that the largest plant genomes are in plants that have distinct and short periods of growth followed by long periods of 0.8

large dormancy (e.g. geophytes in the monocot orders and Liliales).

0.7 14 to 35 Fig 2: Genome size distribution according to the Life Fig 3: Genome size distribution according to Life 0.6 Growth form (Raunkier 1934). cycle types.

0.5 intermediate .Ph = Phanérophyte, CH= Chaméphyte, G= Géophyte, T = A= annual; B = biennial; P = perennial, small 3.5 to 14 Thérophyte, H= Hémicryptophyte. 1.4 to 3.5 0.4

0.3 Cumulative Frequency Cumulative

0.2 very small <1.4 0.1 Fritillaria alfredae Hypericum thymifolium

0.0 0 1 10 100 1C (pg)

For many species present around the Mediterranean, taxa present in Lebanon are considered as varieties and are found to be polyploid when compared to the widely distributed species. It VS = Very small, S = Small, Int = Intermediate, L = Large, VL = Very large was the case for example for Puschkinia.

Paeonia species we found that they were polyploid with 2n=4x=20 chromosomes. Paeonia mascula seems to have different ploidy levels. It was reported by (Mulry M 1999) as diploid 2n=10 chromosomes while (Tzanoudakis 1983) found Greek taxa as teraploid. For Paeonia mascula, (Mulry and L. 1999) found 17.75 pg/C for 10 chromosomes. The same so called species in Lebanon have 29.25 pg/C with 20 chromosomes. Karyotype analysis showed that this species is 2n=4x=20. Moreover based on chromosome morphology, this plant seems to be an allopolyploid plant. Endemic to Lebanon The endemic Paeonia species, Paeonia keserwanensis, is also tetraploid with a geome size significantly different from Paeonia macula. Harboring the same chromosome number, more invetigations must be done to elucidate this genome size vaiation Puschkinia scilloides libanotica 2n=2x=10 chromosomes, 13,25pg/2C 2n=4x=20 chromosomes, 27,13 pg/2C

Conslusions Paeonia macula Paeonia keserwanensis 2n=4x=20 chromosomes, 58pg/2C 2n=4x=20 chromosomes, 65pg/2C

Genome doubling through auto- or allopolyploidy, contributing to speciation and evolution, is a well-documented phenomenon occurring in the plant world. In some plant groups polyploidy occurs frequently and represents the major force in divergence. Allopolyploidy is a primary force of hybridization that affects manifold genome size variation through series of different ploidy levels. Our results show that polyploidy has had a significant role in shaping the Lebanese flora and contributes to the speciation process Genome size assessment constitutes a rapid tool to highlight polyploidisation and other genome organization in some species which is known to create new species. In this study genome size evaluation by flow cytometry was used as a tool to make a quick screening in the Lebanese flora. Based on obtained data, some taxonomic groups like Astragalus, Iris, Cyclamen, Pancratium and others are actually profoundly studied using molecular markers and cytogenetic advanced techniques.

This project is supported by the Lebanese Ministry of environment, the Lebanease NCSR (National Center for Scientific Research), a fellowships from Unesco-L’Oréal for Women in Science program, HP-UNESCO Barin gain initiative program and Réseau BioVEG of the AUF (Agence Universitaire de la Francophonie).