Abstract: Background and Objective: Teucrium in Libya is represented by 11 species, five of which are endemic and among them problems at different taxonomic levels. Besides, Libya suffers from both human and environmental pressures and population fragmentation, which in turn lead to the loss of genetic diversity. There is an urgent need to assess this diversity therefor the aim of this study was to investigate the diversity among and within Teucrium as a prerequisite for conservation programming. Materials and Methods: The genetic diversity was assessed for 12 Teucrium taxa by RAPD and ISSR. Both the total and specific bands, polymorphism percentage, Shannon index (H) and Simpson index (D) were determined and the data was processed with cluster and principal component analyses. Results: Teucrium taxa were characterized with relatively low genetic diversity, particularly T. polium subsp. flavovirens. It was noted that the taxa of each section were not grouped together and the dendrogram resulted in a distinct sorting among the taxa at 0.545 similarity level. Both cluster and PCA analyses were clearly separated between T. capitatum and T. polium at the level of dissimilarity on par with other species. Conclusion: The study accepted T. capitatum and T. polium as two separate species and T. polium subsp. flavovirens as T. luteum subsp. flavovirens and for both Girian and Siert taxa, there was a doubt that they were assembled under T. polium which need to be confirmed by matching with other Teucrium species.
INTRODUCTION
Teucrium L. is the second considerable genus in Ajugoideae, which is often described as a contentious in demarcation at different taxonomic levels. It comprises more than 300 species of cosmopolitan distribution, especially in the Mediterranean region1-5. Teucrium species are rich in phenolic compounds and used for digestive and respiratory disorders and as diuretic, anti-septic, anti-rheumatic, anti-inflammatory, anti-oxidative, anti-microbial, anti-diabetic, hepatoprotective, hypolipidemic and anti-cancer6,7.
Teucrium is represented in Libya by three sections-Chamaedrys (Mill.) Schreb., Teucrium L. and Polium (Mill.) Schreb. and eleven species. Five out of them were endemic; T. apollinis Maire and Weiller, T. barbeyanum Asch. and Taub. ex E.A. Durand and Barratte, T. davaeanum Coss., T. lini-vaccarii Pamp. and T. zanonii Pamp.8,9.
The most problematic and heterogeneous section was Polium which contained more than 50% of the species and 82% of the endemicones1,10. It was classified into four subsections-Polium, Simplicipilosa, Pumilum and Rotundifolia-that were reduced to three by the eliminating of Rotundifolia1,11. The main taxonomic problem was under the subsection Polium, especially between T. polium and T. capitatum and among T. polium subspecies. The Euro+Med and WCPS12,13 accepted five subspecies of T. polium in the Mediterranean region-aurasiacum (Maire) Greuter and Burdet, clapae S. Puech, polium, purpurascens (Benth.) S. Puech and rupestricola (Sennen) T. Navarro and Rosua. They also recognized the subspecies capitatum and flavovirens as T. capitatum and T. luteum subsp. flavovirens; respectively. Many authors interpreted this diversity as adaptive radiation and differentiation through polyploidy as a result of drought stress and regional establishment of the Mediterranean climate that facilitated the genus speciation2,14-16.
Libya is a good example of representing both human and environmental pressures and population fragmentation that cause genetic bottle necks and promote genetic drift, which in turn leads to further loss of genetic diversity. Therefore there is an urgent need to assess the genetic diversity of these populations with particular attention to both endemic and polymorphic species. These assessments were crucial for planning effective conservation strategies17,18. Boulila et al.2, Ding et al.19, Saini et al.20,21 and Sharma et al.22 clarified that both RAPD and ISSR were valuable tools for these purposes. The RAPD featured many advantages such as low cost and speed, but with some disadvantages such as low reproducibility. Thus, mixing with other data from ISSR was very promising for distinguishing among species due to its high reproducibility23.
The intention of the study is to assess the taxonomic relations and genetic diversity among Libyan Teucrium species-specially within the subsection Polium-based on both RAPD and ISSR, as a prerequisite for the conservation programming.
MATERIALS AND METHODS
Twelve Teucrium taxa were collected from its natural habitats in Libya in two flowering seasons; 2009 and 2010 (Table 1). The voucher specimens were deposited in the herbaria at Alexandria University, Egypt (ALEX) and Omar Al-Mukhtar University, Libya.
For both RAPD and ISSR analyses, genomic DNA was extracted from 10-20 mg dry seeds using GeneJET Genomic DNA purification kit (Thermo-Scientific). Ready-To-Go Analysis Beads (GE Healthcare Life Sciences, 27-9502-01 with primers) kit was performed for amplification reaction.
For RAPD analysis, six decamer oligonucleotide arbitrary primers were reproducible; RAPD-1 (GGTGCGGGAA), RAPD-2 (GTTTCGCTCC), RAPD-3 (GTAGACCCGT), RAPD-4 (AAGAGCCCGT), RAPD-5 (AACGCGCAAC) and RAPD-6 (CCCGTCAGCA).
| Table 1: | Studied Teucrium species (*endemic), location, altitude, coordinates and habitat |
Gene Amp Polymerase Chain Reaction (PCR) system cycler was achieved at 94°C for 7 min followed by 30 cycles at 94°C for 1 min, 35°C for 1 min and 72°C for 2 min and final extension step at 72°C for 6 min.
For ISSR analysis, six oligonucleotide arbitrary primers were reproducible; ISSR-1 (GAG[CAA]5), ISSR-2 ([GA]8T), ISSR-3 ([AG]8T), ISSR-4 ([AC]8C), ISSR-5 ([AGC]6G) and ISSR-6 ([TG]8A). Gene Amp Polymerase Chain Reaction (PCR) system cycler was achieved at 94°C for 5 min followed by 35 cycles at 94°C for 30 sec, 52°C for 45 sec and 72°C for 2 min and final extension step at 72°C for 5 min.
The amplified products from RAPD and ISSR was separated on 2% agarose gel, visualized under UV transilluminator and photographed via gel documentation system (Digi-doc, UVP Company, England). The discrete bands were scored as binary characters and UVP Doc-It® LS Image Analysis Software was used for molecular weights determination through 1000 base pair DNA ladder of Thermo scientific Co.
The genetic diversity among taxa was appraised using three estimators:
| • | The percentage of polymorphism = 100 (number of polymorphic bands/number of total bands) |
| • | Shannon index (H) = -∑pilog2pi, where pi is the frequency of a given band24 |
| • |
where, ni is the number of bands of species i and N is the total number of bands25.
The Shannon index was in direct relation with both the richness (number of bands in a species) and the evenness (comparing the number of bands among species). On the other hand, Simpson index was approached to one where every species has a unique genotype. Due to the confusing of richness and evenness in the Shannon index, the combination between direct estimate of species richness (Shannon index) with some measure of dominance or evenness (Simpson index) were appropriate26.
Both cluster analysis, using the unweighted pair group method with arithmetic mean (UPGMA) based on Jaccard distance and principal component analysis (PCA) were performed through PAST program27.
RESULTS
The RAPD and ISSR patterns obtained from genomic DNA of Teucrium species using RAPD-1 and ISSR-1 were represented in Fig. 1.
| Fig. 1(a-b): | RAPD and ISSR patterns obtained from genomic DNA of Teucrium species using RAPD-1 (GGTGCGGGAA) and ISSR-1; GAG(CAA)5, (a) RAPD-1 and (b) ISSR-1 |
| Fig. 2: | Total number of bands (TNB), percentage of polymorphism (P %) and specific bands (SB) of Teucrium species generated from RAPD |
| Table 2: | Shannon index (H) and simpson index (D) for RAPD and ISSR analyses |
For RAPD analysis, 94 of 107 amplified fragments were polymorphic by 90.65%. The generated bands from each primer ranged from 73-171, with averages from 6.08-14.25. While the averages per species for the total number of bands, polymorphism (%) and specific bands were 51.92, 28.63 and 1.8, respectively. Teucrium zanonii attained the highest total produced bands and both Shannon’s and Simpson’s indices in RAPD, while T. polium subsp. flavovirens recorded the lowest values for all genetic diversity parameters (Fig. 2, Table 2). For ISSR analysis, the polymorphic bands were 210 from 226 generated fragments with 92.92%. The produced bands per primer varied from 102-152 with averages from 8.5-12.67. The averages of total number of bands, polymorphism (%) and specific bands per species were 63.91, 21.26 and 7.75, respectively. The highest total number of generated bands and polymorphism (%) was found in T. campanulatum, while T. polium of Siert achieved the lowest values in the same parameters along with the values of Shannon’s and Simpson’s indices. On the other hand, the specific bands varied from 3 in T. brevifolium to 16 in T. zanonii (Fig. 3, Table 2). For both RAPD and ISSR, the highest values of Shannon’s and Simpson’s indices were in T. capitatum and the lowest in T. polium subsp. flavovirens (Table 2). The discrimination among studied taxa in the non-hierarchical form by PCA accounted 41.604% of variance for the three first components, with 17.966, 12.839 and 10.799% for the 3 axes respectively. It was noted that the taxa of each section were not grouped together, especially those belonging to section Polium (Fig. 4). Within this section, both T. capitatum and T. polium were clearly separated and also T. polium from Siert was kept apart from other taxa of the same species. The dendrogram from UPGMA resulted in a distinct sorting among the taxa at 0.545 similarity level (Fig. 5). The T. capitatum and T. polium were distinguished at the lowest level of similarity (0.38).
| Fig. 3: | Total number of bands (TNB), percentage of polymorphism (P %) and specific bands (SB) of Teucrium species generated from ISSR |
| Fig. 4: | PCO of Teucrium species based on both RAPD and ISSR analyses |
| *Species of section polium | |
Both T. polium subsp. flavovirens and the taxon of Girian were detached from that of Siert at 0.41similarity level, while T. apollinis and both T. barbeyanum and T. zanonii were segregated at 0.425 and 0.461 similarity levels, respectively.
DISCUSSION
Libya suffers from the destruction of biodiversity and the risk of genetic erosion due to desertification and several environmental and human crisis28,29. Therefore, the study evaluated the genetic diversity among Libyan Teucrium that is the second largest genus composed of endemic species which will assist in its conservation approach.
| Fig. 5: | Dendrogram of Teucrium species based on both RAPD and ISSR analyses |
Teucrium is well adapted to these pressures that has led to its speciation16,30 and the present study confirms this idea by revealing relatively high Simpson’s and Shannon’s indices and stated why species were not grouped under the accepted sections. The genetic diversity assessment through different molecular markers and the combining of more than one technique were important and both RAPD and ISSR are suitable tools for these evaluations particularly for endemic species2,31-33. The ISSR is more discriminative than RAPD which appeared through total number of bands, specific bands and both Simpson’s and Shannon’s indices for different species. The primers RAPD-4 and ISSR-4 record the highest number of common bands among taxa and the study recommends that they be genus specific. Luan et al.34 pointed out that endemic species and those with small geographic ranges were characterized by low genetic diversity. These projections are evident in T. apollinis, which maintains low values for the total number of bands, the percentage of polymorphism, both Shannon’s and Simpson’s indices and relatively high specific bands as an average for both RAPD and ISSR-compared to other endemic species. These values may be related with the reduced population sizes and the geographical region of dry stony flat hills with relatively low humidity (38.9%) and high altitude (356 m). On the other hand, T. zanonii and T. davaeanum accomplish relatively high genetic diversity that can be interpreted through their distinctive habitats; loamy sand, relatively high humidity (72%) and low altitude (9 m) for the first and the presence of the second at the slope of Wadi El Quttarh35. Luan et al.34, Maguire and Sedgley36 and Zawko et al.37 were pointed to a link between high genetic diversity in rare plants and the recent decline in population size, insufficient isolation time or wide gene flow. Therefore, the study recommends that priority be given to in situ and ex situ conservation for both species, particularly for vulnerable populations. Both T. polium and T. capitatum were among the most controversial species of taxonomic problems in Teucrium1. Many authors treated T. capitatum as a subspecies38-40 and others referred to T. polium regardless of their subspecies10,41-44. The dendrogram and PCA are clearly separated between them and at the level of dissimilarity on par with other species, so the study accepts the level of species for both taxa. This is consistent with Marzouk et al.8,9 for the distinctiveness of each species with specific nutlet and pollen micro-morphological attributes. In T. capitatum, the nutlet epidermal cells were specified with depressed anticlinal and convex external periclinal walls and the pollen with verrucate-scabrate sculpture. While T. polium was characterized by nutlet cells of raised, wavy anticlinal and concave external periclinal walls and pollen with verrucate-perforate sculpture. The three studied taxa of T. polium share 36 common bands of RAPD and ISSR, while 12, 11 and 6 of the finger-printing bands are distinctive in Girian, Siert and subsp. flavovirens; respectively. The taxon of Girian is closely related to the subsp. flavovirens through 29 common bands from RAPD and ISSR. Despite the segregation of Siert taxon, it shares 24 common bands with Girian and 8 bands with the subspecies. The great diversity in karyotyping pattern and ploidal levels within T. polium played an important role in its speciation2,14-16,45. Meanwhile, Askar35 identified 12 essential oils in Libyan Teucrium; α-Cadinol, Camphene, β-Caryophyllene, α-Copaene, β-Elemene, Germacrene-B, Germacrene-D, Isoborneol, Limonene, Linalool, α-Pinene and Terpinen. He confirmed the absence of α-Cadinol and Isoborneol in Girian taxon, Linalool and Terpinen in Siert taxon and α-Cadinol, α-Pinene and Terpinen in the subsp. flavovirens. Thus, the study agrees with WCPS13 to accept T. polium subsp. flavovirens as T. luteum subsp. flavovirens. For the other two taxa; Girian and Siert, there is a doubt that they are assembled under one species which need to be confirmed by matching with other Teucrium species.
CONCLUSION
Teucrium were characterized with relatively low genetic diversity and about 42% of its species were endemic that require conservation priority, especially for the populations of critical sizes or those subjected to human and climatic pressures. Also, the study highlights the validity of both RAPD and ISSR in the assessment of genetic diversity and the discrimination among and within Teucrium.
SIGNIFICANCE STATEMENT
This study evaluated the genetic diversity among Libyan Teucrium, which will assist in the conservation approach of these species suffering from environmental and human crisis. The taxa achieved a low level of genetic diversity. Both dendrogram and PCA were clearly separated between T. capitatum and T. polium, so the study accepted the species level for each. The results also confirmed the reallocation of T. polium subsp. flavovirens as T. luteum subsp. flavovirens. For the two taxa of T. polium from Girian and Siert, there was a doubt that they were gathered under one species.