Original Research

AFLP based analysis of genetic diversity within Section Pseudobaccatae and Alatae of the genus Ephedra in Western Himalaya

Prabha Sharma1*, Prem Lal Uniyal 1, Shashi Bhushan Tripathi2, Madan Singh Negi2 Axel Schwekendiek3

1 Department of Botany, University of Delhi, Delhi – 110 007
2Biotechnology and Management of Bioresources Division The Energy Research Institute, Darbari Seth Block India Habitat Centre, Lodhi Road New Delhi- 110003
3 Department of Biology, University of Northern Iowa, Cedar Falls, IA 50614

J Innov Biol (2014) Volume 2, Issue 4: Pages: 245-249

Abstract: The present AFLP based molecular analysis of the fifteen populations of Ephedra occurring in the Western Himalayan range gives an insight on the distinct taxonomical structure of the interpopulational differentiation of the Section Pseudobaccatae and Alatae. This is the first hand work reflecting genetic diversity in Western Himalayan Ephedra species alongwith the current geographical distribution of the Indian Ephedra species in Western Himalaya. On the basis of the molecular data four new species and one variety (E. yurtungensis Sharma & Uniyal sp. nov., E. yurtungensis var. lutea Sharma & Uniyal comb. nov. E. Lamayuruensis Sharma & Uniyal sp. nov., E. sheyensis Sharma & Uniyal sp. nov., E. khardongensis Sharma & Uniyal sp. nov.) have also been discovered.

Received: 13 September 2015
Accepted: 28 September 2015
Published Online: 10 October 2015

Corresponding Author:
Sharma P.,
Department of Botany, University of Delhi, Delhi – 110 007
email: sharmaprabha3@gmail.com

Keywords: AFLP; Ephedra; Evolution; Genetic diversity; Pseudobaccatae; Western Himalaya


Ephedra L. (Ephedraceae) is a distinct gnetalean genus of ca. 50–65 species of shrubs, vines, or rarely small trees, widely distributed in temperate areas of Eurasia, northern Africa, southwestern North America, and western South America, where it is often abundant in dry and open habitats such as deserts, rocky slopes, grasslands, and maritime areas with a Mediterranean climate (Stevenson, 1993; Price, 1996; Sharma and Uniyal, 2008a,b; Sharma et al. 2010). The distribution of Indian species of Ephedra extends from Sikkim in Eastern Himalaya to north – west in Uttarakhand, Himachal Pradesh and Kashmir. Eleven species occur in the Indian sub-continent. E. foliata Boiss, occurs in the Punjab, Sind and Rajasthan, E. gerardiana Wall. ex Stapf, in the semiarid alpine and temperate Himalaya from Kashmir to Kumaon, E. intermedia Schr. & Meyer in Kashmir, Himachal and Garhwal, E. nebrodensis Tineo in Kashmir and Himachal Pradesh, E. pachyclada Boiss in Jammu and Kashmir, E. saxatilis (Stapf) Royle ex Florin in Uttarakhand and var. sikkimensis (Stapf) Florin in Sikkim, E. regeliana Florin in Ladakh and E. przewalskii Stapf, restricted to Nanga Parbat in Kashmir and in the bed of Shyok River in Ladakh. E. sumlingensis Sharma & Uniyal sp. nov., E. khurikensis Sharma & Uniyal sp. nov. and E. kardangensis Sharma & Uniyal sp. nov. have been discovered from the remote area of Lahaul and Spiti ( Sharma and Uniyal, 2008a,b; Sharma et al. 2010).
Multiple introductions and subsequent gene flow between populations may maintain part or all genetic variation present in native populations (Pairon et al. 2010). Evaluation of genetic diversity using molecular techniques provide useful baseline information for breeding programs. Ephedrine (Eph) is an alkaloid extracted from Ephedra sinica (Ma huang), has been known for effects on the central nervous system, cardiovascular system, and smooth muscles. The economic importance of Ephedra has generated wide interest in understanding the genetic diversity of the species as an initial step towards selection and breeding of superior genotypes for drug targeting. The use of AFLP methodology has been reliable for studies on diversity, phylogeny, genomic linkage mapping, and identification of varieties (Maughan et al. 1996; Xu et al. 2000). This is the first hand work relating AFLP depicting genetic diversity in Western Himalayan Ephedra species. Another objective of the present study is to document the current geographical distribution of the Indian Ephedra species and to determine the extent of continuity, disjunction, overlapping of the populations in Western Himalaya (Fig. 1).
Sampling wild Ephedra populations
Fifteen populations of Ephedra were collected from Yurtung, Shey, Ganglas, Khardong, Shyok river, Lamayuru and Kurbathang in Western Himalaya (Table 1, Fig.1).
DNA extraction
Genomic DNA was extracted from fresh or dry leaves using the method of Doyle & Doyle (1990), with slight modifications. Addition of 3% PVP in the extraction buffer (for removal of excess polyphenols and polysaccharides) and inclusion of a second washing step with cold ethanol were found to be necessary for extraction of genomic DNA of high quality.
AFLP analysis
The conventional AFLP protocol (Vos et al. 1995) was employed with minor modifications, using AFLP Kit I (Invitrogen Inc.). The DNA (approx. 200-250ng) was digested in 25 μl volume using 5 μl of 5X restriction ligation buffer containing ATP, 2.5 μl of 0.5 M NaCl, 1.25 μl of BSA (1mg/ml) 2.5 unit of Mse I and 5 unit of Eco RI at 37˚C for 2 hours followed by enzyme inactivation at 70˚C for 10 minutes. Ligation was done at 20˚C for 2 hours by adding 5 pMole of Eco RI adaptor, 50 pMole of Mse I adaptor and 1 unit of T4 DNA ligase to the digestion reaction. The ligation mix was diluted 1:10 times in TE buffer (10mM Tris 0.1 mM EDTA) and 2 μl of diluted ligation mix was used as template for amplification with adapter specific primers Eco RI + A and Mse I + C in a total of 20 μl volume (Table 2). The PCR reaction was performed in a Gene Amp PCR 9700 thermal cycler using the following cycling parameters: 20 cycles of 30s at 94˚C, 60 s at 56˚C and 60s at 72˚C. The pre-amplified mix was diluted 50 fold for selective amplification. Selective amplification was done using Eco RI (labeled with 32P) and Mse I primers with 3 selective nucleotides in a total of 10 μl reaction volume. The PCR parameters were: 1 cycle of 30s at 94˚C, 30s at 65˚C and 72˚C for 60s. The annealing temperature was reduced by 1˚C per cycle during the first 11 cycles, and then 23 cycles were performed at 94˚C for 30s, 56˚C for 30s and 72˚C for 60s. The samples were size-fractionated on 6% polyacrylamide gel and the fragments were detected by autoradiography.
Data analysis
AFLP profile data is summarized by (i) total number of amplified fragments (or bands) (ii) number of polymorphic bands per assay unit; each AFLP primer-pair combination is defined as one assay unit (iii) mean number of fragments per assay unit (Multiplex ratio) and (iv) mean number of polymorphic bands per assay unit (Effective Multiplex Ratio). For each AFLP primer-pair combination (Table 2), the number of polymorphic bands were counted. Only distinct bands with strong intensity (size range: 50-400 bp) were manually scored. Presence of a band denoted as ‘1’ and absence as ‘0’ and included in the binary data matrix analysis to estimate genetic similarity coefficient (Table 3) using Jaccard coefficient [GSJ=a/(a+b+c)]; where GS is the measure of genetic similarity between individuals i and j, a is the number of polymorphic bands (Table 4) that are shared by i and j, b is the number of bands present in i and absent in j, c is the number of bands present in j and absent in i. The similarity matrix was subjected to UPGMA (Unweighted Pair Group Method of Arithmetic Averages) method of clustering in order to construct the phenetic dendrogram (Fig. 2). The above mentioned statistical analyses were performed using NTSYS-pc software (version 2.02, Rohlf, 1998).
Outcome of Molecular data
Four new species and one variety are discovered as E. yurtungensis Sharma & Uniyal sp. nov., E. yurtungensis var. lutea Sharma & Uniyal comb. nov., E. Lamayuruensis Sharma & Uniyal sp. nov., E. sheyensis Sharma & Uniyal sp. nov., E. khardongensis Sharma & Uniyal sp. nov. on the basis of of morpho – molecular analysis (Fig. 2, Table 1,4).
AFLP polymorphism and cluster analysis
The six primer pair combinations are used for AFLP analysis that generated 447 fragments for the 15 accessions, of which 387 are polymorphic (86.57%; Table 4). The large number of polymorphic fragments at the inter- and intra- specific level demonstrated that there is a high level of genetic variation and provided insight into the taxonomic relationships, evolution and the proposed current taxonomic treatment (Fig. 2, Table 1).
A distinct relationship is found between AFLP groups (Fig. 2). Occurrence of more than one species at a particular locality indicate sympatric speciation which brought out four new species and a variety in Ladakh range of Western Himalaya (Figs. 1-2, Table 1). We consider this molecular distinctness to be the most important criteria for future ranking decisions at the specific and infraspecific levels.
AFLP marker has also been used to study the evolutionary relationships at the species or genus level and is considered as an effective tool to highlight phylogenetic relationships (Brouat et al. 2004; Stefenon et al. 2003). Indeed AFLP has been found very useful in establishing the genetic relationships among Ephedra species. The dendrogram constructed by using UPGMA method revealed two main clusters (Fig. 2). The first one comprised of E. yurtungensis sp. nov. (YU0451), E. yurtungensis var. lutea (YU0457), E. regeliana (YR0452), E. sheyensis sp. nov. (SH0460). Cluster II consisted of nine species and one variety viz. E. intermedia (YU0465), E. intermedia var. tibetica (KU0504), E. pachyclada (SH0468), E. nebrodensis (SH 0467), E. gerardiana (GA0469), E. lamayuruensis sp. nov. (KH0485, LA0499), E. khurikensis (KH0481), E. sumlingensis (SH0466), E. khardongensis sp. nov. (KH0474) and E. przewalskii (SY0495).
Historical events of past fragmentation and range expansion, associated with glaciation, may have shaped the phylogeographic patterns of Section Pseudobaccatae and Alatae. Recession of lakes and subsequent desertification occurred in the Himalayas as evident by the river impressions marked at various places in Lahaul & Spiti (Himachal Pradesh) and Ladakh region. Therefore, the diverse Ephedra species may have originated after the formation of the recent deserts and E. regeliana is showing recency in origin as compared to rest of the investigated species in the Himalayan range.
High genetic diversity verses altitude range
Outcrossed, animal-dispersed, long-lived and late successional species with wide distribution ranges show higher genetic diversity than selfed, gravity-dispersed, short-lived and early successional species with small distribution ranges (Thiel-Egenter et al. 2009). In the present study genetic diversity was found to be high suggesting that high-mountain populations possess mechanisms to ensure genetic diversity over the long term. The standardized sampling across the whole range of four different mountain regions and the application of the molecular marker type brought out the significant findings that genetic diversity is species, population specific. One could therefore conclude that in alpine plants neither gene flow nor sexual reproduction seems to be restricted in high-elevation as compared to low-elevation habitats (Plüss and Stöcklin, 2004).
We thank Kumar Shantanu and Saira Parveen, for their kind help in the collection of plant material. This work was supported by the Indian Institute of Science, Bangalore, Department of Biotechnology, Govt. of India and University of Delhi.

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