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Integrated approaches for identification of promising populations of Valeriana jatamansi in West Himalaya
Journal of Asia-Pacific Biodiversity 9 (2016) 152e159
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Original article
Integrated approaches for identification of promising populations of Valeriana jatamansi in West Himalaya Arun Kumar Jugran, Indra Dutt Bhatt*, Ranbeer Singh Rawal G.B. Pant Institute of Himalayan Environment and Development, Kosi-Katarmal, Almora, Uttarakhand, India
a r t i c l e i n f o
a b s t r a c t
Article history: Received 27 September 2015 Received in revised form 1 November 2015 Accepted 27 February 2016 Available online 3 March 2016
Biodiversity conservation and management is a global issue of concern for biologists keeping in view the rapid rate of degradation. The present study focuses on Valeriana jatamansi, a medicinally important herb used in traditional and modern medicines. The raw material of the species is collected from the wild, which puts pressure on its natural habitat; hence, data on diversity attributes including morphological, phytochemical, antioxidant activity, and genetic diversity of V. jatamansi are analyzed to identify promising sources and to develop conservation and management strategies for target species. Neighbor joining cluster and principal component analysis was used to identify elites based on all these attributes. Based on neighbor joining cluster and principal component analysis two populations, Dunagiri and Tharali, were identified as elites and promising sources for raw material collection and conservation purposes. However, individually, each attribute prioritizes different populations. The approach of the present study can be further utilized for developing conservation planning and management of different medicinal plants of the region. This will also promote the interest of researchers in future studies and will aid in collecting plant material from a choice of population. Copyright Ó 2016, National Science Museum of Korea (NSMK) and Korea National Arboretum (KNA). Production and hosting by Elsevier. This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/).
Keywords: antioxidant activity conservation genetic diversity phytochemicals Valeriana jatamansi
Introduction Setting priorities for conservation and utilization of threatened plants is a major concern of researchers. The conservation efforts often require balance between human needs and available resources, which may at times create a direct conflict with conservation (Vucetich et al 2013). This conflict has become more severe in cases of plant species of commercial importance. In this context, prioritization of the populations/individual is considered one of the best options for future conservation and utilization of a species (Allendorf et al 2010). In cases of medicinal plants, investigations on habitat protection, possibility of dispersal, and evolution via gene flow with naturally selected surrounding populations is considered the best method for preserving vulnerable populations at particular habitats/ecosystems where selection holds special importance. Conserving the habitat of a species is considered to conserve all the morphological, genetic, and phytochemical attributes; thus, ensuring the long-term conservation of the species. Studies
* Corresponding author. Tel.: þ91 5962 241041; fax: þ91 5962 241014. E-mail addresses: [email protected], [email protected] (I.D. Bhatt). Peer review under responsibility of National Science Museum of Korea (NSMK) and Korea National Arboretum (KNA).
recommend conservation of biological diversity at three levels: genes, species, and ecosystem (McNeely et al 1990) and all have their own significance. For instance, conserving biological diversity at ecosystem level is best because the losses of habitat and ecological processes have immediate impacts on species survival. However, it is not possible to conserve all habitats and ecosystems. Therefore, prioritization of representative habitats/populations is an alternative to conserve maximum diversity in an ecosystem, which will ultimately conserve the diversity of a species. Moreover, conservation at species level is an alternative method to conserve the population of choice, which has some direct ecological and economic significance. This is rather easy as it involves less complexity than the ecosystem level. It is more important in the Himalayan perspective where variation in environmental conditions is prevalent. Conservation of a species based on a combination of personal experience, ecological understanding, and political expediency are being used for setting priorities; however, these are not applicable for all species. In addition, conclusions drawn on the basis of a few selected attributes do not provide full mechanistic insights of a species for conservation and management. Therefore, analysis and assemblage of multiple attributes in respect to different dimensions is a prerequisite for the conservation planning of a species (Oliver et al 2004). The present study focuses on
http://dx.doi.org/10.1016/j.japb.2016.02.009 pISSN2287-884X eISSN2287-9544/Copyright Ó 2016, National Science Museum of Korea (NSMK) and Korea National Arboretum (KNA). Production and hosting by Elsevier. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
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Table 1. Site characteristics and accession number of selected populations of Valeriana jatamansi. S. no.
Population
Altitude (m asl)
Latitude (N )
Longitude (E )
Habitat
Accession number
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
Daulaghat (DAG) Katarmal (KAT) Tharali (THA) Dolti (DOL) Berinag (BER) Majkhali (MAJ) Talwari (TAL) Khirshu (KHR) Didihat (DID) Pithoragarh (PIT) Sitlakhet (SIT) Gwaldam (GWL) Kausani (KAU) Ukhimath (UKH) Camel back (CMB) Jaberkhet (JAB) Joshimath (JHM) Dunagiri (DNG) Buranskhanda (BRK) Nainital (NNT) Makku band (MKB) Munsyari (MUN) Malyadaur (MLD) Dwali (DWL) Surkanda (SRK)
1215 1250 1330 1626 1672 1702 1785 1810 1850 1872 1900 1923 1925 1985 2000 2080 2100 2125 2150 2176 2240 2240 2350 2730 2775
29 460 000 0 29 380 250 0 30 040 060 0 30 020 560 0 29 430 170 0 29 400 200 0 30 010 460 0 29 510 170 0 29 460 160 0 29 360 140 0 29 350 400 0 30 000 240 0 29 470 510 0 30 310 000 0 30 270 430 0 30 270 180 0 29 470 370 0 29 470 370 0 30 270 220 0 29 230 340 0 30 340 0.00 0 30 030 390 0 30 080 160 0 30 100 380 0 30 240 210 0
80 180 000 0 79 370 200 0 79 300 080 0 79 290 500 0 80 020 140 0 79 310 470 0 79 300 590 0 79 350 460 0 80 170 590 0 80 110 400 0 79 320 420 0 79 330 300 0 79 260 120 0 80 050 000 0 78 040 260 0 78 060 500 0 79 270 380 0 79 270 400 0 78 050 590 0 79 270 450 0 79 130 000 0 80 140 360 0 79 570 560 0 79 590 460 0 78 170 210 0
Grassy Oak Oak Oak Pine Pine Oak Oak Oak Pine Pine Oak Grassy Pine Grassy Grassy Mixed Grassy Oak Mix Grassy Mixed Mix Mix Mixed
113261 BSD-112793 113206 GBPI-3201-2 113257 113209 113205 113212 113253 113214 GBPI-3201-1 113213 113210 113258 113200 113260 113211 113254 113207 113208 113262 113202 113255 113204 113259
asl, above sea level; S., species.
Valeriana jatamansi Jones (common name “Tagar” or “Indian valerian”; family Valerianaceae), one of the most important medicinal plants of the Himalayan region. This species remains a subject of interest due to its uses in traditional and modern medicine, variability in morphology, reproductive behavior, essential oil content, and antioxidant properties. The species is dioecious, perennial, polygamous, or occasionally polygamo monocious (Prakash 1999) and grows wild in the temperate Himalayan region between 1000 m above sea level (asl) and 3000 m asl. The species is used in the treatment of various ailments and comprises over 150 chemical compounds (Jugran 2013). V. jatamansi is used as a substitute of Valeriana officinalis, a member of same family of European origin (Singh et al 2010). Although the species is widely distributed, at a regional scale the species is vulnerable due to overexploitation and habitat destruction (CAMP 2003; Jugran et al 2013a; Jugran et al 2013b). Therefore, there is an urgent need to investigate the species at different dimensions for setting priorities for conservation and utilization. The present study is an attempt to: (1) assess the relationship among morphological, phytochemical, and genetic attributes in different populations; and (2) develop an approach for prioritizing the promising populations based on these attributes. The result of the present study will thus be helpful in developing suitable strategies for conservation and management of V. jatamansi.
Materials and methods V. jatamansi (family Valerinaceae) was collected from 25 different populations of Uttarakhand within an altitudinal range from 1000 m asl to 3000 m asl (Table 1; Figure 1). Different morphological and genetic attributes were analyzed across populations of V. jatamansi (Jugran et al 2013b; Jugran et al 2015). A total of 125 plants (5 plants/population) from 25 distant populations of V. jatamansi were subjected to detailed morphological analysis and various morphological attributes like leaf number, leaf area, plant height, above ground fresh weight (AGFW), below ground fresh weight (BGFW), above ground dry weight (AGDW),
below ground dry weight (BGDW), rhizome length (RL), and rhizome diameter (RD) were evaluated. Similarly, DNA was isolated from 151 genotypes belonging to 25 distant populations of V. jatamansi and polymerase chain reaction amplification was performed using 20 different inter simple sequence repeat primers. For phytochemical analysis plant material was collected from all 25 populations, washed thoroughly with tap water followed by washing with distilled water, and divided into two portions namely, aerial and root portion. Phytochemical (valerenic acid, total phenolics, flavonoids, and tannins) and antioxidant activity of all populations were investigated across the populations (Jugran 2013). Data drawn from different attributes (morphological, phytochemical, and genetic) were used for detailed analysis and prioritization of populations. Identification of promising sources/populations with higher morphological, phytochemical, and genetic attributes was performed using different sets of statistical procedures. Neighbor joining cluster analysis was performed using Paleontological Statistics Software Package for Education and Data Analysis packages, USA (Hammer et al 2001) for clustering of V. jatamansi populations based on all studied attributes. Neighbor joining clustering is considered an alternative method for hierarchical clustering analysis (Saitou and Nei 1987) for small data sets (Jianfu et al 2011). The data were further subjected for principal component analysis (PCA) to identify the major population groups with higher ranking of each attributes using SPSS version 16 (SPSS Inc. 1989e1996, Chicago, IL, USA).
Results Data pertaining to different morphological (leaf number, leaf area, plant height, AGFW, BGFW, AGDW, BGDW, RL, and RD), phytochemical (total phenolics, flavonoids, tannins, valerenic acid, and antioxidant activity), and molecular attributes (Nei’s genetic diversity and %polymorphism) recorded among targeted populations revealed wide variations among the populations (Jugran et al 2013a; Jugran et al 2015). Different populations behave differently for each attribute. For example, in the morphological
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Not to scale
Indian Himalayan Region (IHR)
NEPAL BHUTAN
78°
79 °
80 °
81 °
Figure 1. Locality map of Valeriana jatamansi showing the distribution of 25 studied populations in Uttarakhand.
attributes, when considering RL (3.62 0.43 cm), the Surkunda population was the best whereas the Talwari population for RD (1.84 0.02 cm) was the best. Similarly, the Katarmal population was best for AGFW (8.88 1.72 g) and BGFW (4.70 1.87 g) but the Surkunda population for AGDW (3.36 0.98 g) and BGDW (1.57 0.52 g) was the best. Phytochemical analysis exhibited significantly higher valerenic acid content in Katarmal (aerial portion: 0.57 0.04%) and Joshimath populations (root portion: 1.80 0.12%); total phenolic content in the aerial [25.23 0.03 mg gallic acid equivalent (GAE)/g dry
weight (dw)] and root (27.54 0.06 mg GAE/g dw) portions of the Munsyari population; and flavonoid contents in the aerial [32.67 0.34 mg quercetin equivalent (QE)/g dw] and root (29.91 0.47 mg QE/g dw) portion of the Jaberkhet and Talwari populations. Ukhimath and Gwaldam populations exhibited significantly higher tannin content [aerial portion: 8.09 0.04 mg TAE/g dw; root portion: 7.91 0.60 mg tannic acid equivalent (TAE)/ g dw]. Significant difference (p < 0.05) was observed in antioxidant activity across the populations of V. jatamansi (aerial and root portion) measured by 2,20 -azino-bis(3-ethylbenzothiazoline-6-
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Figure 2. Elite identification based on neighborhood joining cluster analysis. A, morphological attributes; B, phytochemical and antioxidant activity; C, genetic attributes (polymorphic loci % and Nei genetic diversity index); D, total studied attributes.
sulphonic acid), 2,2-diphenylpicrylhydrazyl, and ferric reducing antioxidant power assays. Different populations, such as the Didihat and Katarmal populations, showed maximum activity in aerial and root portions, respectively, using 2,20 -azino-bis(3ethylbenzothiazoline-6-sulphonic acid) activity. In the case of the 2,2-diphenylpicrylhydrazyl assay, the Talwari population exhibited the maximum [16.80 0.03mM ascorbic acid equivalent (AAE)/ 100g dw] antioxidant activity in the aerial portion, whereas the root portion of the Didihat population exhibited maximum antioxidant activity (17.53 0.04mM AAE/100g dw). The results using ferric reducing antioxidant power assay indicated that the population from Didihat showed significantly higher antioxidant activity in aerial portions and the Ukhimath population in root portions (12.71 0.04mM AAE/100g dw). Genetic diversity analysis based on inter simple sequence repeat marker analysis generated 159 clear and reproducible bands in
studied populations of V. jatamansi, and 125 of them were polymorphic. With the mean of 78.62%, the percentage of polymorphic loci ranged from 65.41% (Dwali) to 91.19% (Pithoragarh). Nei genetic diversity index ranged from 0.25 (Surkunda) to 0.37 (Pithoragarh) with a mean value of 0.31. Neighbor joining cluster analysis was performed for determining the relationship among different populations of V. jatamansi depending on morphological, phytochemical (in aerial and root portions), and genetic attributes. Neighbor joining cluster analysis based on morphological attributes placed Surkunda and Dunagiri in the top order (Figure 2A), based on phytochemicals, Gwaldam and Munsyari (Figure 2B), and based on molecular attributes Pithoragarh population (Figure 2C). Taking together all the attributes, Dunagiri and Tharali populations came up in the top order (Figure 2D). PCA based on the correlation matrix of morphological attributes across the populations grouped all populations into three
156 AK Jugran et al. / Journal of Asia-Pacific Biodiversity 9 (2016) 152e159 Figure 3. Projection of different populations of Valeriana jatamansi based on principal component analysis. A, morphological attributes; B, phytochemicals and antioxidant activity; C, genetic attributes based on genetic distance (Source: Jugran et al 2013); D, total studied attributes. Circles show clustering of populations with same attributes.
AK Jugran et al. / Journal of Asia-Pacific Biodiversity 9 (2016) 152e159
major groups. Morphological attributes among all populations revealed 89.88% of the total variability in the first component and 8.36% in the second component while remaining variability is explained by Component 3 and Component 4 (Figure 3A; Table 2). Results indicated Katarmal, Nainital, and Munsyari as promising populations based on morphological attributes. PCA analysis revealed three major groups based on phytochemicals (valerenic acid, total phenols, and tannins) and antioxidant activity. Group I includes populations from Shitlakhet and Munsyari which showed high levels of all phytochemicals while Group II and Group III included all other remaining populations of V. jatamansi (Figure 3B; Table 3). PCA analysis based on Nei genetic similarity and distance matrix revealed three major groups of populations. Group I included the populations Berinag, Majkhali, Talwari, Didihat, Kausani, and Pithoragarh, which exhibited the highest genetic diversity compared with the remaining populations. These populations are considered a promising source harboring most of the variation residing within the species (Figure 3C). Taking together all the attributes, PCA analysis categorized all 25 populations into two major Groups; Group I includes Tharali, Berinag, Dunagiri, Joshimath, Dolti, and Surkunda populations whereas Group II includes other remaining populations of V. jatamansi (Figure 3D; Table 4).
Discussion Studies on morphological attributes across the populations are considered an important criterion to assess growth performance and biomass of the species. In the present study, Katarmal, Nainital, and Munsyari populations showed the best performance based on morphological attributes; therefore, these populations can be further utilized for mass multiplication to obtain a higher biomass of the species. Significant variations in morphological parameters among different populations might be due to differences in ecotypes, which are often indicative of genetic differences in many genes (Jugran 2013). However, a population with only higher
Table 2. Total variance explained using principal components of the morphological attributes of Valeriana jatamansi. Component Initial eigenvalues Total
Extraction sums of squared loadings
% of variance Cumulative % Total
1 22.47 89.88 2 2.09 8.36 3 0.38 1.52 4 0.05 0.22 5 0.01 0.02 6 0.00 0.01 7 0.00 0.00 8 0.00 0.00 9 0.00 0.00 10 0.00 0.00 11 0.00 0.00 12 0.00 0.00 13 0.00 0.00 14 0.00 0.00 15 0.00 0.00 16 0.00 0.00 17 0.00 0.00 18 0.00 0.00 19 0.00 0.00 20 0.00 0.00 21 0.00 0.00 22 0.00 0.00 23 0.00 0.00 24 0.00 0.00 25 0.00 0.00
89.88 98.24 99.75 99.97 99.99 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00
% of variance Cumulative %
22.47 89.88 2.09 8.36
89.88 98.24
157
Table 3. Total variance explained using principal components based on phytochemical and antioxidant activities in Valeriana jatamanai. Component Initial eigenvalues Total
Extraction sums of squared loadings
% of variance Cumulative % Total
1 20.21 80.83 2 2.04 8.17 3 0.95 3.79 4 0.71 2.86 5 0.45 1.78 6 0.20 0.80 7 0.17 0.69 8 0.14 0.55 9 0.06 0.25 10 0.04 0.17 11 0.02 0.08 12 0.01 0.03 13 0.00 0.01 14 0.00 0.00 15 0.00 0.00 16 0.00 0.00 17 0.00 0.00 18 0.00 0.00 19 0.00 0.00 20 0.00 0.00 21 0.00 0.00 22 0.00 0.00 23 0.00 0.00 24 0.00 0.00 25 0.00 0.00
80.83 89.00 92.78 95.64 97.42 98.22 98.91 99.46 99.71 99.88 99.96 99.99 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00
% of variance Cumulative %
20.21 80.83 2.04 8.17
80.83 89.00
growth performance cannot be considered elite as it should possess a higher amount of the major active ingredient along with maintaining its genetic diversity, which can be utilized for future breeding programs of V. jatamansi. Considering the availability of phytochemical contents, the present investigation revealed that the mean values for total phenolics and tannins in V. jatamansi were considerably higher compared with earlier reports on the same
Table 4. Total variance explained using principal components base on morphological, phytochemical, and genetic attributes (polymorphic loci % and Nei genetic diversity index only). Component Initial eigenvalues Total
Extraction sums of squared loadings
% of variance Cumulative % Total
1 24.12 96.48 2 0.28 1.11 3 0.18 0.71 4 0.12 0.48 5 0.10 0.39 6 0.07 0.29 7 0.04 0.16 8 0.03 0.13 9 0.02 0.08 10 0.02 0.06 11 0.01 0.04 12 0.01 0.04 13 0.00 0.01 14 0.00 0.01 15 0.00 0.00 16 0.00 0.00 17 0.00 0.00 18 0.00 0.00 19 0.00 0.00 20 0.00 0.00 21 0.00 0.00 22 0.00 0.00 23 0.00 0.00 24 0.00 0.00 25 0.00 0.00
96.48 97.59 98.30 98.79 99.17 99.46 99.62 99.75 99.83 99.89 99.93 99.97 99.98 99.99 99.99 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00
% of variance Cumulative %
24.12 96.482 0.277 1.109
96.482 97.591
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species (Bhatt et al 2012) and other related species of the family, e.g. Nardostachys jatamansi and V. officinalis (Surveswaran et al 2007). Although it was lower than the value reported by Kalim et al (2010). Similarly, antioxidant activity reported in the present study (Jugran et al 2015) by all three assays was higher compared with other plants like N. jatamansi and V. officinalis. Reports are also available on the antioxidant activity in extracts and essential oils of V. jatamansi (Kalim et al 2010; Das et al 2011; Bhatt et al 2012; Jugran et al 2013b). However, the reason for variation in antioxidant activity (either lower or higher) could be due to different genetic backgrounds and sources of samples. Various factors such as harsh climatic conditions (light, chilling, pollution, etc.; Valentine et al 2003) and environmental stresses (Pennycooke et al 2005; Close and McArthur 2002; Reddy et al 2004) are reported to be responsible for these variations. Further studies on valerenic acid quantification in V. jatamansi (Singh et al 2006) and V. officinalis (another species of same family) have been performed (Bos et al 1998; Singh et al 2006; Ebrahimzadeh et al 2008; Wills and Shohet 2009). The reason for variation in phytochemical content might be the increased phenylalanine ammonia lyase activity. Phenylalanine ammonia lyase is a key and regulating enzyme in the secondary metabolic pathway leading to the synthesis of phenolic compounds (Jones 1984). Genetic diversity is of fundamental importance for the adaptation of the species to future environmental changes. Highest genetic diversity in the Pithoragarh population (1872 m asl) and lowest genetic diversity in the Dwali (2730 m asl) population indicates that there is limited genetic diversity of V. jatamansi at higher altitudes. Various factors like the inherent breeding system mode of reproduction, pollination, seed dispersal, stylar movement (Khajuria et al 2011; Jugran et al 2013a), and ploidy level (Soltis and Soltis 2000) in the species is reported to be responsible for genetic variations and contributes to the maintenance of high genetic diversity. Morphological, phytochemicals, antioxidant activity, and genetic attributes showed that the Tharali, Dunagiri, and Munsyari populations are the most promising sources and can be designated as the best morphotype/chemotype/genotypes of V. jatamansi. Morphological and genetic attributes were, for the first time, used in this study for prioritization; however, studies are available on chemotyping of the species based of different chemical attributes. For example, two chemotypes of V. jatamansi based on essential oil composition were identified, one with patchouli alcohol as a major compound and the other with maaliol as a major compound (Mathela et al 2005). Recently, 16 accessions of V. jatamansi using essential oil compounds were classified in three chemotypes (Sundaresan et al 2012). Chemotypic variability in various other species based on essential oil composition have also been reported, e.g. Origanum vulgare (Mockute et al 2001), Hippophae rhamnoides (Tian et al 2004), Lychnophora ericoides (Curado et al 2006), Liptospermum scorparium (Douglas et al 2004), Anemopsis californica (Medina et al 2008), and Artemisia dracunculus (Chauhan et al 2010). Four (phytochemical based) and three (antioxidant activity based) populations identified in this study could be due to a large sample size of the species, which covered almost the entire Uttarakhand area compared with the previous one. This can be used for classifying and selecting populations based on different compounds. These populations could be further explored for focusing a particular group on chemical production and to obtain the desired phytochemicals with maximized yield. The present study suggests that screening of multilocational plants/populations for different attributes (morphological, phytochemical, antioxidant, and genetic) is essential for obtaining promising stock containing optimum levels of these attributes. Based on neighbor joining and PCA, Tharali and Dunagiri populations are found best for all studied attributes. This is the first
report of prioritization of promising populations based on different parameters and multilocational sample collection; this may promote the interest of researchers for further investigation of the species. Similarly, promising populations (Tharali and Dunagiri) identified by all studied attributes harbor maximum genetic variation within the species. Therefore, cultivation and crop improvement under the plant breeding program of the populations from these areas can be recommended. These populations could be used as a promising stock for mass multiplication of the target species and can be informative for conservation purposes. As such, protection of a particular population with high genetic variation is unlikely to protect all the variation in the species. Therefore, several populations with diverse locations from the entire range should be considered for conservation. However, it is not practically feasible to conserve all populations; therefore, conserving populations that harbor maximum variations in all aspects could be a viable option. As the population size of V. jatamansi is gradually reducing due to habitat fragmentation by human disturbance or natural calamities (Alyemeni and Sher 2010), this might contribute to population extinction and loss of genetic variation. Therefore, desired clones or populations can be mass multiplied and used for long-term use, exploration, and conservation. Acknowledgments We thank Dr P.P. Dhyani, Director G. B. Pant Institute of Himalayan Environment and Development (GBPIHED) for facilities and encouragement. Colleagues of Biodiversity Conservation and Management Theme are thanked for their cooperation and help during the study. Financial support from the SERB-DST-FTYS (NO. SB/YS/LS-162/2013) scheme is acknowledged. References Allendorf FW, Hohenlohe PA, Luikart G. 2010. Genomics and the future of conservation genetics. Nature Reviews Genetics 11:697e709. Alyemeni MN, Sher H. 2010. Impact of human pressure on the population structure of Persicaria amplexicaule, Valeriana jatamansi and Viola serpens the naturally growing medicinal plants in Malam Jaba, Swat, Pakistan. Journal of Medicinal Plant Research 4:2080e2091. Bhatt ID, Dauthal P, Rawat S, et al. 2012. Characterization of essential oil composition, phenolic content, and antioxidant properties in wild and planted individuals of Valeriana jatamansi Jones. Scientia Horticulture 136:61e68. Bos R, Woerdenbag HJ, Van Putten FMS, et al. 1998. Seasonal variation of the essential oil, valerenic acid and derivatives, and valepotriates in Valeriana officinalis roots and rhizomes and the selection of plants suitable for phytomedicines. Planta Medica 64:143e147. CAMP. 2003. Conservation assessment and management prioritization: For the medicinal plants of Himachal Pradesh, Jammu & Kashmir and Uttarakhand. Proceedings of the Workshop held at Shimla. Bangalore, India: Hosted by Foundation for Revitalization of Local Health Traditions (FRLHT). Chauhan RS, Kitchlu S, Ram G, et al. 2010. Chemical composition of Capillene chemotypes of Artemissia dracunculus L. from North West Himalaya. Industrial Crops and Products 31:546e549. Close DC, McArthur C. 2002. Rethinking the role of many plant phenolicsdprotection from photodamage not herbivores? Oikos 99:166e172. Curado MA, Carolina BA, Oliveira JGJ, et al. 2006. Environmental factors influence on chemical polymorphism of the essential oils of Lychnophora ericoides. Phytochemistry 67:2363e2369. Das J, Mao AA, Handique PJ. 2011. Terpenoid composition and antioxidant activities of two Indian Valerian oils from the Khasi Hills of north- east India. Natural Products Community 6:129e132. Douglas MH, Van Klink JW, Smallfield BM, et al. 2004. Essential oils from New Zealand manuka: triketone and other chemotypes of Leptospermum scoparium. Phytochemistry 65:1225e1264. Ebrahimzadeh H, Radjabian T, Ekhteraei Tousi S, et al. 2008. Evaluation of some Iranian wild species from Valerianaceae as commercial sources of valepotriates. Journal of Biological Science 8:549e555. Hammer O, Harper DAT, Ryan PD. 2001. PAST: Paleontological statistics software package for education and data analysis. Palaeontologia Electronica 4:1e9. Jianfu LI, Jianshuang LI, Huaiqing HE. 2011. A simple and accurate approach to hierarchical clustering. Journal of Computer Information Systems 7:2577e2584. Jones DH. 1984. Phenylalanine ammonia-lyase: regulation of its induction, and its role in plant development. Phytochemistry 23:1349e1359.
AK Jugran et al. / Journal of Asia-Pacific Biodiversity 9 (2016) 152e159 Jugran AK, Bhatt ID, Rawal RS, et al. 2013a. Patterns of morphological and genetic diversity of Valeriana jatamansi Jones in different habitats and altitudinal range of West Himalaya, India. Flora-Morphology, Distribution, Functional Ecology of Plants 208:13e21. Jugran A, Rawat S, Dauthal P, et al. 2013b. Association of ISSR markers with some biochemical traits of Valeriana jatamansi Jones. Industrial Crops and Products 44: 671e676. Jugran AK. 2013. Fingerprinting phytochemical and genetic variations in Valeriana jatamansi and V. himalayana in Uttarakhand. Nainital, Uttarakhand, India: Kumaun University. PhD Thesis (in Biotechnology). Jugran AK, Bhatt ID, Rawal RS. 2015. Identification of ISSR markers associated with valerenic acid and antioxidant activity in Valeriana jatamansi Jones in Western Himalaya. Molecular Breeding 35:73. http://dx.doi.org/10.1007/s11032-015-0241-5. Kalim MD, Bhattacharya D, Banerjee A, et al. 2010. Oxidative DNA damage preventive activity and antioxidant potential of plants used in Unani system of medicine. BMC Complementary and Alternative Medicine 10:77. Khajuria A, Verma S, Sharma P. 2011. Stylar movement in Valeriana wallichii DC.da contrivance for reproductive assurance and species survival. Current Science 100:1143e1144. Mathela CS, Sati S, Chanotiya CS. 2005. Comparative investigations of the leaf and root oils of Valeriana wallichii DC from Northwestern Himalaya. Journal of Essential Oil Research 17:408e409. McNeely JA, Miller KR, Reid WV, et al. 1990. Conserving the World’s Biological Diversity. Washington, DC: IUCN, World Resources Institute, Conservation International, WWF-US, World Bank. Medina HA, Holguin OF, Micheletto S, et al. 2008. Chemotypic variation of essential oils in the medicinal plant, Anemopsis californica. Phytochemistry 69:919e927. Mockute D, Bernotiene G, Judzentiene A. 2001. The essential oil of Origanum vulgare L spp. Vulgare growing wild in Vilnius district (Lithuania). Phytochemistry 57: 65e69. Oliver I, Holmes A, Dangerfield JM, et al. 2004. Land systems as surrogates for biodiversity in conservation planning. Ecological Applications 14:485e 503.
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Pennycooke JC, Cox S, Stushnoff C. 2005. Relationship of cold acclimation, total phenolic content and antioxidant capacity with chilling tolerance in Petunia. Environmental and Experimental Botany 53:225e232. Prakash V. 1999. Indian Valerianaceae a monograph on medicinally important family. Jodhpur, India: Scientific Publishers. Reddy AR, Chaitanya KV, Vivekanandan M. 2004. Drought-induced responses of photosynthesis and antioxidant metabolism in higher plants. Journal of Plant Physiology 161:1189e1202. Saitou N, Nei M. 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Molecular Biology and Evolution 4:406e425. Singh N, Gupta AP, Singh B, Kaul VK. 2006. Quantification of valerenic acid in Valeriana jatamansi and Valeriana officinalis by HPTLC. Chromatographia 63: 209e213. Singh RD, Gopichand Meena RL, Sharma B, et al. 2010. Seasonal variation of bioactive components in Valeriana jatamansi from Himanchal Pradesh, India. Industrial Crops and Products 32:292e296. Soltis ED, Soltis PS. 2000. Contributions of plant molecular systematic to studies of molecular evolution. Plant Molecular Biology 42:45e75. Sundaresan V, Shani G, Verma RS, et al. 2012. Impact of geographic range on genetic and chemical diversity of Indian Valerian (Valeriana jatamansi) from northwestern Himalaya. Biochemical Genetics 50:797e808. Surveswaran S, Cai Y, Corke H, et al. 2007. Systematic evaluation of natural phenolic antioxidants from 133 Indian medicinal plants. Food Chemistry 102:938e953. Tian C, Nan P, Chen J, et al. 2004. Volatile composition of Chinese Hippophae rhamnoides and its chemotaxonomic implications. Biochemical Systematics and Ecology 32:431e441. Valentine IK, Maria VK, Bruno B. 2003. Phenolic cycle in plants and environment. Journal of Molecular Cell Biology 2:13e18. Vucetich JA, Peterson RO, Nelson MP. 2013. Discernment and precaution: A response to Cochrane and Mech. The George Wright Forum 30:333e340. Wills RBH, Shohet D. 2009. Changes in valerenic acids content of valerian root (Valeriana officinalis L. s.l.) during long-term storage. Food Chemistry 115: 250e253.
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Original article
Integrated approaches for identification of promising populations of Valeriana jatamansi in West Himalaya Arun Kumar Jugran, Indra Dutt Bhatt*, Ranbeer Singh Rawal G.B. Pant Institute of Himalayan Environment and Development, Kosi-Katarmal, Almora, Uttarakhand, India
a r t i c l e i n f o
a b s t r a c t
Article history: Received 27 September 2015 Received in revised form 1 November 2015 Accepted 27 February 2016 Available online 3 March 2016
Biodiversity conservation and management is a global issue of concern for biologists keeping in view the rapid rate of degradation. The present study focuses on Valeriana jatamansi, a medicinally important herb used in traditional and modern medicines. The raw material of the species is collected from the wild, which puts pressure on its natural habitat; hence, data on diversity attributes including morphological, phytochemical, antioxidant activity, and genetic diversity of V. jatamansi are analyzed to identify promising sources and to develop conservation and management strategies for target species. Neighbor joining cluster and principal component analysis was used to identify elites based on all these attributes. Based on neighbor joining cluster and principal component analysis two populations, Dunagiri and Tharali, were identified as elites and promising sources for raw material collection and conservation purposes. However, individually, each attribute prioritizes different populations. The approach of the present study can be further utilized for developing conservation planning and management of different medicinal plants of the region. This will also promote the interest of researchers in future studies and will aid in collecting plant material from a choice of population. Copyright Ó 2016, National Science Museum of Korea (NSMK) and Korea National Arboretum (KNA). Production and hosting by Elsevier. This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/).
Keywords: antioxidant activity conservation genetic diversity phytochemicals Valeriana jatamansi
Introduction Setting priorities for conservation and utilization of threatened plants is a major concern of researchers. The conservation efforts often require balance between human needs and available resources, which may at times create a direct conflict with conservation (Vucetich et al 2013). This conflict has become more severe in cases of plant species of commercial importance. In this context, prioritization of the populations/individual is considered one of the best options for future conservation and utilization of a species (Allendorf et al 2010). In cases of medicinal plants, investigations on habitat protection, possibility of dispersal, and evolution via gene flow with naturally selected surrounding populations is considered the best method for preserving vulnerable populations at particular habitats/ecosystems where selection holds special importance. Conserving the habitat of a species is considered to conserve all the morphological, genetic, and phytochemical attributes; thus, ensuring the long-term conservation of the species. Studies
* Corresponding author. Tel.: þ91 5962 241041; fax: þ91 5962 241014. E-mail addresses: [email protected], [email protected] (I.D. Bhatt). Peer review under responsibility of National Science Museum of Korea (NSMK) and Korea National Arboretum (KNA).
recommend conservation of biological diversity at three levels: genes, species, and ecosystem (McNeely et al 1990) and all have their own significance. For instance, conserving biological diversity at ecosystem level is best because the losses of habitat and ecological processes have immediate impacts on species survival. However, it is not possible to conserve all habitats and ecosystems. Therefore, prioritization of representative habitats/populations is an alternative to conserve maximum diversity in an ecosystem, which will ultimately conserve the diversity of a species. Moreover, conservation at species level is an alternative method to conserve the population of choice, which has some direct ecological and economic significance. This is rather easy as it involves less complexity than the ecosystem level. It is more important in the Himalayan perspective where variation in environmental conditions is prevalent. Conservation of a species based on a combination of personal experience, ecological understanding, and political expediency are being used for setting priorities; however, these are not applicable for all species. In addition, conclusions drawn on the basis of a few selected attributes do not provide full mechanistic insights of a species for conservation and management. Therefore, analysis and assemblage of multiple attributes in respect to different dimensions is a prerequisite for the conservation planning of a species (Oliver et al 2004). The present study focuses on
http://dx.doi.org/10.1016/j.japb.2016.02.009 pISSN2287-884X eISSN2287-9544/Copyright Ó 2016, National Science Museum of Korea (NSMK) and Korea National Arboretum (KNA). Production and hosting by Elsevier. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
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Table 1. Site characteristics and accession number of selected populations of Valeriana jatamansi. S. no.
Population
Altitude (m asl)
Latitude (N )
Longitude (E )
Habitat
Accession number
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
Daulaghat (DAG) Katarmal (KAT) Tharali (THA) Dolti (DOL) Berinag (BER) Majkhali (MAJ) Talwari (TAL) Khirshu (KHR) Didihat (DID) Pithoragarh (PIT) Sitlakhet (SIT) Gwaldam (GWL) Kausani (KAU) Ukhimath (UKH) Camel back (CMB) Jaberkhet (JAB) Joshimath (JHM) Dunagiri (DNG) Buranskhanda (BRK) Nainital (NNT) Makku band (MKB) Munsyari (MUN) Malyadaur (MLD) Dwali (DWL) Surkanda (SRK)
1215 1250 1330 1626 1672 1702 1785 1810 1850 1872 1900 1923 1925 1985 2000 2080 2100 2125 2150 2176 2240 2240 2350 2730 2775
29 460 000 0 29 380 250 0 30 040 060 0 30 020 560 0 29 430 170 0 29 400 200 0 30 010 460 0 29 510 170 0 29 460 160 0 29 360 140 0 29 350 400 0 30 000 240 0 29 470 510 0 30 310 000 0 30 270 430 0 30 270 180 0 29 470 370 0 29 470 370 0 30 270 220 0 29 230 340 0 30 340 0.00 0 30 030 390 0 30 080 160 0 30 100 380 0 30 240 210 0
80 180 000 0 79 370 200 0 79 300 080 0 79 290 500 0 80 020 140 0 79 310 470 0 79 300 590 0 79 350 460 0 80 170 590 0 80 110 400 0 79 320 420 0 79 330 300 0 79 260 120 0 80 050 000 0 78 040 260 0 78 060 500 0 79 270 380 0 79 270 400 0 78 050 590 0 79 270 450 0 79 130 000 0 80 140 360 0 79 570 560 0 79 590 460 0 78 170 210 0
Grassy Oak Oak Oak Pine Pine Oak Oak Oak Pine Pine Oak Grassy Pine Grassy Grassy Mixed Grassy Oak Mix Grassy Mixed Mix Mix Mixed
113261 BSD-112793 113206 GBPI-3201-2 113257 113209 113205 113212 113253 113214 GBPI-3201-1 113213 113210 113258 113200 113260 113211 113254 113207 113208 113262 113202 113255 113204 113259
asl, above sea level; S., species.
Valeriana jatamansi Jones (common name “Tagar” or “Indian valerian”; family Valerianaceae), one of the most important medicinal plants of the Himalayan region. This species remains a subject of interest due to its uses in traditional and modern medicine, variability in morphology, reproductive behavior, essential oil content, and antioxidant properties. The species is dioecious, perennial, polygamous, or occasionally polygamo monocious (Prakash 1999) and grows wild in the temperate Himalayan region between 1000 m above sea level (asl) and 3000 m asl. The species is used in the treatment of various ailments and comprises over 150 chemical compounds (Jugran 2013). V. jatamansi is used as a substitute of Valeriana officinalis, a member of same family of European origin (Singh et al 2010). Although the species is widely distributed, at a regional scale the species is vulnerable due to overexploitation and habitat destruction (CAMP 2003; Jugran et al 2013a; Jugran et al 2013b). Therefore, there is an urgent need to investigate the species at different dimensions for setting priorities for conservation and utilization. The present study is an attempt to: (1) assess the relationship among morphological, phytochemical, and genetic attributes in different populations; and (2) develop an approach for prioritizing the promising populations based on these attributes. The result of the present study will thus be helpful in developing suitable strategies for conservation and management of V. jatamansi.
Materials and methods V. jatamansi (family Valerinaceae) was collected from 25 different populations of Uttarakhand within an altitudinal range from 1000 m asl to 3000 m asl (Table 1; Figure 1). Different morphological and genetic attributes were analyzed across populations of V. jatamansi (Jugran et al 2013b; Jugran et al 2015). A total of 125 plants (5 plants/population) from 25 distant populations of V. jatamansi were subjected to detailed morphological analysis and various morphological attributes like leaf number, leaf area, plant height, above ground fresh weight (AGFW), below ground fresh weight (BGFW), above ground dry weight (AGDW),
below ground dry weight (BGDW), rhizome length (RL), and rhizome diameter (RD) were evaluated. Similarly, DNA was isolated from 151 genotypes belonging to 25 distant populations of V. jatamansi and polymerase chain reaction amplification was performed using 20 different inter simple sequence repeat primers. For phytochemical analysis plant material was collected from all 25 populations, washed thoroughly with tap water followed by washing with distilled water, and divided into two portions namely, aerial and root portion. Phytochemical (valerenic acid, total phenolics, flavonoids, and tannins) and antioxidant activity of all populations were investigated across the populations (Jugran 2013). Data drawn from different attributes (morphological, phytochemical, and genetic) were used for detailed analysis and prioritization of populations. Identification of promising sources/populations with higher morphological, phytochemical, and genetic attributes was performed using different sets of statistical procedures. Neighbor joining cluster analysis was performed using Paleontological Statistics Software Package for Education and Data Analysis packages, USA (Hammer et al 2001) for clustering of V. jatamansi populations based on all studied attributes. Neighbor joining clustering is considered an alternative method for hierarchical clustering analysis (Saitou and Nei 1987) for small data sets (Jianfu et al 2011). The data were further subjected for principal component analysis (PCA) to identify the major population groups with higher ranking of each attributes using SPSS version 16 (SPSS Inc. 1989e1996, Chicago, IL, USA).
Results Data pertaining to different morphological (leaf number, leaf area, plant height, AGFW, BGFW, AGDW, BGDW, RL, and RD), phytochemical (total phenolics, flavonoids, tannins, valerenic acid, and antioxidant activity), and molecular attributes (Nei’s genetic diversity and %polymorphism) recorded among targeted populations revealed wide variations among the populations (Jugran et al 2013a; Jugran et al 2015). Different populations behave differently for each attribute. For example, in the morphological
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Not to scale
Indian Himalayan Region (IHR)
NEPAL BHUTAN
78°
79 °
80 °
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Figure 1. Locality map of Valeriana jatamansi showing the distribution of 25 studied populations in Uttarakhand.
attributes, when considering RL (3.62 0.43 cm), the Surkunda population was the best whereas the Talwari population for RD (1.84 0.02 cm) was the best. Similarly, the Katarmal population was best for AGFW (8.88 1.72 g) and BGFW (4.70 1.87 g) but the Surkunda population for AGDW (3.36 0.98 g) and BGDW (1.57 0.52 g) was the best. Phytochemical analysis exhibited significantly higher valerenic acid content in Katarmal (aerial portion: 0.57 0.04%) and Joshimath populations (root portion: 1.80 0.12%); total phenolic content in the aerial [25.23 0.03 mg gallic acid equivalent (GAE)/g dry
weight (dw)] and root (27.54 0.06 mg GAE/g dw) portions of the Munsyari population; and flavonoid contents in the aerial [32.67 0.34 mg quercetin equivalent (QE)/g dw] and root (29.91 0.47 mg QE/g dw) portion of the Jaberkhet and Talwari populations. Ukhimath and Gwaldam populations exhibited significantly higher tannin content [aerial portion: 8.09 0.04 mg TAE/g dw; root portion: 7.91 0.60 mg tannic acid equivalent (TAE)/ g dw]. Significant difference (p < 0.05) was observed in antioxidant activity across the populations of V. jatamansi (aerial and root portion) measured by 2,20 -azino-bis(3-ethylbenzothiazoline-6-
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sulphonic acid), 2,2-diphenylpicrylhydrazyl, and ferric reducing antioxidant power assays. Different populations, such as the Didihat and Katarmal populations, showed maximum activity in aerial and root portions, respectively, using 2,20 -azino-bis(3ethylbenzothiazoline-6-sulphonic acid) activity. In the case of the 2,2-diphenylpicrylhydrazyl assay, the Talwari population exhibited the maximum [16.80 0.03mM ascorbic acid equivalent (AAE)/ 100g dw] antioxidant activity in the aerial portion, whereas the root portion of the Didihat population exhibited maximum antioxidant activity (17.53 0.04mM AAE/100g dw). The results using ferric reducing antioxidant power assay indicated that the population from Didihat showed significantly higher antioxidant activity in aerial portions and the Ukhimath population in root portions (12.71 0.04mM AAE/100g dw). Genetic diversity analysis based on inter simple sequence repeat marker analysis generated 159 clear and reproducible bands in
studied populations of V. jatamansi, and 125 of them were polymorphic. With the mean of 78.62%, the percentage of polymorphic loci ranged from 65.41% (Dwali) to 91.19% (Pithoragarh). Nei genetic diversity index ranged from 0.25 (Surkunda) to 0.37 (Pithoragarh) with a mean value of 0.31. Neighbor joining cluster analysis was performed for determining the relationship among different populations of V. jatamansi depending on morphological, phytochemical (in aerial and root portions), and genetic attributes. Neighbor joining cluster analysis based on morphological attributes placed Surkunda and Dunagiri in the top order (Figure 2A), based on phytochemicals, Gwaldam and Munsyari (Figure 2B), and based on molecular attributes Pithoragarh population (Figure 2C). Taking together all the attributes, Dunagiri and Tharali populations came up in the top order (Figure 2D). PCA based on the correlation matrix of morphological attributes across the populations grouped all populations into three
156 AK Jugran et al. / Journal of Asia-Pacific Biodiversity 9 (2016) 152e159 Figure 3. Projection of different populations of Valeriana jatamansi based on principal component analysis. A, morphological attributes; B, phytochemicals and antioxidant activity; C, genetic attributes based on genetic distance (Source: Jugran et al 2013); D, total studied attributes. Circles show clustering of populations with same attributes.
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major groups. Morphological attributes among all populations revealed 89.88% of the total variability in the first component and 8.36% in the second component while remaining variability is explained by Component 3 and Component 4 (Figure 3A; Table 2). Results indicated Katarmal, Nainital, and Munsyari as promising populations based on morphological attributes. PCA analysis revealed three major groups based on phytochemicals (valerenic acid, total phenols, and tannins) and antioxidant activity. Group I includes populations from Shitlakhet and Munsyari which showed high levels of all phytochemicals while Group II and Group III included all other remaining populations of V. jatamansi (Figure 3B; Table 3). PCA analysis based on Nei genetic similarity and distance matrix revealed three major groups of populations. Group I included the populations Berinag, Majkhali, Talwari, Didihat, Kausani, and Pithoragarh, which exhibited the highest genetic diversity compared with the remaining populations. These populations are considered a promising source harboring most of the variation residing within the species (Figure 3C). Taking together all the attributes, PCA analysis categorized all 25 populations into two major Groups; Group I includes Tharali, Berinag, Dunagiri, Joshimath, Dolti, and Surkunda populations whereas Group II includes other remaining populations of V. jatamansi (Figure 3D; Table 4).
Discussion Studies on morphological attributes across the populations are considered an important criterion to assess growth performance and biomass of the species. In the present study, Katarmal, Nainital, and Munsyari populations showed the best performance based on morphological attributes; therefore, these populations can be further utilized for mass multiplication to obtain a higher biomass of the species. Significant variations in morphological parameters among different populations might be due to differences in ecotypes, which are often indicative of genetic differences in many genes (Jugran 2013). However, a population with only higher
Table 2. Total variance explained using principal components of the morphological attributes of Valeriana jatamansi. Component Initial eigenvalues Total
Extraction sums of squared loadings
% of variance Cumulative % Total
1 22.47 89.88 2 2.09 8.36 3 0.38 1.52 4 0.05 0.22 5 0.01 0.02 6 0.00 0.01 7 0.00 0.00 8 0.00 0.00 9 0.00 0.00 10 0.00 0.00 11 0.00 0.00 12 0.00 0.00 13 0.00 0.00 14 0.00 0.00 15 0.00 0.00 16 0.00 0.00 17 0.00 0.00 18 0.00 0.00 19 0.00 0.00 20 0.00 0.00 21 0.00 0.00 22 0.00 0.00 23 0.00 0.00 24 0.00 0.00 25 0.00 0.00
89.88 98.24 99.75 99.97 99.99 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00
% of variance Cumulative %
22.47 89.88 2.09 8.36
89.88 98.24
157
Table 3. Total variance explained using principal components based on phytochemical and antioxidant activities in Valeriana jatamanai. Component Initial eigenvalues Total
Extraction sums of squared loadings
% of variance Cumulative % Total
1 20.21 80.83 2 2.04 8.17 3 0.95 3.79 4 0.71 2.86 5 0.45 1.78 6 0.20 0.80 7 0.17 0.69 8 0.14 0.55 9 0.06 0.25 10 0.04 0.17 11 0.02 0.08 12 0.01 0.03 13 0.00 0.01 14 0.00 0.00 15 0.00 0.00 16 0.00 0.00 17 0.00 0.00 18 0.00 0.00 19 0.00 0.00 20 0.00 0.00 21 0.00 0.00 22 0.00 0.00 23 0.00 0.00 24 0.00 0.00 25 0.00 0.00
80.83 89.00 92.78 95.64 97.42 98.22 98.91 99.46 99.71 99.88 99.96 99.99 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00
% of variance Cumulative %
20.21 80.83 2.04 8.17
80.83 89.00
growth performance cannot be considered elite as it should possess a higher amount of the major active ingredient along with maintaining its genetic diversity, which can be utilized for future breeding programs of V. jatamansi. Considering the availability of phytochemical contents, the present investigation revealed that the mean values for total phenolics and tannins in V. jatamansi were considerably higher compared with earlier reports on the same
Table 4. Total variance explained using principal components base on morphological, phytochemical, and genetic attributes (polymorphic loci % and Nei genetic diversity index only). Component Initial eigenvalues Total
Extraction sums of squared loadings
% of variance Cumulative % Total
1 24.12 96.48 2 0.28 1.11 3 0.18 0.71 4 0.12 0.48 5 0.10 0.39 6 0.07 0.29 7 0.04 0.16 8 0.03 0.13 9 0.02 0.08 10 0.02 0.06 11 0.01 0.04 12 0.01 0.04 13 0.00 0.01 14 0.00 0.01 15 0.00 0.00 16 0.00 0.00 17 0.00 0.00 18 0.00 0.00 19 0.00 0.00 20 0.00 0.00 21 0.00 0.00 22 0.00 0.00 23 0.00 0.00 24 0.00 0.00 25 0.00 0.00
96.48 97.59 98.30 98.79 99.17 99.46 99.62 99.75 99.83 99.89 99.93 99.97 99.98 99.99 99.99 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00
% of variance Cumulative %
24.12 96.482 0.277 1.109
96.482 97.591
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AK Jugran et al. / Journal of Asia-Pacific Biodiversity 9 (2016) 152e159
species (Bhatt et al 2012) and other related species of the family, e.g. Nardostachys jatamansi and V. officinalis (Surveswaran et al 2007). Although it was lower than the value reported by Kalim et al (2010). Similarly, antioxidant activity reported in the present study (Jugran et al 2015) by all three assays was higher compared with other plants like N. jatamansi and V. officinalis. Reports are also available on the antioxidant activity in extracts and essential oils of V. jatamansi (Kalim et al 2010; Das et al 2011; Bhatt et al 2012; Jugran et al 2013b). However, the reason for variation in antioxidant activity (either lower or higher) could be due to different genetic backgrounds and sources of samples. Various factors such as harsh climatic conditions (light, chilling, pollution, etc.; Valentine et al 2003) and environmental stresses (Pennycooke et al 2005; Close and McArthur 2002; Reddy et al 2004) are reported to be responsible for these variations. Further studies on valerenic acid quantification in V. jatamansi (Singh et al 2006) and V. officinalis (another species of same family) have been performed (Bos et al 1998; Singh et al 2006; Ebrahimzadeh et al 2008; Wills and Shohet 2009). The reason for variation in phytochemical content might be the increased phenylalanine ammonia lyase activity. Phenylalanine ammonia lyase is a key and regulating enzyme in the secondary metabolic pathway leading to the synthesis of phenolic compounds (Jones 1984). Genetic diversity is of fundamental importance for the adaptation of the species to future environmental changes. Highest genetic diversity in the Pithoragarh population (1872 m asl) and lowest genetic diversity in the Dwali (2730 m asl) population indicates that there is limited genetic diversity of V. jatamansi at higher altitudes. Various factors like the inherent breeding system mode of reproduction, pollination, seed dispersal, stylar movement (Khajuria et al 2011; Jugran et al 2013a), and ploidy level (Soltis and Soltis 2000) in the species is reported to be responsible for genetic variations and contributes to the maintenance of high genetic diversity. Morphological, phytochemicals, antioxidant activity, and genetic attributes showed that the Tharali, Dunagiri, and Munsyari populations are the most promising sources and can be designated as the best morphotype/chemotype/genotypes of V. jatamansi. Morphological and genetic attributes were, for the first time, used in this study for prioritization; however, studies are available on chemotyping of the species based of different chemical attributes. For example, two chemotypes of V. jatamansi based on essential oil composition were identified, one with patchouli alcohol as a major compound and the other with maaliol as a major compound (Mathela et al 2005). Recently, 16 accessions of V. jatamansi using essential oil compounds were classified in three chemotypes (Sundaresan et al 2012). Chemotypic variability in various other species based on essential oil composition have also been reported, e.g. Origanum vulgare (Mockute et al 2001), Hippophae rhamnoides (Tian et al 2004), Lychnophora ericoides (Curado et al 2006), Liptospermum scorparium (Douglas et al 2004), Anemopsis californica (Medina et al 2008), and Artemisia dracunculus (Chauhan et al 2010). Four (phytochemical based) and three (antioxidant activity based) populations identified in this study could be due to a large sample size of the species, which covered almost the entire Uttarakhand area compared with the previous one. This can be used for classifying and selecting populations based on different compounds. These populations could be further explored for focusing a particular group on chemical production and to obtain the desired phytochemicals with maximized yield. The present study suggests that screening of multilocational plants/populations for different attributes (morphological, phytochemical, antioxidant, and genetic) is essential for obtaining promising stock containing optimum levels of these attributes. Based on neighbor joining and PCA, Tharali and Dunagiri populations are found best for all studied attributes. This is the first
report of prioritization of promising populations based on different parameters and multilocational sample collection; this may promote the interest of researchers for further investigation of the species. Similarly, promising populations (Tharali and Dunagiri) identified by all studied attributes harbor maximum genetic variation within the species. Therefore, cultivation and crop improvement under the plant breeding program of the populations from these areas can be recommended. These populations could be used as a promising stock for mass multiplication of the target species and can be informative for conservation purposes. As such, protection of a particular population with high genetic variation is unlikely to protect all the variation in the species. Therefore, several populations with diverse locations from the entire range should be considered for conservation. However, it is not practically feasible to conserve all populations; therefore, conserving populations that harbor maximum variations in all aspects could be a viable option. As the population size of V. jatamansi is gradually reducing due to habitat fragmentation by human disturbance or natural calamities (Alyemeni and Sher 2010), this might contribute to population extinction and loss of genetic variation. Therefore, desired clones or populations can be mass multiplied and used for long-term use, exploration, and conservation. Acknowledgments We thank Dr P.P. Dhyani, Director G. B. Pant Institute of Himalayan Environment and Development (GBPIHED) for facilities and encouragement. Colleagues of Biodiversity Conservation and Management Theme are thanked for their cooperation and help during the study. Financial support from the SERB-DST-FTYS (NO. SB/YS/LS-162/2013) scheme is acknowledged. References Allendorf FW, Hohenlohe PA, Luikart G. 2010. Genomics and the future of conservation genetics. Nature Reviews Genetics 11:697e709. Alyemeni MN, Sher H. 2010. Impact of human pressure on the population structure of Persicaria amplexicaule, Valeriana jatamansi and Viola serpens the naturally growing medicinal plants in Malam Jaba, Swat, Pakistan. Journal of Medicinal Plant Research 4:2080e2091. Bhatt ID, Dauthal P, Rawat S, et al. 2012. Characterization of essential oil composition, phenolic content, and antioxidant properties in wild and planted individuals of Valeriana jatamansi Jones. Scientia Horticulture 136:61e68. Bos R, Woerdenbag HJ, Van Putten FMS, et al. 1998. Seasonal variation of the essential oil, valerenic acid and derivatives, and valepotriates in Valeriana officinalis roots and rhizomes and the selection of plants suitable for phytomedicines. Planta Medica 64:143e147. CAMP. 2003. Conservation assessment and management prioritization: For the medicinal plants of Himachal Pradesh, Jammu & Kashmir and Uttarakhand. Proceedings of the Workshop held at Shimla. Bangalore, India: Hosted by Foundation for Revitalization of Local Health Traditions (FRLHT). Chauhan RS, Kitchlu S, Ram G, et al. 2010. Chemical composition of Capillene chemotypes of Artemissia dracunculus L. from North West Himalaya. Industrial Crops and Products 31:546e549. Close DC, McArthur C. 2002. Rethinking the role of many plant phenolicsdprotection from photodamage not herbivores? Oikos 99:166e172. Curado MA, Carolina BA, Oliveira JGJ, et al. 2006. Environmental factors influence on chemical polymorphism of the essential oils of Lychnophora ericoides. Phytochemistry 67:2363e2369. Das J, Mao AA, Handique PJ. 2011. Terpenoid composition and antioxidant activities of two Indian Valerian oils from the Khasi Hills of north- east India. Natural Products Community 6:129e132. Douglas MH, Van Klink JW, Smallfield BM, et al. 2004. Essential oils from New Zealand manuka: triketone and other chemotypes of Leptospermum scoparium. Phytochemistry 65:1225e1264. Ebrahimzadeh H, Radjabian T, Ekhteraei Tousi S, et al. 2008. Evaluation of some Iranian wild species from Valerianaceae as commercial sources of valepotriates. Journal of Biological Science 8:549e555. Hammer O, Harper DAT, Ryan PD. 2001. PAST: Paleontological statistics software package for education and data analysis. Palaeontologia Electronica 4:1e9. Jianfu LI, Jianshuang LI, Huaiqing HE. 2011. A simple and accurate approach to hierarchical clustering. Journal of Computer Information Systems 7:2577e2584. Jones DH. 1984. Phenylalanine ammonia-lyase: regulation of its induction, and its role in plant development. Phytochemistry 23:1349e1359.
AK Jugran et al. / Journal of Asia-Pacific Biodiversity 9 (2016) 152e159 Jugran AK, Bhatt ID, Rawal RS, et al. 2013a. Patterns of morphological and genetic diversity of Valeriana jatamansi Jones in different habitats and altitudinal range of West Himalaya, India. Flora-Morphology, Distribution, Functional Ecology of Plants 208:13e21. Jugran A, Rawat S, Dauthal P, et al. 2013b. Association of ISSR markers with some biochemical traits of Valeriana jatamansi Jones. Industrial Crops and Products 44: 671e676. Jugran AK. 2013. Fingerprinting phytochemical and genetic variations in Valeriana jatamansi and V. himalayana in Uttarakhand. Nainital, Uttarakhand, India: Kumaun University. PhD Thesis (in Biotechnology). Jugran AK, Bhatt ID, Rawal RS. 2015. Identification of ISSR markers associated with valerenic acid and antioxidant activity in Valeriana jatamansi Jones in Western Himalaya. Molecular Breeding 35:73. http://dx.doi.org/10.1007/s11032-015-0241-5. Kalim MD, Bhattacharya D, Banerjee A, et al. 2010. Oxidative DNA damage preventive activity and antioxidant potential of plants used in Unani system of medicine. BMC Complementary and Alternative Medicine 10:77. Khajuria A, Verma S, Sharma P. 2011. Stylar movement in Valeriana wallichii DC.da contrivance for reproductive assurance and species survival. Current Science 100:1143e1144. Mathela CS, Sati S, Chanotiya CS. 2005. Comparative investigations of the leaf and root oils of Valeriana wallichii DC from Northwestern Himalaya. Journal of Essential Oil Research 17:408e409. McNeely JA, Miller KR, Reid WV, et al. 1990. Conserving the World’s Biological Diversity. Washington, DC: IUCN, World Resources Institute, Conservation International, WWF-US, World Bank. Medina HA, Holguin OF, Micheletto S, et al. 2008. Chemotypic variation of essential oils in the medicinal plant, Anemopsis californica. Phytochemistry 69:919e927. Mockute D, Bernotiene G, Judzentiene A. 2001. The essential oil of Origanum vulgare L spp. Vulgare growing wild in Vilnius district (Lithuania). Phytochemistry 57: 65e69. Oliver I, Holmes A, Dangerfield JM, et al. 2004. Land systems as surrogates for biodiversity in conservation planning. Ecological Applications 14:485e 503.
159
Pennycooke JC, Cox S, Stushnoff C. 2005. Relationship of cold acclimation, total phenolic content and antioxidant capacity with chilling tolerance in Petunia. Environmental and Experimental Botany 53:225e232. Prakash V. 1999. Indian Valerianaceae a monograph on medicinally important family. Jodhpur, India: Scientific Publishers. Reddy AR, Chaitanya KV, Vivekanandan M. 2004. Drought-induced responses of photosynthesis and antioxidant metabolism in higher plants. Journal of Plant Physiology 161:1189e1202. Saitou N, Nei M. 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Molecular Biology and Evolution 4:406e425. Singh N, Gupta AP, Singh B, Kaul VK. 2006. Quantification of valerenic acid in Valeriana jatamansi and Valeriana officinalis by HPTLC. Chromatographia 63: 209e213. Singh RD, Gopichand Meena RL, Sharma B, et al. 2010. Seasonal variation of bioactive components in Valeriana jatamansi from Himanchal Pradesh, India. Industrial Crops and Products 32:292e296. Soltis ED, Soltis PS. 2000. Contributions of plant molecular systematic to studies of molecular evolution. Plant Molecular Biology 42:45e75. Sundaresan V, Shani G, Verma RS, et al. 2012. Impact of geographic range on genetic and chemical diversity of Indian Valerian (Valeriana jatamansi) from northwestern Himalaya. Biochemical Genetics 50:797e808. Surveswaran S, Cai Y, Corke H, et al. 2007. Systematic evaluation of natural phenolic antioxidants from 133 Indian medicinal plants. Food Chemistry 102:938e953. Tian C, Nan P, Chen J, et al. 2004. Volatile composition of Chinese Hippophae rhamnoides and its chemotaxonomic implications. Biochemical Systematics and Ecology 32:431e441. Valentine IK, Maria VK, Bruno B. 2003. Phenolic cycle in plants and environment. Journal of Molecular Cell Biology 2:13e18. Vucetich JA, Peterson RO, Nelson MP. 2013. Discernment and precaution: A response to Cochrane and Mech. The George Wright Forum 30:333e340. Wills RBH, Shohet D. 2009. Changes in valerenic acids content of valerian root (Valeriana officinalis L. s.l.) during long-term storage. Food Chemistry 115: 250e253.