Journal of Genetics (2019)98:100 Ó Indian Academy of Sciences

https://doi.org/10.1007/s12041-019-1147-5 (0123456789().,-volV)(0123456789().,-volV)

RESEARCH ARTICLE

Narrow gene pool can threaten the survival of nagbettai R. R. Fernald & Dey: a highly, endemic dioecious species in the Western Ghats of India

SUMA ARUN DEV1* , SWATHI BALAKRISHNAN1, ANOJA KURIAN1 and V. B. SREEKUMAR2

1Forest Genetics and Biotechnology Division, Kerala Forest Research Institute, Peechi, Thrissur 680 653, India 2Forest Ecology and Biodiversity Conservation Division, Kerala Forest Research Institute, Peechi, Thrissur 680 653, India *For correspondence. E-mail: [email protected].

Received 21 January 2019; revised 6 August 2019; accepted 16 August 2019

Abstract. , the spiny climbing palms of (Palmae) family exhibit high endemism to the biodiversity hot spots in India. Of the five rattan genera, Calamus is the only found in peninsular India with 15 of 21 species, endemic to the Western Ghats. The extensive utilization of rattans owing to their strength, durability and huge demand has resulted in depletion of their natural resources. Of the 15 endemic species, C. nagbettai is the most affected species on account of endemism, low population size and restricted distribution with fragmented populations. The present study revealed high amount of genetic diversity in the surviving scattered populations of the species using microsatellite markers. High gene flow (Nm = 1.498) observed across the populations resulted in low genetic differentiation (14%). A clear genetic admixture could be seen in Kerala as well as one of the Karnataka’s populations while the remaining two populations were genetically distinct. UPGMA, PCoA and STRUCTURE analyses showed significantly different genetic composition in Kerala population compared to other populations. Kerala and Karnataka populations of C. nagbettai were also unique in their genetic structure and allelic composition. Therefore, effective management and conservation strategies have to be implemented to preserve the rare alleles with adaptive potential to protect this economically valuable Calamus species from endangerment. Overexploitation, low seed set and poor regeneration, as well as habitat fragmentation can further threaten the survival of this endemic, narrowly distributed dioecious rattan species in the Western Ghats region.

Keywords. rattans; overexploitation; simple sequence repeat; genetic diversity; conservation.

Introduction Western Ghats and the Andaman and Nicobar Islands. Of all rattans, genus Calamus alone occurs in peninsular India, with a Rattans are spiny climbing palms with scaly fruits representation of 21 with 15 species endemic to the Western belonging to the family Arecaceae (Palmae). It comprises Ghats (Renuka 1992, 1999; Ravikanth et al. 2002;Uma of about 650 species in 22 genera distributed around the Shaanker et al. 2004). Kodagu region of the Western Ghats world and of which, Calamus is the largest genus with 375 harbours nine species of rattans with five endemic species (Uma species (Uhl and Dransfield 1987;Govaertset al. 2015). In Shaanker et al. 2004). Climate, topography and edaphic factors India, rattans are represented by five genera, namely Cala- play a vital role in phenological behaviour of the dioecious wind mus, Plectocomia, , and Salacca pollinating Calamus species (Manohara et al. 2007). with a total of about 60 species (Basu 1992; Renuka Rattans are highly treasured for their economic and 1992, 1995; Uma Shaanker et al. 2004). Rattans are highly medicinal values. They are one of the major nontimber endemic with 43 of 60 species endemic to India and are forest products (NWFPs) in South and Southeast Asia mainly distributed in three regions, namely northeast India, (Lyngdoh et al. 2005). In Southeast Asia, approximately half a million population are engaged directly in rattan All authors contributed equally to this work. trade(UmaShaankeret al. 2004). The global trade in 100 Page 2 of 10 Suma Arun Dev et al. rattans approximates to USD two billion, a year (Rattan- such as Korthalsia, Daemonorops, Salacca and Calamus land 2011). Calamus species owing to its high strength, (Rao et al. 2007). flexibility and durability are extensively considered for The present study aims at identifying the genetic diversity furniture making, handicrafts and sports articles. They are status of C. nagbettai, an endemic peninsular rattan species, also used in Ayurvedic systems of medicine for the treat- for effective conservation and management of the available ment of various diseases like cold, rabies, among others gene pool. (Lakshmana 1993). Calamus oil extracted from the root is used for perfuming and flavouring liquors, and tender shoots of some species like C. erectus Roxb.,C.flori- Materials and methods bundus Griff. and C. latifolius Roxb. are edible (Singh et al. 2004). Population sampling and DNA extraction Of the 15 endemic species of Calamus in the Western Ghats, C. nagbettai regionally known as ‘nagbetha’ is one All available mature individuals of C. nagbettai from the of the endemic rattans confined to the Karnataka and Kerala only existing natural populations representing two geo- regions of the Western Ghats. This species is considered graphic areas (Kerala and Karnataka) of the Western Ghats sacred and worshipped in Karnataka and is mainly found in were sampled namely Subramanya (nine individuals), Kar- Subramanya forests of Dakshina Kannada district and also ikke (nine individuals), Charmadi (15 individuals) from parts of Hassan and Kodagu districts of Karnataka in Karnataka part and Umayar (15 individuals) from Kerala central Western Ghats. The species is highly threatened part (table 1). Fresh leaves were collected and dried on silica owing to their high economic and aesthetic values (Uma gel. Total DNA was extracted from 20 mg of leaf tissue Shaanker et al. 2004;Rameshaet al. 2007). In Kerala, using the modified Cetyl trimethyl ammonium bromide distributionofthespeciesishighlyrestrictedwithonlyfew (CTAB) method (Doyle and Doyle 1987). surviving individuals in the Umayar forest (Renuka and Sreekumar 2012). C. nagbettai is a high climbing, robust clustering rattan around 25-m long with cirrate leaf and SSRs attains reproductive maturity in about 16–20 years (direct observation-KFRI Palmetum). The sheaths are yellowish Fourteen microsatellite loci reported from C. simplicifolius green to green with spiny lower half. Male and female (Li et al. 2013) were considered for the present study inflorescences are up to 30 cm and 70 cm long, respec- (table 2). The forward primers were labelled with a flour- tively. Fruits are oblong–ovate with ruminate endosperm. ochrome (6-FAM). Polymerase chain reaction (PCR) was Destruction of habitat, change of land use pattern and forest performed using 20 lL reaction containing 50–100 ng DNA, fire along with increase in demand for raw material has 10x Taq buffer with 1.5 mM MgCl2, 200 lM dNTPs, 10 pm resulted in overexploitation and consequent erosion of of each primer and 2 U Taq DNA polymerase (Invitrogen, diversity in C. nagbettai. Endemism, low population size Bengaluru). The PCR reaction conditions included an initial and restricted distribution limits the genetic diversity and denaturation at 95°C for 5 min, followed by 35 cycles at fitness of the surviving individuals and the species weigh 94°C for 45 s, specific annealing temperatures for 60 s up as ‘vulnerable’ in the Red Data Book (Ahmedullah and (table 2), an extension period at 72°C for 45 s, followed by a Nayar 1986; Ravikanth et al. 2010). final extension period at 72°C for 10 min. SSR genotyping Among the available genetic markers, simple sequence was performed using ABI 3730XL sequencer (Applied repeats (SSRs) or microsatellites are codominant, locus- Biosystems) with an internal size standard of Gene Scan specific, ubiquitous, experimentally reproducible, multiple 500LIZ (Applied Biosystems). The SSR allele size was allele markers with high amount of polymorphism (Selkoe evaluated using GENEMAPPER software v4.0 (Applied and Toonen 2006; Zhang et al. 2014; Harris-Shultz et al. Biosystems). 2014; Chen and Okie 2015; Kumbhar et al. 2015; Mason 2015). SSR markers have been particularly used to distin- guish between closely related genotypes owing to their high degree of variability making them ideal for population Table 1. Details of C. nagbettai population in the Western Ghats. genetics (Smith and Devey 1994). SSR markers were employed to analyse genetic diversity and population No. of Longitude structure of palms such as Pheonix dactylifera L. (Billotte Locality samples Latitude (N) (E) et al. 2004; Elshibli and Korpelainen 2009), Elaeis Umayar, Kerala 15 8°52001.200N77°13008.100E guineensis Jacq. (Billotte 2001), Cocos nucifera L. (Perera Subramanya, 912°40013.200N75°37015.800E et al. 2000; Teulat et al. 2000; Meerow et al. 2003), Cala- Karnataka mus thwaitesii Becc. (Kurian et al. 2013) and C. simplici- Karikke, Karnataka 9 12°26027.900N75°25044.700E 0 00 0 00 folius Wei. (Li et al. 2013). Cross-species amplification of Charmadi, 15 13°03 56.0 N75°26 37.1 E Karnataka SSR markers was recently reported in four genera of rattans Endemic dioecious rattan species in the Western Ghats of India Page 3 of 10 100

Table 2. Microsatellite markers used for PCR amplification (Li et al. 2013).

Locus Repeat motifs Primer sequence Annealing temperature (°C)

CALeSSR080 (GA)6 F:(6-FAM)GCCTTTCCTCCACCTTGC 57.3 R:ATGCCACTGGTTGCCTCT CALeSSR082 (TC)5TT(TA)8 F:(6-FAM)ACAATCGAATGTAGCCAAGT 56.2 R:AGGCCCGGAAATAATGAA CALeSSR083 (GAGC)4 F:(6-FAM)GGGCCTTGGATAGCGTCTT 57.5 R:ACGGGTGAAGGAAACCAAAGAG CALeSSR084 (TCCTGA)4 F:(6-FAM) GCCTTCTTCTCGCTTCCT 57.5 R:TTCAACGGCTCATCCACT CALeSSR094 (CAG)8 F:(6-FAM)TCAGCCCAAGCCAACTCC 60 R: CGGGTGGGTAACGTAATGGT CALeSSR095 (CT)11 F(6-FAM)TATTAGTAGTGGCTTCAGTTCC 55.8 R: ACCAGACGCCCGATCCTT CALeSSR098 (TCC)8 F:(6-FAM)ATGGCGAACGAATGAGTG 54 R:ACATCGGCGTAGACCTGA CALeSSR099 (TGAGAA)4 F:(6-FAM)CCCCACAGACTGAAAGAA 58 R: TAGACTGCTCCACTCCAA CALeSSR100 (TACCA)3 F:(6-FAM)ATGGTTGGGAGTGAGAAA 53 R:TTAGCGTGAGGTCTGAAA CALeSSR102 (TG)8 F:(6-FAM)ACGCCGGTCCTCAAATCT 64 R:TTGGTGGGGGCTTGGAAA CALeSSR103 (GCA)5 F:(6-FAM)ACAGGCTTATCCACAACA 59 R: TACCAGCTAACACTCAGAAC CALeSSR105 (AGA)4 F:(6-FAM) GGCTACTGCTTGGTGCTT 49.8 R:TCTCCTGCGTTACCTCAT CALeSSR106 (TC)8 F:(6-FAM)GCGTTTCTTCTTCCTCGTT 53.8 R: GCCTGCCCACTGTTGTAG CALeSSR107 (TC)7 F:(6-FAM)TTTAGAGGGAGCGAAGCC 61 R:CTGCACCACCGAAGTCAA

Data analysis 25 independent runs for each simulated values of K. Optimal K value was determined using stimulation method The polymorphic loci were analysed for assessing the (Evanno et al. 2005) in the software Structure Harvester genetic diversity. Genetic diversity among four populations vA.2 (Earl 2012). Outputs of 25 iterations at K 1-6 were was estimated using the following parameters, namely run in CLUMPP v.1.1.2 (Jakobsson and Rosenberg 2007) observed number of alleles (Na), effective number of alleles to align clustering results, and visualized using DISTRUCT (Ne), number of private alleles (Np), observed heterozygosity v.1.1 (Rosenberg 2004). (Ho), expected heterozygosity (He), Shannon’s genetic diversity index (I) (Lewontin 1972), genetic differentiation coefficient (Fst), gene flow (Nm), Nei’s genetic distance (Gd), Result principal component analysis (PCoA) and Mantel test using GenALEx 6 software (Peakall and Smouse 2006). Unbiased Genetic diversity status genetic distance was used to construct dendrogram using unweighted pair group arithmetic mean with arithmetic Of the selected 14 SSR primers, two were found to be average (UPGMA) as implemented in POPGENE v1.3.1 monomorphic and others were polymorphic. The number of (Yeh et al. 1999) and Power Marker v3.25 (Liu and Muse observed alleles (Na) per locus ranged from 2.00 to 3.625 2005). with a mean of 2.656. Effective number of alleles (Ne) was Allelic profile data was analysed to determine subpop- 1.837 and ranged from 1.539 to 2.455. Shannon’s informa- ulations of C. nagbettai genotypes, using an admixture tion index (I) ranged from 0.424 to 0.887 with an average of model in the Bayesian analysis tool Structure v2.3. This 0.573. Average observed and expected heterozygosity were was used to analyse the separation of total individuals into 0.323 and 0.310, respectively (table 3). A total of 25 private clusters (K) that represent the number of gene pools alleles (Np) were identified. Genetic diversity was highest in (Pritchard et al. 2010). A burn-in period of 105 and 106 the Umayar (Kerala) and lowest in the Karikke (Karnataka) Markov chain Monte Carlo (MCMC) iterations was set populations. Percentage of polymorphic loci was found to be with an admixture model. For each run, Hardy–Weinberg around 65.63%. equilibrium, correlated allele frequency and independent The genetic differentiation among populations was mea- loci were used. K values were set from 1 to 6 followed by sured by Fst analysed by AMOVA. The pair-wise Fst 100 Page 4 of 10 Suma Arun Dev et al.

Table 3. Population diversity parameters in the existing populations of C. nagbettai.

Population Na Ne Ho He IFFst

Umayar 3.625 2.455 0.324 0.465 0.887 0.301 Subramanya 2.125 1.694 0.389 0.281 0.482 -0.389 Karikke 2.000 1.660 0.33 0.244 0.424 -0.352 Charamadi 2.875 1.539 0.228 0.252 0.500 0.095 Mean 2.656 1.837 0.323 0.310 0.573 -0.094 0.143

Na, number of different alleles; Ne, number of effective alleles; Ho, observed heterozygosity; He, expected heterozygosity; I, Shannon’s information index; F, fixation index; Fst, inbreeding coefficients among populations. estimated ranged from 0.000 to 0.206, with an average of 0.143 showing 14% of variation among populations and 86% within populations. High gene flow (Nm = 1.498) was also observed between the populations.

Genetic relationship and population structure

The optimal K value was found to be K = 2 (figure 1), indicating that the two subpopulations showed highest probability of population clustering. A total of 48 genotypes were inferred and assigned into two subpopulations A and B (figure 2). Admixture of genotypes was found in Karnataka (Charmadi) as well as Kerala populations (Umayar). The genotypes of Umayar population (Kerala) clearly differed from the other three populations (Subramanya, Karikke and Charmadi-Karnataka) in their genetic admixture pattern. PCoA distribution of four different populations of C. nag- Figure 1. Bayesian assignment analysis with K = 2. bettai clustered Karnataka populations together separating Kerala population (figure 3). The first co-ordinate axis in the (Hamrick et al. 1991; Karron 1991; Ellstrand and Elam PCoA analysis accounted for 96.53% of the variance while 1993) and are more prone to extinction (Barrett and Kohn second accounted for 3.47%. 1991). However, outcrossing species are inclined to possess Genetic grouping of the analysed individuals (genotypes) high level of genetic diversity (Hamrick et al. 1992; Bartish constructed using Power Marker v3.25 showed a clear et al. 1999). Although the studied rattan species is endemic overlap between populations and the separation was also in to Western Ghats, their dioecious nature of flowering ensures accordance with PCoA and Structure analysis (figure 4). cross fertilization and maintains high levels of genetic UPGMA tree constructed using Popgene formed two major diversity (Renuka et al. 1998). High level of genetic diver- clusters: Karnataka populations clustered together very dis- sity (I = 0.341) with moderate level of genetic differentia- tinctly from the Kerala population. Karnataka populations tion (0.184) was earlier reported in the endemic rattan were further divided into two clusters; Subramanya and species C. garuba in (Meena et al. 2018). The Karikke, being much more similar were placed in a single percentage of total genetic differentiation (18. 4%) among clade sister to Charmadi (figure 5). Mantel test performed to the four populations is only slightly above the mean Gst infer correlation between genetic distance and geographical value (0.129) reported in many other tropical species distance showed positive correlation but with a very low R2 (Hamrick et al. 1992). Similarly, high levels of polymor- value of 0.0047 (figure 6). phism, as observed (65.63%) in the present study, is also suggestive of the outcrossing nature of the resident geno- types. High percentage of polymorphic loci were reported in Discussion other rattan species like C. manan (66.67–76.67%) in Sumatra, 70.5% in C. subinermis in Malaysia (Bon 1995), C. Some of the key factors influencing genetic diversity within thwaitesii (82.77%) (Ravikanth et al. 2001) and C. met- population is the viable population size, mode of reproduc- zianus (95%) (Sreekumar et al. 2006) in India. As per the tion, pattern of seed dispersal and its geographical distribu- reports, genetic polymorphism detected in calamoid palms tion (Hamrick et al. 1991). Endemic species’ with very can be compared to that of other allogamous and perennial narrow distribution have generally low genetic diversity species (Wachira et al. 1995; Forapani et al. 2001; Kapteyn Endemic dioecious rattan species in the Western Ghats of India Page 5 of 10 100

Figure 2. Population structure pattern of four populations of C. nagbettai using DISTRUCT for K = 2 and K = 4.

Figure 3. Two dimensional plot of PCoA of four C. nagbettai populations.

and Simon 2002; Li and Luukkanen 2001; Sinha et al. Karnataka (Subramanya and Karikke) populations also 2018). Average Shannon diversity index of the species was indicates presence of maximum heterozygotes within the also high (0.573) suggesting high gene diversity of C. population. Similarly, negative fixation index was reported nagbettai indicating high fitness of the small populations. by Kurian et al. (2018a) in all the seven economically Similar inference was made in the small population of C. important Calamus species studied. Most of the loci anal- brandisii with moderate to high genetic diversity (Kurian ysed in the Subramanya and Karikke populations also et al. 2018a). deviated from Hardy–Weinberg equilibrium suggesting a The populations had observed heterozygosity ranging positive selection of heterozygotes mostly to cope up with from 0.228 to 0.389 suggesting high genetic variation within changes in fragmented population (Kang et al. 2005). population. C. nagbettai showed mean Ho value of 0.325 Moreover, these two populations also lack rare alleles indi- compared to that reported by Kurian et al. (2018a)in cating loss of alleles. Presence of few to no rare alleles could Calamus species like C. brandisii, C. gamblei, C. hookeri- be an aftermath of fragmentation as fragmented population anus, C. metzianus, C. thwaitesii, C. travancoricus and C. experience stochastic loss of alleles owing to the presence of nagbettai. Negative fixation index value of the two a small portion of gene pool (Young et al. 1996). Habitat 100 Page 6 of 10 Suma Arun Dev et al.

Figure 4. UPGMA dendrogram individuals per population clustering pattern using Powermarker (Can, Umayar; CNb, Subramanya; CNc, Karikke; CNd, Charmadi).

fragmentation could lead to the consequent decline in allelic differentiation among populations (14%) and high variation richness rather than reduction of heterozygotes (Kang et al. within the population (86%). Since Nm was greater than 1.0, 2005) as evident from the present study. the populations are expected to maintain genetic stability Study of gene flow is essential to deduce the level of over time (Lowe et al. 2004; Zong et al. 2015; Matesanz genetic differentiation among and between the populations et al. 2017). Assessment of genetic diversity in C. vattayila (Gerber et al. 2014). According to McDermott and reported restricted gene flow or inbreeding (Priya et al. McDonald (1993) Nm [ 1 indicates little differentiation 2016). High gene flow was reported in Centaurea hyssopi- among population and migration more than genetic drift. folia, a narrow endemic gypsophile with fragmented distri- Further, movement of at least a single individual per gen- bution (Matesanz et al. 2017). Gene flow is mainly mediated eration can prevent significant divergence between popula- through pollen and seed dispersal. In the genus Calamus, tions (Wright 1969). C. nagbettai populations showed high birds and small mammals are reported as the main agents for gene flow (Nm = 1.498) because of it being dioecious and seed dispersal (Renuka 1995). However, information on outcrossing (Loveless and Hamrick 1984; Selkoe and Too- pollination, seed dispersal and fruiting of C. nagbettai nen 2006). High gene flow indicates low genetic specifically is not known. Here the high gene flow between Endemic dioecious rattan species in the Western Ghats of India Page 7 of 10 100

changing specific allele frequencies (Brown 1992). Further, no information is available on the origin of the C. nagbettai species in either of the geographical areas. But, the popu- lation structure revealed similar genetic makeup among the individuals in Karnataka populations and remarkably dif- ferent genetic makeup of Kerala population thereby dividing it into two diverse populations. The study also revealed a positive correlation between genetic and geographic dis- tances albeit a very low R2 value (0.0047), indicating neg- ligible or insignificant role of geographic distance in determining genetic structure of population. Although all four surviving populations of C. nagbettai showed high gene diversity, their fragmented populations with a very low seed set and regeneration rate are major concerns. Lower regen- eration of endemic species may be attributed to their restricted distribution, reproductive constraints and low competitive ability (Gitzendanner and Soltis 2000; Gibson Figure 5. UPGMA dendrogram based population clustering et al. 2008). According to Manohara et al. (2007) in certain pattern. canes the phenology, maturity and receptivity of stigma and production of pollen grains are affected by adverse climatic factors which could directly influence the seed set and nat- ural regeneration. Further, low seed set may be due to inadequate pollen discharge for fertilization, probably because of inappropriate male–female sex ratio within the population (Agren et al. 1986; Elmqvist et al. 1993; House 1992; Carlsson-Graner et al. 1998). Natural distribution of rattans in the Western Ghats is patchy, mostly due to heavy exploitations of the species and changes in land use pattern or by human interference (Chandran 1997). Apart from being economically important, they are endemic with a very narrow distributional range and Figure 6. Relationship between genetic distance and geographic distance of the four C. nagbettai populations. high gene diversity. The few surviving genotypes in the existing populations may drastically decline the evolutionary potential of the species. This may affect long-term survival the populations may be due to the human mediated intro- and extinction of the species if there is no timely restoration gression of genotypes in the past to enrich the available of genotypes (Gilpin and Soule` 1986; Jump and Penuelas germplasm of this endemic rattan resources. 2005; Willi et al. 2006). Recently, a study on rattans of the Structure analysis placed individuals from the four pop- Western Ghats identified natural distribution zones and ulations into two subpopulations A and B. Umayar popu- conservation values of Calamus species along with habitat lation (Kerala) had maximum individuals belonging to restoration of 15 endemic rattan species (Joshi 2017). The subpopulation A, whereas Charmadi population had maxi- developmental activities in the Western Ghats region is mum individuals belonging to subpopulation B while pointed out as the reason for loss of habitat (Jha et al. 2000), Subramanya and Karikke had almost all individuals which can equally threaten the survival of these endemic, belonging to subpopulation B. Two Karnataka populations narrowly distributed rattan species. In order to protect and (Karikke and Subramanya) showed a similar pattern with no preserve economically valuable Calamus species, effective or very little admixture. Dendrogram and PCoA analysis strategies for conservation and management have to be also clustered these two populations. Similar patterns were implemented. Further, knowledge of male–female sex ratio derived using ISSR markers in the case of C. thwaitessi could aid in the long-term survival of established planta- (Sreekumar and Renuka 2006) and C. guruba (Meena et al. tions, thereby emphasizing the need for early sex determi- 2018). Admixture of genotypes could be seen in Kerala’s nation of the species (Kurian and Sabu 2017). Studies for populations as well as one of the Karnataka’s populations identifying sex linked DNA markers in few rattans has also (Charmadi). This could be due to human mediated intro- been reported (Sarmah and Sarma 2011; Sarmah et al. 2016; gression wherein seeds from Kerala population might have Kurian et al. 2018b). The protected areas can also serve as been introduced in the Karnataka population (Charmadi) and refugia for resource enrichment of this threatened rattan vice versa. Many human activities alter genetic structure species as earlier reported for C. thwaitesii (Ramesha et al. either by disrupting the level of genetic diversity, or by 2007). 100 Page 8 of 10 Suma Arun Dev et al.

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