Genetic Structure, Diversity and Long Term Viability of a Medicinal Plant, Nothapodytes nimmoniana Graham. (Icacinaceae), in Protected and Non-Protected Areas in the Western Ghats Biodiversity Hotspot

Background and Question The harvesting of medicinal plants from wild sources is escalating in many parts of the world, compromising the long-term survival of natural populations of medicinally important plants and sustainability of sources of raw material to meet pharmaceutical industry needs. Although protected areas are considered to play a central role in conservation of plant genetic resources, the effectiveness of protected areas for maintaining medicinal plant populations subject to intense harvesting pressure remain largely unknown. We conducted genetic and demographic studies of Nothapodytes nimmoniana Graham, one of the extensively harvested medicinal plant species in the Western Ghats biodiversity hotspot, India to assess the effectiveness of protected areas in long-term maintenance of economically important plant species. Methodology/Principal Findings The analysis of adults and seedlings of N. nimmoniana in four protected and four non-protected areas using 7 nuclear microsatellite loci revealed that populations that are distributed within protected areas are subject to lower levels of harvesting and maintain higher genetic diversity (He = 0.816, Ho = 0.607, A = 18.857) than populations in adjoining non-protected areas (He = 0.781, Ho = 0.511, A = 15.571). Furthermore, seedlings in protected areas had significantly higher observed heterozygosity (Ho = 0.630) and private alleles as compared to seedlings in adjoining non-protected areas (Ho = 0.426). Most populations revealed signatures of recent genetic bottleneck. The prediction of long-term maintenance of genetic diversity using BOTTLESIM indicated that current population sizes of the species are not sufficient to maintain 90% of present genetic diversity for next 100 years. Conclusions/Significance Overall, these results highlight the need for establishing more protected areas encompassing a large number of adult plants in the Western Ghats to conserve genetic diversity of economically and medicinally important plant species

A conserved amino acid residue critical for product and substrate specificity in plant triterpene synthases

Triterpenes are structurally complex plant natural products with numerous medicinal applications. They are synthesized through an origami-like process that involves cyclization of the linear 30 carbon precursor 2,3-oxidosqualene into different triterpene scaffolds. Here, through a forward genetic screen in planta, we identify a conserved amino acid residue that determines product specificity in triterpene synthases from diverse plant species. Mutation of this residue results in a major change in triterpene cyclization, with production of tetracyclic rather than pentacyclic products. The mutated enzymes also use the more highly oxygenated substrate dioxidosqualene in preference to 2,3-oxidosqualene when expressed in yeast. Our discoveries provide new insights into triterpene cyclization, revealing hidden functional diversity within triterpene synthases. They further open up opportunities to engineer novel oxygenated triterpene scaffolds by manipulating the precursor supply

Membership of each pre-defined population in each of the two (K = 2) clusters generated by CLUMPP based on the results of STRUCTURE v2.2 analysis of SSR data.

<p>Populations contributing >50% of their ancestry to a single cluster are highlighted in bold.</p><p>Membership of each pre-defined population in each of the two (K = 2) clusters generated by CLUMPP based on the results of STRUCTURE v2.2 analysis of SSR data.</p

Analysis of molecular variance (AMOVA) for 195 individuals of <i>Nothapodytes nimmoniana</i> in protected (PA) and non-protected (NPA) areas.

<p>Analysis of molecular variance (AMOVA) for 195 individuals of <i>Nothapodytes nimmoniana</i> in protected (PA) and non-protected (NPA) areas.</p

Image of A) <i>N. nimmoniana</i> plant and chemical structure of B) Camptothecine extracted from wood of <i>N. nimmoniana</i> C) Irinotecan and D) Topotecan, two clinically used drugs synthesized from Camptothecine as a precursor.

<p>Image of A) <i>N. nimmoniana</i> plant and chemical structure of B) Camptothecine extracted from wood of <i>N. nimmoniana</i> C) Irinotecan and D) Topotecan, two clinically used drugs synthesized from Camptothecine as a precursor.</p

Map showing the distribution and sampling locations of <i>Nothapodytes nimmoniana</i> in the Western Ghats, India.

<p>Map showing the distribution and sampling locations of <i>Nothapodytes nimmoniana</i> in the Western Ghats, India.</p

Map showing genetic diversity parameters at sampling locations of <i>Nothapodytes nimmoniana</i> from Western Ghats, India A) Nei’s gene diversity B) Allelic richness.

<p>Note: Regions represented in dark red indicate areas of high genetic diversity and allelic richness.</p

Location, geographical coordinates, sample size (N) and private alleles (A_P) in eight populations of <i>Nothapodytes nimmoniana</i>.

<p>Note: PA = Protected area, NPA = Non-protected area.</p><p>Location, geographical coordinates, sample size (N) and private alleles (A_P) in eight populations of <i>Nothapodytes nimmoniana</i>.</p

Predicted genetic diversity in eight populations of <i>Nothapodytes nimmoniana</i> sampled from protected (PA) and non-protected (NPA) areas of Western Ghats, India over next 100 years using BOTTLESIM.

<p>The current population size is unable to maintain 90% of current genetic diversity over the next 100 years in almost all sampled populations. A) Agumbe (PA) B)Vadagere (NPA) C)Dandeli (PA) D) Joida (NPA) E) Kemmangundi (PA) F) Multavara (NPA) G) Talakaveri (PA) and H) Bondikadu (NPA). Note: The observed numbers of alleles (OA) were projected to decline (Sex ratio 1∶1 F:M).</p

Analysis of historical and recent genetic bottleneck based on stepwise mutation model (SMM) of microsatellite evolution and Mode shift test for allele frequency distribution.

<p>Note: – = data absent. *P, <0.05; **P, <0.01. A = adults, S = seedlings.</p><p>The significance of gene diversity excess (He. Heq) is an indication of recent effective population size reductions (bottlenecks). The significance was tested using Sign test and Wilcoxon signed ranks test <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112769#pone.0112769-Luikart1" target="_blank">[44]</a> based on 5000 replications.</p><p>Analysis of historical and recent genetic bottleneck based on stepwise mutation model (SMM) of microsatellite evolution and Mode shift test for allele frequency distribution.</p