Introduction

Phyllanthus is one of the most species-rich genera of the family Phyllanthaceae, comprising over 800 species worldwide and over 50 species in India (Ravikanth et al. 2011). They are characterized by diverse growth forms, including shrubs, trees, annual or biennial herbs, and are distributed throughout the tropical and subtropical regions of both hemispheres. Among the species found in India, P. amarus, P. debilis, P. fraternus, P. urinaria, P. kozhikodianus, P. maderaspatensis, P. acidus, P. emblica and P. indofischeri are widely used in preparation of herbal medicines, and some of these species are cultivated in southern India (Srirama et al. 2010; Ravikanth et al. 2011).

P. emblica L. and P. indofischeri Bennet, commonly known as the Indian gooseberries, form an important non-timber product species (NTFP) in southern India. The fruits of these species are rich in vitamin C and used for treating digestive disorders, constipation, fever, cough, asthma and to stimulate hair growth (Ravikanth et al. 2011). The fruits of both the species are traded as ‘Amla’ in India, and the extracts of fruits have been reported to have antioxidant, analgesic, anti-inflammatory and chemoprotective properties (Khopde et al. 2001; Srirama et al. 2012). They are also used in the preparation of pickles, jams, juices and also used in cosmetics, hair dye and shampoo industries (Siva 2003; Ganesan and Setty 2004). Both the species are medium-sized trees widely distributed in the forests of south India, where, P. emblica is commonly found in the evergreen to moist deciduous forests, and P. indofischeri is often seen in the dry deciduous forests of southern India.

The fruits of these species form a major source of livelihood for many forest-dwelling communities in India (Ganesan and Setty 2004). Besides these species, a number of Phyllanthus species are used in the herbal industries as a source for many Ayurvedic preparations (Ravikanth et al. 2011; Srirama et al. 2012). The predicted annual industrial consumption of dry Amla by the herbal industries is 17,000 MT, which correlates to 85,000 MT of green fruits (Babu 2010). It is also estimated that the forest-dwelling communities obtain 65% of the fruits from the wild (Ganesan and Setty 2004; Ved and Goraya 2008). Such enormous usage of NTFP can have a detrimental effect on the wild populations of both P. emblica and P. indofischeri leading to the lower regeneration and reduction in population size (Ravikanth and Siddappa Setty 2017). Reduced population size and fragmentation could result in mating among closely related individuals, leading to inbreeding and alteration of the genetic structure of the populations and finally to local extinction of populations (Ravikanth and Siddappa Setty 2017). Understanding how harvesting of fruits of these species modifies the genetic structure could provide important information in developing sustainable harvesting practices. Secondly, for effective utilization and conservation of genetic resources of Phyllanthus species, critical information on the spatial distribution of genetic variability of the species is crucial. Mapping the distribution of genetic variability could aid in the identification of genetic hot spots for in situ conservation, as well as to assist in designing germplasm collections (Ravikanth et al. 2001).

Pandey and Changtragoon (2012) reported the characterization of six microsatellite markers of P. emblica, of which five were polymorphic. These five microsatellite markers are however extremely low to address the impacts of harvesting on the genetic structure of these two important NTFP species, to study the population genetic variability or to assess the gene flow across the populations. In this study, we additionally developed 15 polymorphic microsatellite markers of P. emblica to understand the population genetic diversity of both P. emblica and P. indofischeri. We also show the cross-amplification of these markers with other related species of Phyllanthus. These primers can be useful in assessing the genetic diversity, gene flow and spatial genetic structure of P. emblica, P. indofischeri and related species.

Materials and methods

Young leaves from 20 individuals each of P. emblica and P. indofischeri were collected from Biligiri Rangaswamy Temple Wildlife Sanctuary (BRT WLS) (\(11{^{\circ }}59'38''\hbox {N}\): \(77{^{\circ }}8'26''\hbox {E}\)), India. Two to three individuals each of the related species were also collected from School of Ecology and Conservation Garden, GKVK, Bengaluru. Genomic DNA was extracted from the leaves using the DNeasyPlant Mini kit (Qiagen, Hilden, Germany). Purified genomic DNA of P. emblica was used for microsatellite development using the hybridization capture approach, described by Glenn and Schable (2005) with minor modifications. The genomic DNA (\(20\hbox { }{\mu }\text {g}\)) of P. emblica was initially digested by the RsaI and XmnI restriction digestion enzymes (New England Biolabs, Massachusetts, USA) for 1 h at \(37{^{\circ }}\hbox {C}\). The digested DNA was ligated to double-stranded Super SNX linkers (SNX-F- 5\('\)-GTTTAAGGCCTAGCTAGCAGAATC and SNX-R- 5\('\)-ATTCTGCTAGCTAGGCCTTAAACAAAA) and the ligated DNA was amplified with the SNX forward primer. The amplified products were hybridized with the Biotin labelled oligonucleotide probes containing the microsatellite repeats (AC, GA, AA, CTT, AGG and ACAG). The hybridized microsatellite rich products were captured magnetically using streptavidin-coated Dynabeads (Invitrogen, Oslo, Norway). These enriched fragments were amplified using the SNX linker primer and cloned to pTZ57R/T vector using the Thermo Scientific TA cloning kit. The cloned vector was transformed into the competent E. coli cells (\(\hbox {CB5}\upalpha \), Chromous Biotech, Bengaluru). The recombinant or the positive clones were identified as white colonies by the blue/white screening procedure on LB Agar plates containing Ampicillin and X-gal. These positive colonies were further amplified from the plasmid DNA using the M13 primers and inserted length that was more than 150 bp were selected. Based on this criterion, 132 colonies were selected and sequenced using ABI PRISM 3100 Genetic Analyzer Applied Biosystems (Chromous Biotech, Bengaluru). The sequences were screened for the presence of microsatellite repeat motifs using the web-based SSR Finder program (Martins et al. 2009). Of the 132 colonies, 41 had sufficient SSRs and the forward and reverse primers were designed using a web-based Primer3 program (http://primer3.wi.mit.edu/; Rozen and Skaletsky 2000).

These 41 primers were initially screened with five individuals of P. emblica. Of the 41 primers, 15 primer pairs were selected based on the length variations in the agarose gel (table 1). The selected primers were labelled using different fluorescent label dyes (6-FAM, HEX, NED, PET) at the 5\(^{\prime }\) end of the forward primers (Invitrogen, California, USA; table 1). Twenty individuals each of P. emblica and P. indofischeri from the BRT WLS population were amplified with the labelled primers. The PCR reaction for the amplification was carried out using 5–10 ng DNA template, 1X polymerase buffer, 1 mM dNTPs, 5 pmol each of forward and reverse primer and 1 unit of Taq polymerase (Bangalore Genei, India). The PCR’s cycling conditions were 5 min at \(95{^{\circ }}\hbox {C}\), followed by 38 cycles of 30 s at \(94{^{\circ }}\hbox {C}\), 45 s at the annealing temperature of designed specific primer (47.7–52.8\({^{\circ }}\hbox {C}\)) and 45 s at \(72{^{\circ }}\hbox {C}\), with an extension of 10 min at \(72{^{\circ }}\hbox {C}\) in the final cycle. The labelled products were genotyped on an Applied Biosystems 3730 Genetic Analyzer with a GeneScan 500 LIZ (-250) Size Standard (SciGenom, Cochin, India). The genotype results were evaluated and scored for the allele sizes using Peak Scanner ver. 2 (Applied Biosystems).

Table 1 List of 15 polymorphic microsatellite primers developed for P. emblica with their GenBank numbers.
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Table 2 Characterization of 15 polymorphic loci for P. emblica and P. indofischeri.
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Table 3 Cross-amplification of P. emblica microsatellite markers with closely related Phyllanthus species.
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The data was analysed for the genetic diversity indices such as observed heterozygosity (\(H_{\mathrm{o}}\)), expected heterozygosity (\(H_{\mathrm{e}}\)), number of alleles per locus (\(N_{\mathrm{a}}\)), effective number of alleles (\(N_{\mathrm{e}}\)); fixation index (F) and deviation from Hardy–Weinberg equilibrium (HWE). The analysis was carried out in Cervus 3.0 (Kalinowski et al. 2007) and Genealex 6.5 (Peakall and Smouse 2012). The presence of null alleles and allele dropouts were assessed using Microchecker 2.2.3 (Van Oosterhout et al. 2004). All the 15 primers were also analysed for cross-amplification with seven related Phyllanthus species, namely P. amarus, P. debilis, P. tenellus, P. kozhikodianus, P. polyphyllus, P. baillonianus and P. acidus.

Results

Twenty individuals each of both the species P. emblica and P. indofischeri were assessed for their genetic variability using 15 microsatellite markers. Of these 15 primers, two were mononucleotide, seven dinucleotides and six trinucleotide repeat motifs (table 1). All the microsatellite markers were found to be polymorphic and the number of alleles per locus was in the range of 2–9 in P. emblica and 2–11 in the case of P. indofischeri with an average of 4 and 4.2, respectively. The effective number of alleles was in the range of 1.63–5.02 for P. emblica and 1.69–5.59 for P. indofischeri with an overall mean of 2.70 and 2.53, respectively. The observed and expected heterozygosity for P. emblica was in the range of 0–1 and 0.401–0.825, respectively. Similarly, the observed and expected heterozygosity of P. indofischeri was in the range of 0.5–1 and 0.366–0.842, respectively. The overall mean for observed and expected heterozygosity for both the species were 0.805 and 0.607 (P. emblica) and 0.813 and 0.582 (P. indofischeri), respectively (table 2). The presence of null alleles and allele dropouts in all the loci was not detected using Microchecker at the confidence level of 95%, based on Van Oosterhout et al. (2004). The fixation index for P. emblica ranged from 1 to −1 with an overall mean of −0.376. Similarly, the fixation index for P. indofischeri ranged from 0.087 to −0.909 with an overall mean of −0.448. Four of the 15 primers showed significant deviation from HWE (\(P{<}0.05\)) in the case of P. emblica and six of the 15 primers showed significant deviation from HWE for P. indofischeri.

The primers developed also cross-amplified with all the seven related Phyllanthus species. However, not all the primers successfully amplified in all the seven species. In case of P. debilis, P. tenellus, P. kozhikodianus and P. polyphyllus, 11 primers were found to cross amplify (table 3); however, only four primers in P. amarus, five in P. baillonianus and six in P. acidus cross-amplified (table 3).

Discussion

Only a few studies have investigated the genetic diversity of Phyllanthus species (Uma Shaanker and Ganeshaiah 1997; Padmini et al. 2001). However, these studies using isozymes provided limited information on the spatial genetic structure as well as on gene flow across the populations. Our initial study with the markers in one population for each of the two species revealed a mean observed and expected heterozygosity of 0.805 and 0.607 (P. emblica); and 0.813 and 0.582 (P. indofischeri). The results indicate that the primers PE8, 9, 11 and 15 in case of P. emblica and PE 3, 9, 12, 27, 34 and 35 in case of P. indofischeri showed significant deviation from HWE (table 2). One of the possible reasons for this deviation from HWE could be due to higher levels of inbreeding in the sampled population of both the species. There has been a long history of extraction of fruits of these species in sites sampled (Ganesan and Setty 2004). Besides, over the years, there has been increased mortality and reduced fruit set of both the species due to the infestation of mistletoe, Taxillus tomentosus (Rist et al. 2011). The spread of invasive species such as Lantana camara in recent decades, mistletoe infection and harvesting, have led to poor regeneration causing a significant reduction in population size of these species. Reduced population size could significantly increase inbreeding in the population.

In an earlier study, of the six microsatellite loci five showed polymorphism with the number of alleles ranging from four to seven and the observed and expected heterozygosity ranging from 0.360 to 0.760 and 0.499 to 0.806 similar to the observed and expected heterozygosity found in our study (Pandey and Changtragoon 2012).

In addition to the markers reported by Pandey and Changtragoon (2012), the microsatellite markers developed in this study could be used to study the population genetic structure, gene flow and genetic diversity of P. emblica, P. indofischeri and other Phyllanthus species. Besides P. emblica and P. indofischeri, our study has also shown cross-amplification of these microsatellite markers in a number of Phyllanthus species. Thus, these markers could also aid in assessing the impact of harvesting and other anthropogenic pressures on the genetic structure of other Phyllanthus species, which are also subject to harvesting pressures.