Lodoicea maldivica (J. F. Gmel.) Pers. (Arecaceae; coco de mer) is an evolutionarily and ecologically distinct dioecious palm (Edwards et al., 2002, 2015) that holds several botanical records, among which are the largest female flowers in any palm and the largest seeds in the plant kingdom (Leishman et al., 2000). The species was once widespread across two Seychelles islands, Praslin and Curieuse (Malavois, 1787, quoted in Fauvel, 1909), but now persists in only four main semiconnected populations—at Vallée de Mai, Fond Peper, and Fond Ferdinand on Praslin, and also on Curieuse Island (Fleischer-Dogley et al., 2011).
The total L. maldivica population on Praslin and Curieuse was estimated at 24,376 individuals in 2004, but despite the relatively large population size, reproductive female trees make up only a small proportion (15.6%) of the population (Fleischer-Dogley, 2006). The recent population reduction is due to habitat degradation arising from several serious fires and lumber harvest (Bailey, 1942). Although L. maldivica nut kernel has been listed in the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES), which prohibits exportation without a license, unsustainable harvesting and poaching of nuts continues to threaten the species, as natural regeneration is severely limited (Rist et al., 2010). Fleischer-Dogley et al. (2010) used amplified fragment length polymorphisms to assess genetic diversity in L. maldivica, but the dominant nature of the markers did not permit detailed genetic analyses. By developing microsatellite markers, we provide the foundation for in-depth molecular research on the ecology and population genetics of the species, and a tool for the conservation and sustainable production of L. maldivica nontimber products. This study reports the isolation and characterization of 12 polymorphic and three monomorphic microsatellite loci in L. maldivica.
METHODS AND RESULTS
Size-selected fragments from genomic DNA were enriched for simple sequence repeat (SSR) content using magnetic streptavidin beads and biotin-labeled CT and GT repeat oligonucleotides. The SSR-enriched library was made by the company ecogenics (Balgach, Switzerland) and analyzed on a Roche 454 platform using the GS FLX Titanium reagents (454 Life Sciences, a Roche Company, Branford, Connecticut, USA). The 6607 reads had an average length of 143 base pairs. Of these, 617 contained a microsatellite insert with a tetra- or a trinucleotide of at least six repeat units or a dinucleotide of at least 10 repeat units. Primer design was done using the Primer3 core (Rozen and Skaletsky, 1999). Suitable primer design was possible in 212 reads. Seventy-eight primer pairs were tested, and the most reliable polymorphic candidates were optimized. Genomic DNA was extracted from silica gel-dried L. maldivica leaf or flower tissue (n = 1252) following the DNeasy 96 Plant Kit (QIAGEN, Hombrechtikon, Switzerland) manufacturer's protocol, except that grinding was carried out at four cycles of 30 s at 30 Hz, and the first incubation step was extended to 1 h at 65°C. Leaf tissue samples from L. maldivica individuals from each population are located at the Tissue Collection of the Royal Botanic Gardens, Kew, Richmond, Surrey, United Kingdom (Appendix 1).
Characteristics of the 12 polymorphic and three monomorphic microsatellite loci in Lodoicea maldivica.a
Genetic properties of 12 de novo microsatellite markers in the four extant Lodoicea maldivica populations.a,b
Two methods were used for PCR reactions: two multiplex PCRs were used to amplify six primers, and the remainder of the primers were amplified in singleplex. Multiplex PCRs (MP1 and MP2) were carried out using primers labeled with either FAM, ATTO565, ATTO550, or Yakima Yellow (YY) (Microsynth, Balgach, Switzerland) (Table 1). PCR amplifications were carried out in 10.3-µL reactions containing 1× PCR Buffer (colorless Flexi GoTaq PCR buffer), 0.2 mM dNTPs, 3.1 mM MgCl2, 0.05 U/µL Taq Polymerase (all Promega Corporation, Zürich, Switzerland), 0.18 µg/µL bovine serum albumin (BSA; BioConcept, Allschwil, Switzerland), 1.3 µL DNA, labeled forward primers, and unlabeled forward and reverse primers (for primer concentrations see Table 1).
Touchdown PCRs were carried out on a Bio-Rad Dyad Cycler (Bio-Rad Laboratories, Hercules, California, USA) with the following conditions: initial denaturation 95°C/4 min; 12× (denaturation 95°C/30 s, starting annealing temperature 62°C/30 s, decreasing by 0.5°C/cycle, extension 72°C/30 s); 29× (MP1)/28× (MP2) (denaturation 95°C/30 s, annealing 56°C/45 s, extension 72°C/30 s); and final extension 72°C/30 min and storage at 10°C. PCR product (2.5 µL) was added to 10 µL of HIDI formamide and 0.25 µL GeneScan 500 LIZ Size Standard (Applied Biosystems, Waltham, Massachusetts, USA).
The singleplex PCRs used forward primers labeled with M13 tails (5′-TGTAAAACGACGGCCAGT-3′) at the 5′ ends (as described by Schuelke, 2000) (Table 1). PCRs occurred in 11-µL reaction volumes containing 1× PCR Buffer, 0.2 mM dNTPs, 2.5 mM MgCl2, 0.025 U/µL Taq Polymerase, 0.18 µg/µL BSA, 1.0 µL DNA, forward primers with M13 tails, reverse primers and M13-primer universal tails labeled with either FAM, ATTO565, ATTO550, or YY (Microsynth) (for primer concentrations see Table 1). Cycling for singleplex PCRs was as follows: initial denaturation 95°C/5 min; 12× (denaturation 95°C/30 s, starting annealing temperature 62°C/30 s, decreasing by 0.5°C/cycle, extension 72°C/30 s); 25× (denaturation 95°C/30 s, annealing 56°C/45 s, extension 72°C/30 s); 8× (denaturation 95°C/30 s, annealing 53°C/45 s, extension 72°C/45 s); and final extension 72°C/30 min and storage at 10°C. PCR products were combined to create two pseudo-multiplex mixes (Table 1). For each PCR product (Lm4293, Lm2407, Lm6026, and Lm0144 were diluted 20× first), 1.2 µL were added to 10 µL of HIDI formamide and 0.15 µL of GeneScan 500 LIZ Size Standard (Applied Biosystems). Singleplex and multiplex products were denatured for 3 min at 92°C and run on an AB13730xl automatic capillary sequencer (Applied Biosystems). Electropherograms were scored with Gene-Marker 2.6.0 (SoftGenetics, State College, Pennsylvania, USA).
The number of alleles, deviations from Hardy–Weinberg equilibrium (HWE), and observed and expected heterozygosity values were calculated (Table 2) using GenAlEx 6.5 (Peakall and Smouse, 2006). Linkage disequilibrium was tested in GENEPOP (Raymond and Rousset, 1995). The 12 polymorphic loci revealed between five and 21 alleles, with a total of 158 alleles across all L. maldivica individuals (Table 2). Significant deviation from HWE was seen in the majority of loci in all populations (Table 2). Expected heterozygosity values ranged from 0.399–0.896 (mean ± SE: 0.687 ± 0.048) for the polymorphic markers. No significant linkage disequilibrium was detected between loci pairs after sequential Bonferroni correction (α = 0.05) (Holm, 1979). The putative presence of null alleles in 11 loci (all except the monomorphic loci and Lm4716) was detected using MICRO-CHECKER 2.2.3 (van Oosterhout et al., 2004); however, these are unlikely to affect HWE at such low frequencies (Dakin and Avise, 2004). There was no evidence for large allele dropout.
We developed 12 highly polymorphic and three monomorphic loci for L. maldivica, with allele numbers ranging from five to 21 for the polymorphic loci. The pattern of homozygote excess can be observed across almost all loci in all populations. This can likely be explained by high inbreeding levels due to the very clustered growth patterns observed in the species. These markers will provide a useful tool in investigating the natural population structure, seed dispersal patterns, and fine-scale genetic structure of this highly charismatic and important endemic palm species (Morgan et al., in prep.).
 The authors thank Seychelles Islands Foundation, Ravin de Fond Ferdinand Nature Reserve, and Seychelles National Parks Authority and their staff for arrangements on site and field assistance (particularly G. Rose for sample collection); P. Edwards for valuable advice; and the Genetic Diversity Centre of ETH Zürich. Sample collection and export were approved by the Seychelles Bureau of Standards and Department of Environment. This research was funded under grant number ETH-37 12-1 ETH Zürich.