The Anacardiaceae is a family of flowering plants with approximately 81 genera and 800 species. Numerous species within this family are economically important due to the production of agricultural food products such as cashews (Anacardium occidentale L.; Mitchell and Mori, 1987), mangos (Mangifera indica L.; Mukherjee, 1972), pink peppercorns (Schinus molle L.; Barkley, 1944), pistachios (Pistacia vera L.; Al-Saghir and Porter, 2012), “ciruela mexicana” or “jocote” (Spondias purpurea L.), and “jobo” (S. mombin L. and S. radlkoferi Donn. Sm.; Airy Shaw and Forman, 1967). Other species are poisonous, such as Toxicodendron radicans (L.) Kuntze, which contains strong allergens that cause dermatitis to humans and animals (Pell et al., 2011).
Spondias L. consists of 17 species: seven species in the neotropics (Mexico to Brazil) and 10 species in Asia (Miller, 2011; Pell et al., 2011). In Mexico and Central America, S. mombin, S. purpurea, and S. radlkoferi are the most common species, but S. purpurea and S. mombin are the most consumed and cultivated species (Miller, 2011; Pell et al., 2011). The fruits of S. radlkoferi and S. mombin are highly consumed by many bird and mammal species. However, the seeds of both species are relatively large (mean ± SD: S. radlkoferi, 3.11 ± 0.43 cm; S. mombin, 2.10 ± 0.23 cm in length; Benitez-Malvido et al., 2014), which implies that only a few large-sized mammals, such as spider monkeys (Ateles geoffroyi Kuhl), are able to swallow and disperse (through endozoochory) the seeds of these tree species (González-Zamora et al., 2009, 2014; Chaves et al., 2011; Benítez-Malvido et al., 2014). Nevertheless, to date it is largely unknown whether and how seed dispersal by spider monkeys affects the genetic structure and diversity of these tree species. Thus, we developed microsatellite markers for S. radlkoferi to: (1) test the impact that primate seed dispersal may have on the genetic diversity and structure of this tree species, and (2) help future studies on the genetic diversity and population structure of S. radlkoferi and related species.
METHODS AND RESULTS
Genomic DNA of a single individual of S. radlkoferi was isolated (voucher deposited at the herbarium of the Instituto de Ecología, A.C., Pátzcuaro, Mexico [IEB], Appendix 1). Previous to the extraction, a prewash step of leaf tissue was made: washing buffer (10 mM Tris-HCl [pH 8.0], 5 mM EDTA, 0.2 M NaCl, 0.45% of 2-mercaptoethanol, and 1% of poly vinylpyrrolidone [PVP]) was added to the ground leaf tissue and mixed for 1 min. After that, the sample was centrifuged for 5 min at maximal spin. DNA was isolated employing the cetyltrimethylammonium bromide (CTAB) protocol (Doyle and Doyle, 1987). Genomic DNA pyrosequencing was then performed on a Roche 454 GS-FLX Titanium sequencer (454 Life Sciences, a Roche Company, Branford, Connecticut, USA) at the GenoSeq Core of the University of California (Los Angeles, California, USA; http://www.genoseq.ucla.edu). The 454 sequencing reads were assembled into contigs with GS De Novo Assembler software (454 Life Sciences, a Roche Company) at the GenoSeq Core. A database of approximately 40,000 sequences was obtained and employed for screening of putative microsatellite motifs using MSATCOMMANDER version 0.8.2 (Faircloth, 2008). From these 40,000 sequences, 607 contained dinucleotide repeats, 696 trinucleotide repeats, and 126 tetranucleotide repeats. The primers flanking the repeat motifs were automatically designed with Primer3 software (Rozen and Skaletsky, 2000).
Characteristics of 14 microsatellite loci isolated from Spondias radlkoferi.
Genetic diversity of 14 microsatellite loci in S. radlkoferi and S. mombin.
Based on melting temperature, theoretical amplified fragment size, and microsatellite motif, a set of 44 microsatellite sequences were chosen for testing. To evaluate the polymorphisms at each locus, we used genomic DNA from 37 individuals (Appendix 1) collected from the Lacandona rainforest (Marqués de Comillas, Chiapas, Mexico; 16°03′N, 90°45′W). Cross-species amplification was carried out in S. mombin, Rhus aromatica Aiton, and Toxicodendron radicans. PCR amplification was performed in a final reaction volume of 5 µL containing 2× Multiplex PCR master mix (QIAGEN, Valencia, California, USA), 0.4 µM of each forward and reverse primer, and approximately 10 ng of DNA template. PCR amplification was performed in an Eppendorf Mastercycler (Eppendorf, Hamburg, Germany) using the following conditions: first denaturing step 94°C, 15 min; 35 cycles of denaturing 94°C, 30 s; primer annealing at 58°C or 60°C (Table 1), 1 min 30 s; extension 72°C, 1 min, and a final extension at 72°C for 10 min. Loci that were successfully amplified were then tested with a fluorescent forward primer. Fragments were electrophoresed in an ABI PRISM 3130 XL Genetic Analyzer (Applied Biosystems, Foster City, California, USA) with the GeneScan 500 LIZ Size Standard included (Applied Biosystems). PeakScanner software version 1.0 (Applied Biosystems) was used for fragment analysis and final sizing.
In total, 14 polymorphic microsatellite loci were selected (Table 1). Monomorphic primers SPO2, SPO6, SPO7, and SPO9 are also listed in Table 1. The microsatellite sequences have been deposited in GenBank. MICRO-CHECKER (van Oosterhout et al., 2004) was employed for testing scoring errors and null alleles. The number of alleles, effective number of alleles, observed heterozygosity, and expected heterozygosity were determined using GenAlEx version 6.5 (Peakall and Smouse, 2006). In S. radlkoferi, the number of alleles per locus ranged from three to 12, and the number of effective alleles varied from 1.546 to 3.765 (Table 2). The observed heterozygosity varied from 0.382 to 1.00, and was higher than the expected heterozygosity, which ranged from 0.353 to 0.733 (Table 2). Twelve out of 14 microsatellite loci were also successfully amplified in the related S. mombin, nine loci amplified in Rhus aromatica, and eight loci amplified in Toxicodendron radicans (Table 2).
Fourteen new microsatellite markers were isolated and proved to be useful to evaluate the genetic diversity of S. radlkoferi and S. mombin. Cross-amplification of these microsatellite loci in Rhus and Toxicodendron suggests that they can be useful in studies of other species within Anacardiaceae. These microsatellites will be useful in determining the genetic diversity and structure of S. radlkoferi. In particular, we are using these markers to assess the impact that seed dispersal by spider monkeys may have on the genetic diversity of S. radlkoferi in continuous and fragmented tropical rainforest. We will determine the genetic identity of seeds and adults to perform parentage analysis and estimate seed dispersal distances in fragmented tropical landscapes of southeastern Mexico. The dispersal analysis will also help us to determine the movement of spider monkeys within and between forest fragments, a poorly assessed ecological process.