Ecological Divergence and Reproductive Isolation in an Amazonian Tropical Tree: Protium

Premise of the study: The first microsatellite primers were developed for Protium subserratum (Engl.) Engl., a widespread Amazonian tree, to investigate genetic differentiation between populations found on clay, brown-sand and white-sand soils. Methods and Results: Seventeen primer pairs were identified from two individuals of Protium subserratum, found on white-sand and brown-sand soil types. Polymorphism was analyzed in 63 individuals from a total of three populations, each found on a different soil type. The primers amplified tetra-, triand dinucleotide repeats with three to twenty four alleles per locus. Excluding monomorphic loci, observed and expected heterozygosities ranged from 0 to 0.852 and 0.036 to 0.901 respectively. Conclusions: These new microsatellite markers will be useful in studies of genetic diversity, population differentiation and gene flow across habitat types in P. subserratum. INTRODUCTION Protium subserratum (Engl.) Engl. (Burseraceae) is a widespread Neotropical tree found across the lowland Amazon Basin (Daly and Fine, 2011). It is dioecious with flowers that are small, white, nectar-producing and odiferous, indicating generalist pollinator affinities (Daly, 1987). Protium subserratum represents one of the few soil generalist species in the genus (Fine et al., 2005), with two morphologically differentiated sub-populations endemic to the widespread and relatively fertile clay and brown-sand forests as well as to the comparatively rare patches of nutrient poor white-sand forest habitats (Daly and Fine, 2011). A recent phylogeographic study comparing measurements of leaf traits in individuals from all three habitat types demonstrated that P. subserratum found in white-sand habitats differed morphologically in vegetative traits from populations found on terrace and clay soil types. Nuclear sequence data from the same study also showed that populations of P. subserratum from geographically distant clay and terrace soils were more closely related to each other than they were to nearby white sand populations (Fine et al., 2012). While these results are consistent with the idea that adaptations to different soil types may be playing an important role in population divergence, microsatellite markers will provide more powerful tools to examine fine scale population differentiation and gene flow. METHODS AND RESULTS Genomic DNA from two individuals of P. subserratum from a brown-sand population and a white-sand population (Appendix 1.1) were sent to the Savannah River Ecological Laboratories (SREL) at the University of Georgia for microsatellite marker development and primer design. Microsatellite markers were developed according to the protocol developed by Glenn and Schabel (2005). At SREL genomic DNA was combined, digested, ligated with linkers SimpleXL12_U (5'AAAGCTGGCGTCGAAGT -3') and SimpleXL12_Lp (5’pACTTCGACGCCAGC -3’), enriched with biotinylated probes and recovered via polymerase chain reaction (PCR). The enriched library was then sequenced on a 454 using titanium chemistry (454 Life Sciences, a Roche company, Branford CT, USA). A total of 6,123 sequences were obtained. A total of 2,201 reads, identified using MSATCOMMANDER version 0.8.1 (Faircloth, 2008), contained microsatellite repeats suitable for primer design. Primers were