Aphelandra R. Br. is one of the largest genera of Acanthaceae, comprising ca. 175 species of perennial herbs, shrubs, and small trees restricted to the Neotropics (Wasshausen, 1975; Daniel, 1991). Species in this genus have colored flowering spikes (Wasshausen, 1975), and the genus is well known to horticulturists because some species are cultivated for ornamental purposes (Daniel, 1991). However, until now, no studies of molecular genetic diversity in this genus have been carried out. We focus on the understory herb A. aurantiaca (Scheidw.) Lindl., distributed from southern Mexico through Central and South America (Daniel, 1991). In Mexico, its distribution is restricted to regions with abundant rainfall such as Los Tuxtlas rainforest (Daniel, 1991), where it is one of the dominant understory species (Calvo-Irabién, 1997). The region of Los Tuxtlas, considered the northernmost limit of rainforests in the Americas, has been heavily impacted by deforestation and fragmentation (Dirzo and Miranda, 1991; Dirzo and García, 1992). Because fragmentation produces isolation between populations, it could impact their genetic structure (Chávez-Pesqueira et al., 2014), reducing genetic variation and gene flow, and increasing genetic divergence and inbreeding (Young et al., 1996). Aphelandra aurantiaca is a suitable model to study the genetic consequences of rainforest fragmentation due to the life history characteristics of the species. For example, it has a relatively short life span, which means that some generations have passed since the onset of fragmentation, and it depends on canopy cover, which is usually reduced in forest fragments. Furthermore, because A. aurantiaca's attractive, nectar-producing flowers are pollinated by birds (Calvo-Irabién, 1997), its mating system can be affected by habitat fragmentation if this reduces species richness and abundance of pollinators (Aguilar et al., 2006). To date, little is known about its genetic structure, particularly in the context of rainforest fragmentation. Therefore, we aimed to develop variable genetic markers to elucidate the genetic diversity and structure of A. aurantiaca.
METHODS AND RESULTS
Using the DNeasy Plant Mini Kit (QIAGEN, Valencia, California, USA), we extracted genomic DNA from a single individual of A. aurantiaca for use in the isolation of microsatellite loci. A paired-end library was prepared by shearing 1 µg of genomic DNA following the standard protocol of the Illumina TruSeq DNA Library Kit (Illumina, San Diego, California, USA). Illumina sequencing was conducted on the HiSeq (Illumina) with 100-bp paired-end reads. Ten million of the resulting sequences were analyzed with the program PAL_FINDER_v0.02.03 (Castoe et al., 2012), extracting positive reads that contained di-, tri-, tetra-, penta-, and hexanucleotide microsatellites and sending to the program Primer3 (version 2.0.0; Rozen and Skaletsky, 1999) for primer design. To avoid duplicated loci, data were filtered and only primers that occurred one or two times were included; 24 loci out of 1722 that met this criterion were chosen. Primer pairs were tested for amplification and polymorphism using DNA obtained from five different individuals from the four Los Tuxtlas populations sampled (Appendix 1), and amplified PCR products were then separated on 4% Metaphor agarose gels (Lonza, Rockland, Maine, USA). After excluding loci that did not amplify, we selected 14 potential polymorphic loci and marked these with fluorescent labels (Table 1). The PCR amplification was carried out in a 20-µL reaction containing 2 µL of 10× PCR buffer (KCl 500 mM, Tris-HCL pH 8.3, gelatin 100 µg/mL, 1% triton, bovine serum albumin [BSA] 1.5 mg/mL), 1 µL of MgCl2 (30 mM), 2 µL of dNTPs (0.2 mM), 2 µL of DNA, 0.5 µL of each of the two primers (10 mM), 0.5 µL of Taq DNA polymerase (5 U/µL), and 12 µL of water (BIOTECMOL, Mexico City, Mexico), performed on a Thermo Scientific Hybaid Px2 thermal cycler (Thermo Scientific, Waltham, Massachusetts, USA) using the following conditions: 94°C for 10 min; followed by 35 cycles of 94°C for 1 min, at temperatures between 55–61°C for 1 min, and 72°C for 1 min; and a final extension step of 72°C for 7 min.
To encompass the most genetic diversity of A. aurantiaca in the Los Tuxtlas rainforest, we collected leaf tissue of 107 individuals from four populations (Appendix 1). Genomic DNA was extracted following the cetyltrimethylammonium bromide (CTAB) MiniPrep protocol (Doyle and Doyle, 1987). We selected a subset of loci to function well together in four multiplex reactions (QIAGEN Multiplex PCR Kit) with labeled primers (Applied Biosystems, Foster City, California, USA) (Table 1). Each multiplex PCR mixture (10 µL) contained 2 µL of DNA template (20 ng), 0.2 µL of each fluorescent-labeled forward primer (0.2 µM), 0.2 µL of each reverse primer (0.2 µM), 5 µL of QIAGEN Reaction Mix (1×), and 2.6 µL of RNase/DNase-free water (the volume of water varied depending on the number of primers in each multiplex reaction) (QIAGEN). Multiplexed reactions were carried out on a Hybaid Px2 thermal cycler (Thermo Scientific) and a Veriti 96-Well Thermal Cycler (Applied Biosystems). PCRs were performed through touchdown reactions, starting with initial heat activation at 95°C for 10 min, followed by 31 cycles with denaturation of 94°C for 60 s, annealing for 60 s, and 60 s of extension at 72°C. Annealing cycling temperature began at 57°C and decreased 1°C every cycle for six cycles (to 51°C), followed by two stages of 12 cycles each (with annealing temperatures of 55°C and 54°C). To check amplification, 5 µL of the PCR products were subjected to electrophoresis in a 1.5% agarose gel with 1× TBE buffer and stained with ethidium bromide. The remaining PCR products (5 µL) were diluted in 10 µL of water. One or two microliters of these PCR products (20–50 ng) were run on ABI Prism 310 and ABI 3730xl (Applied Biosystems) automated capillary sequencers; allele sizes were scored manually using GeneScan 500 LIZ Size Standard (Applied Biosystems) in GeneMarker version 2.4.0 (SoftGenetics LLC, State College, Pennsylvania, USA).
Characteristics of 14 microsatellite loci developed in Aphelandra aurantiaca.
Of the 14 primers tested, 12 were polymorphic and two were monomorphic with high-quality amplification (Table 2). For each polymorphic locus, we calculated the number of alleles (A), observed heterozygosity (Ho), and expected heterozygosity (He); tests of deviations from Hardy-Weinberg equilibrium (HWE) and linkage disequilibrium (LD) were performed using the software Arlequin version 126.96.36.199 (Excoffier and Lischer, 2010). Fixation indices (FIS) were estimated by GenAlEx version 6.5 (Peakall and Smouse, 2006). The probability of null alleles was estimated using MICRO-CHECKER software (van Oosterhout et al., 2004). We detected higher probabilities of null alleles between two loci (5250 and 1071) as suggested by the general excess of homozygotes (Table 2). There was LD at 10 of 90 paired loci comparisons, and significant departure from HWE was inferred at six loci, although this figure varied depending on the studied population (Table 2). A ranged from two to 12 across the studied populations. Ho and He ranged from 0.22 to 0.96 and from 0.20 to 0.87, respectively, and FIS ranged from −0.41 to 0.44.
We developed and characterized 12 polymorphic and two monomorphic novel microsatellite markers for the herb A. aurantiaca. The primers will be useful for assessing population genetic structure and mating system of A. aurantiaca in both preserved and fragmented rainforest. Likewise, we expect these microsatellite loci could be useful for other Aphelandra species.
Genetic properties of the newly developed polymorphic microsatellite loci of Aphelandra aurantiaca.a
 The authors thank the Los Tuxtlas Biological Research Station for logistics support; S. Lance and the University of Georgia Savannah River Ecology Laboratory for sequencing and developing primers; and L. Márquez-Valdemar, G. Andraca-Gómez, F. Baena-Díaz, and M. Chávez-Pesqueira for assistance in obtaining genetic data. The study was funded by a Universidad Nacional Autónoma de México (UNAM) Programa de Apoyo a Proyectos de Investigación e Innovación Tecnológica (PAPIIT) grant (IN 215111-3). This paper constitutes a partial fulfillment of the Graduate Program in Biological Sciences (UNAM) for P.S.-M., who acknowledges a scholarship and financial support by the Consejo Nacional de Ciencia y Tecnología (CONACyT) and UNAM.