The segregate genus Hexastylis Raf. (Aristolochiaceae), often included in Asarum L., is an enigmatic group of 12 species distributed in the southeastern United States (Blomquist, 1957; Niedenberger, 2010). Hexastylis has been segregated based upon its entirely North American distribution, karyotype (Sugawara, 1981; Soltis, 1984), pollen morphology (Niedenberger, 2010), and several characteristics of flower morphology (Gaddy, 1987). Multiple species complexes have been identified in this genus and this study focuses on the H. heterophylla complex, containing H. heterophylla (Ashe) Small, H. minor (Ashe) H. L. Blomq., and H. naniflora H. L. Blomq. The species in this complex are sympatric over portions of their ranges. Vegetative characters have limited taxonomic value, leaving ephemeral floral morphology as the only diagnosable field character for identification. Previous studies have recognized intermediate floral morphologies in some populations, leading some to question the validity of species circumscriptions. This is particularly problematic in H. naniflora, where land managers and conservation biologists are tasked with protection of this federally threatened species.
Through funding from the North Carolina Department of Transportation, 16 polymorphic microsatellite markers were developed to help distinguish H. naniflora from H. minor and H. heterophylla, to address questions of hybridization, and to identify evolutionarily significant units to aid in the management of these species. These markers have the potential to identify species and hybrids in their vegetative state, allowing land managers to evaluate population value and management strategies throughout the year, instead of only during the short flowering period.
METHODS AND RESULTS
Leaf tissue was collected and preserved on silica gel from plants at 15 sites in North and South Carolina (Appendix 1). Tissue samples from one plant of H. naniflora and one plant of H. heterophylla (selected from geographic ranges where the species do not overlap and confidently identified using flower material) were sent to the Cornell University Evolutionary Genetics Core Facility where total DNA was extracted using a QIAGEN Plant Mini Kit (QIAGEN, Valencia, California, USA). Restriction enzymes AluI, Hpy166II, and RsaI (New England Biolabs, Ipswich, Massachusetts, USA) were used to digest the DNA, which was then ligated to an Illumina Y-adapter (Illumina, San Diego, California, USA) using T4 DNA ligase. The DNA fragments were then hybridized to 3′ biotinylated oligonucleotide repeat probes: (GT)8, (TC)9.5, (TTTTG)1.2, (TTTTC)4.6, (TTC)7, (GTA)8.33, (GTG)4.67, (TCC)5, (GTT)6.33, (TTTC)6, (GATA)7, (TTAC)6.75, (GATG)4.25, (TTTG)5.25, (TTTTG)4.2, (TTTTC)4.6. Enriched fragments were then captured by streptavidin-coated magnetic beads (New England Biolabs) and PCR amplified. Agarose gel and a Qubit 2.0 Fluorometer (Life Technologies, Grand Island, New York, USA) were used to analyze the PCR product, and fragments 300–600 bp were recovered with AMPure beads (Beckman Coulter, Brea, California, USA). Samples were then moved to Cornell Life Sciences Sequencing and Genotyping Facility for sequencing on an Illumina MiSeq. Raw sequence reads were then assembled using SeqMan NGen (v.11, Lasergene Genomics Suite; DNASTAR, Madison, Wisconsin, USA). Contigs containing microsatellite repeats were identified using MSATCOMMANDER version 1.0.3 (Faircloth, 2008), and possible primer pairs were identified.
One hundred fifty-two primer pairs were selected to screen for amplification in eight individuals: six H. naniflora, one H. heterophylla. and one H. minor. PCR amplifications were prepared in a 10-µL reaction consisting of GoTaq Flexi Buffer, 2.5 mM MgCL2, 800 µM dNTPs, 0.5 µM of each primer, 0.5 units of GoTaq Flexi DNA Polymerase, and ∼20 ng of DNA (Promega, Madison, Wisconsin, USA). PCR was completed using a touchdown thermal cycling program on a Techne TC-5000 Thermal Cycler (Bibby Scientific Limited, Stone, Staffordshire, United Kingdom) encompassing a 13°C span of annealing temperatures from 68°C to 55°C. Initial denaturation was at 94°C for 5 min, 13 cycles at 94°C for 45 s, touchdown for 2 min, and 72°C for 1 min; followed by 24 cycles at 94°C for 45 s, 55°C for 1 min, and 72°C for 1 min; followed by a final extension at 72°C for 5 min. The PCR products were examined on a 1% agarose gel and scored for presence or absence of an appropriately sized PCR product. Twenty primer pairs produced repeatable results across all three species (Table 1). These were further screened for polymorphism on a total of 68 individuals, including 44 H. naniflora, 10 H. minor, and 14 H. heterophylla (Appendix 1).
Polymorphism screening PCR reaction conditions were the same as above, except the forward primer concentration was reduced to 0.25 µM, and 0.25 µM of an M13 primer (5′-CACGACGTTGTAAAACGAC-3′), labeled with FAM, VIC, NED, or PET (Life Technologies), was added to the reaction. PCR products labeled with different fluorescent dyes were then pseudo-multiplexed, and 2 µL of the combined reactions were submitted for genotyping on an ABI3730 DNA sequencer using a GeneScan 500 LIZ Size Standard (Life Technologies). Resulting chromatograms were visualized and scored using the software package Geneious Version 7 (Biomatters Ltd., Auckland, New Zealand). The resulting genotypic data were then analyzed with GenAlEx version 6.5 (Peakall and Smouse, 2006, 2012) to obtain standard descriptive statistics and to test for deviations from Hardy–Weinberg equilibrium assumptions (Table 2).
Characteristics of 20 microsatellite primer pairs developed for Hexastylis.
Sixteen of the primer pairs tested were polymorphic, with the number of alleles ranging from two to 23 (mean ∼8.8) in H. naniflora, two to nine (mean ∼4.9) in H. minor, and one to 14 (mean ∼6.1) in H. heterophylla (Table 2). Excessive homozygosity was identified at several of the loci in all three species, and locus Hn00567 was monomorphic in H. heterophylla. A total of 52 private alleles were identified in one of the three species, mostly at low frequencies (<0.05). Three of these private alleles in H. naniflora (Hn7116 [422 bp], Hn01135 [300 bp], and Hn00304 [179 bp]), one in H. minor (Hn00252 [224 bp]), and one in H. heterophylla (Hn00002 [297 bp]) were identified with a frequency greater than 10%, and these can be diagnostic in species identification when morphological characters are unavailable.
Sixteen polymorphic microsatellite markers were developed for H. naniflora, and these primers also amplify in two other species of Hexastylis (H. heterophylla and H. minor). These markers provide a means to assess genetic diversity and to assist in circumscription of the three species in the H. heterophylla complex. This provides the first opportunity to examine species boundaries and hybrids in the complex with molecular tools; application of these tools should lead to a reassessment of distributions and hybrid zones. These markers will also be valuable tools for vegetative identification of new Hexastylis populations when flowers are unavailable. These primers may also be useful in other species of Hexastylis and Asarum.
Standard descriptive statistics for 16 polymorphic microsatellite loci in three species of Hexastylis.
 We acknowledge Margaret Roberts, Taylor Jenson, and George Godsmark for efforts that were vital to the collection of plant material. We also thank the Department of Biology at Appalachian State University, the North Carolina Department of Transportation, and the North Carolina Natural Heritage Program for funding and support.