Sixteen polymorphic microsatellite markers for a federally threatened species, Hexastylis naniflora (Aristolochiaceae), and co-occurring congeners1

and several characteristics of fl ower morphology (Gaddy, 1987). Multiple species complexes have been identifi ed in this genus and this study focuses on the H. heterophylla complex, containing H. heterophylla The species in this complex are sympatric over portions of their ranges. Vegetative characters have limited taxonomic value, leaving ephemeral fl oral morphology as the only diagnosable fi eld character for identifi cation. Previous studies have recognized intermediate fl oral morphologies in some populations, leading some to question the validity of species circumscriptions. This is particularly problematic in H. nanifl ora , 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. nanifl ora from H. minor and H. heterophylla , to address questions of hybridization, and to identify evolutionarily signifi cant 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 fl ow-ering 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. nanifl ora and one plant of H. heterophylla (selected from geographic ranges where the species do not overlap and confi dently identifi ed using fl ower material) were sent to the Cornell University Evolutionary Genetics Core Facility where total DNA was extracted using a QIAGEN Plant Mini Kit Enriched fragments were then captured by streptavidin-coated magnetic beads (New England Biolabs) and PCR amplifi ed. 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 (Beck-man 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 containing microsatellite repeats were identifi ed using MSATCOMMANDER version 1.0.3 (Faircloth, 2008), and possible primer pairs were identifi ed. One hundred fi fty-two primer pairs were selected to screen for amplifi cation in eight individuals: six H. nanifl ora , one H. heterophylla , and one H. minor. • Premise …