Hybridization between rare and widespread species can result in loss of genetic integrity for the rarer species, which can have management and conservation implications. One rare species, Phlox hirsuta, is a serpentine endemic in northern California, and it frequently co-occurs with a widespread congener, P. speciosa. Putative hybrids were recognized based on intermediate morphology, so the possibility of hybridization was explored using floral morphological and molecular data. Ninety-eight individuals of P. hirsuta and P. speciosa were collected from each of three populations, and floral features were measured and compared. Eleven microsatellite loci were amplified for species and putative hybrids, and inter- and intraspecific genetic diversity and relationships were investigated with multiple methods. Variation in morphological and molecular data was recognized. Floral variation was greater for P. hirsuta than P. speciosa. Putative hybrids were genetically allied with P. speciosa, but two individuals of P. hirsuta were resolved to have genetic similarity with P. speciosa. While hybridization is possible between the species, it is uncommon and appears to be primarily unidirectional, with P. speciosa as the hypothesized male parent and P. hirsuta as the hypothesized female parent. The small number of recognized hybrids may be due to ineffective interspecific pollination, early acting inbreeding depression, hybrids being less fit than parents, and/or small sample sizes. Reinforcement does not appear to play a role in secondary contact between species. Both microsatellite loci and floral morphology varied across the small geographic range of P. hirsuta, suggesting local differentiation and adaptation are possible over short distances.

Flowers of 98 individuals of each of Phlox hirsuta and P. speciosa were collected from three populations where both species co-occur: China Hill, Greenhorn, and Soap Creek Ridge (Fig. 1).  Flowers were preserved in 100% EtOH. Flowers were dissected under a Nikon SMZ800N stereomicroscope, and images of the corolla, androecium, and gynoecium were captured using NIS-Elements software with a Nikon DS-Fi2 digital camera.  Using the NIS-Elements software, measurements were made for multiple floral features.

Leaf material of P. hirsuta was collected, in 2013, from 38-42 individuals in each of four putative populations – Cracker Gulch (CG), China Hill (CH), Greenhorn (G), Soap Creek Ridge (SC) (Fig. 1) – and in 2016, leaf material for P. speciosaand putative hybrids of the two species was collected from seven and four individuals from two populations (CH and SC), respectively.  The latitude and longitude were recorded for each collection. Leaf material was dried in silica gel and stored at 4° C.  Leaf material for 186 samples from the four populations of P. hirsuta, two populations of P. speciosa, and putative hybrids of the two species was sent to the biotechnology company Eurofins BioDiagnostics for DNA isolation and microsatellite amplification and sequencing using an ABI 3730XL, with a GeneScan 500LIZ size standard. Twelve microsatellite loci that were identified for related species of Phlox were amplified.