Isolation and characterization of 13 microsatellites for the rare endemic shrub Tetratheca erubescens (Elaeocarpaceae)1

Ap Applicati tions ons in in Pl Plant t Scien Sciences ces Tetratheca erubescens J. P. Bull (Elaeocarpaceae) is a recently described species (Bull, 2007) endemic to the Koolyanobbing Range (a banded ironstone formation) within the Coolgardie Biogeographic Region of the South West Australian Floristic Region (SWAFR). Tetratheca erubescens inhabits rock crevices containing red sandy loam soils among hill crests, steep slopes, cliffs, and associated rocky monoliths at altitudes of 445–450 m a.s.l., within parts of the range. Although the reproductive biology of the species is unknown, fl ower morphology and presentation are similar to the related banded ironstone endemic T. paynterae Alford, which displays buzz pollination by a suite of small native bee species, high outcrossing, and restricted pollen dispersal (Butcher et al., 2011). Seed dispersal is likely to be myrmecochorous due to the presence of a large elaiosome on the seed. Due to its isolation, low total number of individuals (ca. 6000 plants), a narrow distribution limited to less than 2 km, its ecological association with steep cliffs and associated rocky slopes that are rare habitat types in the region, and proximity to mining activity, it is listed as a Rare Flora under the Wildlife Conservation Act 1950 (WA) (Western Australian Minister for Environment, 2013). To assess the potential genetic impact of proposed mining activity on T. erubescens , microsatellite markers were developed to characterize genetic diversity and its spatial structure across the species' range. METHODS AND RESULTS Genomic DNA was extracted from fresh stem material of a single plant sampled at Koolyanobbing using a modifi ed Carlson's method (Carlson et al., 1991) without the addition of β-mercaptoethanol to the lysis buffer and with the additional steps of potassium acetate following lysis incubation and a 5 M NaCl step followed by ethanol precipitation after the isopropanol precipitation step. Next-generation sequencing was performed on a Personal Genome Machine (PGM) semiconductor sequencer (Life Technologies, Carlsbad, Califor-nia, USA) at the Lotterywest State Biomedical Facility Genomics Node in Perth, Western Australia. Briefl y, 100 ng of DNA was sheared to approximately 300–400 bp using an S2 sonicator (Covaris, Woburn, Massachusetts, USA), and a single barcoded library was prepared using a NEBNext Ultra DNA Library Prep Kit (New England Biolabs , Ipswich, Massachusetts, USA). Size selection (insert sizes 330–360 bp) was performed by gel excision (E-Gel; Invi-trogen/Thermo Fisher Scientifi c, Waltham, Massachusetts, USA), and the libraries were assessed and quantifi ed using a …