Abstract The endangered orchid, Microtis angusii, was recently described from a single population consisting of approximately 100 plants. This species is morphologically very similar to close relatives, and taxonomic difficulties have hindered attempts to identify further populations for protection. Here we present a rapid, economical, PCR-based assay for the effective identification of this species based on rDNA sequence variation. Using two single nucleotide sub- stitutions in the internal transcribed spacer (ITS) region of the ribosomal DNA that are diagnostic for M. angusii, we developed an allele-specific PCR that can be easily visualized on a standard agarose gel, avoiding the use of expensive restriction enzymes and DNA sequencing reagents and equipment. Using PCR primer pairs for both the M. angusii, and the alternate allele, we also detected an individual heterozygous for the two alleles, indicating a need for further detailed genetic study. We performed a ‘blind trial’ to confirm the utility of this assay. Mic- rotis angusii samples were successfully discriminated from amongst several congeners, and a further, pre- viously unknown, population of the species was identified. Keywords Species diagnosis Conservation Practical outcomes ITS SNP Amplification refractory mutation system (ARMS) Introduction As species are the common currency for conservation efforts, their accurate identification is essential for the effective preservation of biological diversity. None- theless, some species are difficult to identify through traditional taxonomic methods. Such species include the so-called ‘cryptic’ species—morphologically indis- tinguishable lineages identified only by species-level genetic differences. Other species are difficult for anyone other than an experienced taxonomist to identify. With an increasing deficit of expert taxono- mists (Mace 2004), the identification of a threatened species may be an obstacle to the implementation of conservation measures such as the location of further populations for protection, and the determination of species distributions. In this case, molecular genetic techniques have potential to provide alternative fast and effective means for species identification. How- ever, the use of molecular methods may add consid- erable cost to a conservation program, and so assays N. S. Flanagan (&) R. Peakall School of Botany and Zoology, The Australian National University, Canberra, ACT 0200, Australia e-mail: nicflanagan@fastmail.fm M. A. Clements J. T. Otero Centre for Plant Biodiversity Research, GPO Box 1600, Canberra, ACT 2601, Australia Present Address: J. T. Otero Dept. de Ciencias Agricolas, Universidad Nacional de Colombia, Palmira Valle, Colombia Present Address: N. S. Flanagan Genetics & Biotechnology, Department of Biochemistry, University College Cork, Cork, Ireland Conserv Genet DOI 10.1007/s10592-006-9198-6 123 SHORT COMMUNICATION Identification of the endangered Australian orchid Microtis angusii using an allele-specific PCR assay Nicola S. Flanagan Rod Peakall Mark A. Clements J. Tupac Otero Received: 21 March 2006 / Accepted: 31 July 2006 Ó Springer Science+Business Media B.V. 2006