TECHNICAL NOTE Development of twenty-four novel microsatellite markers for the freshwater crayfish, Geocharax gracilis, using next generation sequencing Craig D. H. Sherman • Daniel Ierodiaconou • Annalise M. Stanley • Kim Weston • Michael G. Gardner • Mark B. Schultz Received: 5 December 2011 / Accepted: 11 December 2011 / Published online: 21 December 2011 Ó Springer Science+Business Media B.V. 2011 Abstract The crayfish Geocharax gracilis is an important inhabitant of natural and agricultural drainage systems in south-eastern Australia. To investigate population structure, genetic diversity and patterns of connectivity in natural and human-altered ecosystems, we isolated and characterised 24 microsatellite loci using next generation sequencing. Loci were initially tested for levels of variation based on 12 individuals from across the species’ geographical range. A further 33 individuals from a single population were used to test for departures from Hardy–Weinberg equilibrium and linkage disequilibrium. We detected high to moderate levels of genetic variation across most loci with a mean allelic richness of 8.42 and observed heterozygosity of 0.629 (all samples combined). We found no evidence for linkage dis- equilibrium between any loci and only three loci (Geo01, Geo24 and Geo47) showed significant deviations from Hardy–Weinberg expectations. These same three loci, plus two additional loci (Geo06 and Geo28), also showed the presence of null alleles. These 24 variable markers will provide an important tool for future population genetic assessments in natural and human altered environments. Keywords Crustacean Á Dispersal Á Genetic structure Á Mating system Á Gene flow Globally, freshwater crayfish are a highly threatened group and are especially vulnerable to anthropogenic-mediated changes to their habitats such as environmental degradation and agricultural development (Crandall et al. 1999; Jones et al. 2007; Schultz et al. 2008). Crayfish are often the largest invertebrates in many freshwater systems and occupy an important place in food webs and are involved in a variety of important ecosystem processes (Crandall et al. 1999; March and Robson 2006; Johnston and Robson 2009). The freshwater crayfish Geocharax gracilis is endemic to Victoria and Tasmania, Australia, and is regarded as a short-range endemic species (Harvey 2002). It is commonly found in freshwaters in lowland coastal, inland and highland zones, and is often associated with agricultural drainage systems (Schultz et al. 2007, 2008). The development of extensive and highly connected irri- gation systems has the potential to drastically alter popu- lation connectivity and subsequent patterns of genetic diversity and structure in this and other freshwater species (Bilton et al. 2001; Lada et al. 2010; Casas et al. 2011). Increased levels of gene flow through drainage systems may result in increased levels of genetic diversity at the population level and allow rapid re-colonisation of extinct populations; however negative effects of genetic swamping C. D. H. Sherman (&) Á A. M. Stanley Á K. Weston School of Life and Environmental Sciences, Centre of Integrative Ecology, Deakin University, Pigdons Road, Waurn Ponds, VIC 3217, Australia e-mail: craig.sherman@deakin.edu.au D. Ierodiaconou Á M. B. Schultz School of Life and Environmental Sciences, Deakin University, Warrnambool, VIC 3280, Australia M. G. Gardner School of Biological Sciences, Flinders University, GPO Box 2100, Adelaide, SA 5001, Australia M. G. Gardner School of Earth and Environmental Science, Australian Centre for Evolutionary Biology and Biodiversity, University of Adelaide, Adelaide, SA, Australia M. B. Schultz Division of Infection and Immunity, Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, VIC 3052, Australia 123 Conservation Genet Resour (2012) 4:555–558 DOI 10.1007/s12686-011-9590-5