Methods
Microsatellite Development and Cross-species utility
MethodsWe screened 23 primer sets developed from eight bird species, including five species from the Petroicidae and three from other families (19 novel primers, plus Pau2 Petroica australis (Townsend et al. 2012), TG02088 Taeniopygia guttata (Dawson et al. 2010), Ase18Acrocephalus sechellensis (Richardson et al. 2000), Ppi2 Pica pica (Martinez et al. 1999) To identify novel microsatellite loci, DNA was extracted from frozen tissue of each robin species using the DNeasy blood and tissue DNA extraction kit (QIAGEN). A total of 16.3 µg of RNAse-treated genomic DNA was used in 1/8 of a plate for pyrosequencing by an external service provider, the Australian Genome Research Facility (www.agrf.com.au), on a Roche GL FLX (454) system. We used the program QDD (Meglécz et al. 2009), to detect microsatellites and subsequently to design primers. We identified 1,412 sequences that contained putative microsatellite motifs with a minimum of 5 repeats and which had sufficiently-long flanking regions free of nanosatellites for which primers could be designed.We selected 19 loci (based on repeat length, repeat motif and PCR product size) for genotyping, and optimized them into three multiplex panels (Supp Table 2). Eight loci were chosen from the Flame Robin (Petroica phoenicea) (our species of special interest), four each from the congeneric Scarlet Robin (P. boodang) and Red-capped Robin (P. goodenovii), and three from the confamilial Eastern Yellow Robin (Eopsaltria australis). To evaluate cross-amplification amongst different species of Petroicidae, blood samples were collected from the brachial vein (Owen 2011) of 25 Flame Robins, 20 Redcapped Robins, 18 Scarlet Robins and 31 Eastern Yellow Robins (including both mitochondrial clades (Pavlova et al. 2013). A further 45 samples of P. phoenicea were collected from four locations spanning 690 km of the species range (see Table 1. for locations and sample sizes). Samples were stored at -20 0 C prior to DNA extraction. All samples were collected under approval of Deakin University Animal Ethics Committee A58-2011, Victoria Department of Environment, Land, Water and Planning Scientific Permit 10005964, Australian Museum Animal Ethics Approval 12-03 and New South Wales Scientific Licence SL100886.DNA samples were sent to the Australian Genome Research Facility, Melbourne, for genotyping. All forward primers incorporated a fluorescent label (FAM, VIC, NED or PET) to allow multiplexed PCR products to be run on an AB 3730xl Sequencer (Applied Biosystems).Genotypes were scored independently by two individuals using GeneMapper version 1.4 (Applied Biosystems). MICROCHECKER 2.2 (Van Oosterhout et al. 2004) was used to check for the presence of null alleles. In addition, significant departures from Hardy-Weinberg and linkage disequilibrium (LD) for each locus site combination were calculated in Arlequin version 3.5.1.2 (Excoffier and Lischer 2010). Significance of tests for each locus or site were assessed using sequential B...