Many orchids are characterized by small, patchily distributed populations. Resolving how they persist is important for understanding the ecology of this hyper-diverse family, many members of which are of conservation concern. Ten populations of the common terrestrial orchid Drakaea glyptodon from Southwest Australia were genotyped with ten nuclear and five chloroplast SSR markers. Levels and partitioning of genetic variation, and effective population sizes (N_e), were estimated. Spatial genetic structure of nuclear diversity, together with chloroplast data, are used to infer the effective number of seed parents per population. We found high genetic diversity, N_e values that generally exceed predictions based on the number of flowering individuals, and moderate levels of gene flow. Two populations were founded by < 5 colonists suggesting some populations are colonized by few seeds, with growth largely resulting from in situ recruitment. A value of 3.65 for m_p /m_s indicates that pollinators play a greater role than seed in introducing genetic diversity to populations via gene flow. Our results highlight that D. glyptodon is highly effective at persisting in patchily distributed populations. However, it is important to examine how insights from this common, widespread species transfer to species that are rare and/or occur in fragmented landscapes.

Total genomic DNA was extracted using a modified CTAB protocol (Doyle & Doyle, 1987). Ten nuclear simple sequence repeat (SSR) loci were amplified with primers developed by Anthony et al. (2010): DgA108, DgA111, DgA114, DgA116, DgB106, DgB109, DgC106, DgC110, DgD3 and DgD102. Four loci (DgC106, DgC110, DgD3, and DgD102) are characterized by trinucleotide repeat motifs while six loci have dinucleotide repeat motifs. PCR amplification was based on a 3-primer protocol using a synthesized CAG-tag sequence (Hauswaldt & Glenn, 2003) whereby one primer in each primer pair was modified on the 5’ end with an engineered CAG-tag sequence [5’-CAGTCGGGCGTCATCA-3’] and a third fluorescently labeled (FAM, HEX or NED) primer that was identical to the CAG-tag was included in the PCR reactions. PCR amplification was carried out in 25 μl reaction volumes containing 1x OneTaq® buffer (New England Biolabs [NEB], Ipswich, Massachusetts, USA), 200 μM of each dNTP (NEB), 0.2 μM of the unlabeled primer, 0.2 μM of the CAG-tagged reverse primer, 0.02 μM of the fluorescently-labelled universal CAG-tagged primer, 0.025 U/μl of OneTaq® hot start DNA polymerase (NEB), 0.8 ng/μl of DNA template, and molecular biology grade water to volume. PCR amplification was performed on an Eppendorf Mastercycler Nexus Gradient thermal cycler and followed the protocol of Anthony et al. (2010), with the modification of a 30 s extension at 68° C, final extension of 15 min at 68° C, and a holding step at 4° C.

Samples were assayed for chloroplast (cpDNA) haplotype diversity using five SSR markers in non-coding regions of the large single copy region of the chloroplast (Ebert, Hayes & Peakall, 2009): ChiloCP01, ChiloCP09, ChiloCP36, ChiloCP39 and ChiloCP68. As with nuclear primers, a 3-primer approach was employed (Hauswaldt & Glenn, 2003). PCR amplification followed protocols of Ebert et al. (2009). PCR was performed in 25 μl reaction volumes using identical concentrations as for the nuclear markers. PCR amplification began with denaturation at 94° C for 3 min, followed by a touchdown series that began with an annealing temperature of 66° C and decreased by 3° C every other cycle until 54° C was reached, followed by 50° C for 30 cycles. Final extension was performed at 68° C for 5 min, followed by a holding step at 4° C (Ebert et al., 2009).

PCR products for up to three loci were combined with GeneScan® 500 ROX dye size standard and subjected to fragment analysis using an ABI 3730xl sequencer (Applied Biosystems) at the Georgia Genomics and Bioinformatics Core at the University of Georgia. Fragments were manually scored while viewing chromatographs in Geneious®.

Nuclear microsatellite data: Missing data are indicated as 000.