DOI: 10.5061/dryad.98sf7m0j8
title: Drakaea glyptodon nuclear microsatellite and chloroplast haplotype data

description: 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 (Ne), 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, Ne values that generally exceed predictions
 based on the number of flowering individuals, and moderate levels of gene
 flow. Two populations were founded by &lt; 5 colonists suggesting some
 populations are colonized by few seeds, with growth largely resulting from
 in situ recruitment. A value of 3.65 for mp /ms 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.
description: 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 <i>et al</i>. (2010):
 <i>Dg</i>A108, <i>Dg</i>A111,
 <i>Dg</i>A114, <i>Dg</i>A116,
 <i>Dg</i>B106, <i>Dg</i>B109,
 <i>Dg</i>C106, <i>Dg</i>C110,
 <i>Dg</i>D3 and <i>Dg</i>D102. Four loci
 (<i>Dg</i>C106, <i>Dg</i>C110,
 <i>Dg</i>D3, and <i>Dg</i>D102) 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 One<i>Taq</i>® 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 One<i>Taq</i>® 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 <i>et
 al</i>. (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): <i>Chilo</i>CP01,
 <i>Chilo</i>CP09, <i>Chilo</i>CP36,
 <i>Chilo</i>CP39 and <i>Chilo</i>CP68. As with
 nuclear primers, a 3-primer approach was employed (Hauswaldt &
 Glenn, 2003). PCR amplification followed protocols of Ebert <i>et
 al</i>. (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 <i>et al.,
 </i>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®.
description: <strong>Nuclear microsatellite data</strong>: Missing
 data are indicated as 000.