DOI: 10.5061/dryad.gf1vhhmqn
title: Genetic structure in patchy populations of a candidate foundation plant: a case study of Leymus chinensis using genetic and clonal diversity

description: PREMISE: The distribution of genetic diversity on the landscape has
 critical ecological and evolutionary implications. This may be especially
 the case on a local scale for foundation plant species since they create
 and define ecological communities, contributing disproportionately to
 ecosystem function. METHODS: We examined the distribution of genetic
 diversity and clones, which we defined first as unique multilocus
 genotypes (MLG), and then by grouping similar MLGs into multilocus
 lineages (MLL). We used 186 markers from inter-simple sequence repeats
 (ISSR) across 358 ramets from 13 patches of the foundation grass Leymus
 chinensis. We examined the relationship between genetic and clonal
 diversities, their variation with patch-size, and the effect of the number
 of markers used to evaluate genetic diversity and structure in this
 species. RESULTS: Every ramet had a unique MLG. Almost all patches
 consisted of individuals belonging to a single MLL. We confirmed this with
 a clustering algorithm to group related genotypes. The predominance of a
 single lineage within each patch could be the result of the accumulation
 of somatic mutations, limited dispersal, some sexual reproduction with
 partners mainly restricted to the same patch, or a combination of all
 three. CONCLUSIONS: We found strong genetic structure among patches of L.
 chinensis. Consistent with previous work on the species, the clustering of
 similar genotypes within patches suggests that clonal reproduction
 combined with somatic mutation, limited dispersal, and some degree
 of sexual reproduction among neighbors causes individuals within
 a patch to be more closely related than among patches.
description: <strong>Sampling
 design</strong> We collected tissue samples in
 August 2010, when the plants were vigorously growing. Thirteen
 <em>L. chinensis</em> patches were selected haphazardly within
 an area of 2000m<sup>2</sup>. Patches were defined naturally
 (by the space occupied by <em>L. chinensis</em> locally), and
 all patches were separated from others by at least 200m. Patches ranged in
 area from 2.8m<sup>2</sup> to 166.8m<sup>2</sup>
 We collected vegetative tillers of <em>L. chinensis</em> at
 distances of 0, 0.5, 1, 2, 4, 8, and 16m from the center of each patch (or
 to the edge of the patch, whichever came first) in each of eight
 directions (N, S, E, W, NE, NW, SE, SW). Consequently, we sampled 17-35
 ramets from each patch (Table 2). The vegetative ramets of <em>L.
 chinensis </em>were harvested at the ground surface and each ramet
 was put into a zip-lock plastic bag with silica gel to preserve the
 tissue. We transported all samples to the laboratory and stored them at
 -20℃. <strong>DNA
 extraction</strong> For each sample, we performed
 extractions of total genomic DNA using a modified CTAB method (Doyle and
 Doyle, 1987). We ground tissue of dried young leaves (0.025 to 0.030g) to
 a fine powder in liquid nitrogen with pure quartz sand in a 2 ml
 centrifuge tube. Then, we added 400 μl of extraction buffer [2% cetyl
 trimethyl ammonium bromide (CTAB); 100 mM Tris (pH=8.0); 20 mM
 ethylenediamine tetra-acetic acid (EDTA); 0.5%
 NaHSO<sub>3</sub>; 1.4 M NaCl; 1% polyvinylpyrrolidone (PVP);
 2% β-mercaptoethanol] to the ground sample and incubated at 65℃ for 30
 minutes. We performed two rounds of extraction with 400 μl of
 tris-saturated phenol. After centrifugation, we performed two more rounds
 of extraction with chloroform: isoamyl alcohol (24:1) and precipitated the
 DNA with 200 μl of 5 M potassium acetate and 1ml of 100% ice-cold ethanol.
 The precipitate was washed with 70% ethanol twice, dried, and resuspended
 in 100ul of 1×Tris-EDTA (TE) buffer (Shimizu et al., 2002). We estimated
 the quantity of DNA by comparing band intensities with known amounts of
 lambda DNA on 0.8% agarose gel. DNA quality was checked by
 spectrophotometry at 260 nm and 280 nm. All the DNA samples were stored at
 -20℃ for ISSR analysis. <strong>ISSR
 amplification</strong> Twenty-one primers from the
 Biotechnology Laboratory, University of British Columbia (UBC set no.9)
 were synthesized by Sangon (Shanghai Sangon Biological Engineering
 Technology and Services Co. Ltd, Shanghai, China) for initial screening.
 Eventually, we selected 11 primers that produced clear bands with good
 reproducibility and high polymorphism (Table 1). We performed ISSR amplification in
 a volume of 10 μl containing 10ng template DNA, 1μl 10×PCR Buffer, 20 mM
 MgCl<sub>2</sub>, 10 mM dNTPs, 1.5 μM primer, and 0.4U of Taq
 DNA polymerase. All amplifications were carried out in a Veriti 96 Well
 Thermal Cycler (Applied Biosystems, USA). For primers 6, 10, 13, 807, 818,
 827, 834, 842, 848 and 855, the PCR reaction procedure was as followed: an
 initial denaturation step of 2 min at 94℃, 35 cycles of 1 min at 94℃, 1
 min at respective <em>T</em><sub>m</sub> values
 (Table 1), and 1 min at 72℃, and a final extension step of 10 min at 72℃.
 For primer 811, the amplifications began with initial denaturation for 5
 min at 94℃, followed by 45 cycles of 30 s at 94℃, 45 s at 56℃, and 2 min
 at 72℃, and a final extension at 72℃ for 5 min. The amplification products
 obtained were separated on 2% agarose gels containing 0.1% ethidium
 bromide at 120 V for 2h, and photographed under UV light. The molecular
 weight of every amplified band was evaluated by using the DL 2000 Marker
 (Beijing Dingguo Changsheng Biotechnology Co. Ltd, Beijing, China). Each
 reaction was repeated twice for each sample with each primer. Only those
 primers which generated reproducible bands in both reactions for each
 sample were used for the data analysis.
description: Methods are completely described in the paper. R code is
 provided.