DOI: 10.5061/dryad.kd51c5b8x
title: Epidendrum radicans – x, y coordinates and genetic data of individuals within 5 focal populations

description: Colonization is a fundamental ecological process that is important for the
 persistence of species, particularly when a changing environment
 necessitates range shifts. Vacant habitats available for colonization
 often arise from landscape disturbance. Colonization and population
 expansion processes can be inferred by examining the levels and spatial
 distribution of genetic variation of plant populations with known
 disturbance histories. Samples (N = 690) of the terrestrial orchid,
 Epidendrum radicans, were collected from five lava flow sites on the
 slopes of Volcán Arenal in Costa Rica that last experienced major
 eruptions in 1968 and 1992. Individuals were also sampled (N = 188) from
 four regional populations. Samples were characterized using 15 nuclear
 genetic markers and analyzed using population genetics statistics. Genetic
 diversity within sites was moderate (He = 0.092–0.192). Contrary to
 expectation, diversity tended to be lower on the older lava flows (0.131
 versus 0.172) which may reflect their more sheltered topography that
 restricted pollen/seed immigration, and/or greater intra- and
 interspecific competition. Genetic diversity measures indicate that the
 lava flows were colonized by numerous individuals that likely originated
 from multiple sources while spatial genetic structure (SGS) statistics
 indicate that most recruitment in the study sites subsequent to
 colonization resulted from in situ reproduction and localized seed
 deposition. Younger sites had significantly greater SGS over larger
 distances which reflects fewer reproductive events, and less spatial and
 temporal overlap of seed shadows relative to the older sites. Clones were
 also generally larger on the older sites (≤ 8m versus ≤ 3m).
description: <strong>Study sites and sampling</strong>
 Samples were obtained from five <em>E</em>.
 <em>radicans</em> collection sites growing on lava flows of
 different ages. At all five sample sites, we made every effort to avoid
 collection of multiple samples from obviously clonal individuals. The two
 Arenal Slope (AS) sites are located on the southwestern slope of Volcán
 Arenal where lava, rocks and ash were deposited during the 1992 eruption
 (Soto & Sjoböhm, 2005; Fig. 1). The two Tabacón Springs (TS) sites
 occur on an older lava flow last disturbed by bomb and block bombardment,
 and ash fall in 1968 (Soto & Sjoböhm, 2005; Fig. 1). The TS sites
 are separated from the AS sites by approximately 4.7 km. The Arenal 1968
 (SE) site is on a lava field deposited during the 1968 eruption (Fig. 1).
 This site is ~2.3 km from AS and ~2.7 km from TS. 
 <em>Arenal Slope Sites</em> (<em>AS</em>)
 - In April 2004, leaves were collected at the first Arenal Slope site
 (10.4504° N and 84.7250° W) from 96 individuals (AS-1). In April 2010,
 leaves were collected at the second Arenal Slope site from 103 individuals
 (AS-2). The two AS sample sites are separated by ~90 m. From a central
 point for each of the two AS collections, a transect line was placed
 successively in a radial pattern and a leaf was collected from every
 individual, with its distance and angle relative to the central point
 recorded.  <em>Tabacón</em>
 <em>Springs</em> <em>Sites</em>
 (<em>TS</em>) - At the Tabacón Springs location (10.4924° N
 and 84.7197° W), samples were collected from 209 individuals in April 2005
 (TS-1) and 166 individuals in April 2010 (TS-2). These two sites are
 separated by ~45 m. Due to the higher density of individuals, the TS
 collections represented a sub-sample of the total number of
 <em>E</em>. <em>radicans</em> individuals. For
 TS-1 a baseline was established and 8m long transect lines were placed
 perpendicular to the baseline at 0.8m intervals. At 0.8m intervals along
 each of 21 transect lines, a leaf was collected from the nearest
 individual and its distance from the 0.8m interval mark on the transect
 line was recorded. For TS-2, because of differing terrain, 11m long
 transect lines were placed perpendicular to the baseline at 0.8m
 intervals. At 1m intervals along each of these 20 transect lines, a leaf
 was collected from the nearest individual and its distance from the 1m
 interval mark on the transect line was recorded.  
 <em>Arenal 1968 Site</em> (<em>SE</em>) -
 At this site (10.4705° N and 84.7317° W), because of the more challenging
 terrain, 116 samples were collected from the nearest
 <em>E</em>. <em>radicans</em> encountered on
 either side of a 20m transect line and the distance of each plant from the
 transect line was recorded. To compare the genetic
 composition of lava flow populations with populations in the surrounding
 landscape, additional regional populations were sampled. A mean of 47
 individuals (range 42–50) was collected from each of four
 <em>E</em>. <em>radicans</em> populations located
 within 1.5 to 26.8 km (mean = 17.4 km) of the study sites. While we did
 not survey the surrounding landscape comprehensively, we collected from
 every population found in the vicinity. 
 <strong>Genotyping</strong> Leaves
 were snap frozen in liquid nitrogen within a few hours of collection and
 sent to the University of Georgia in an ultra-cold dry shipper. Leaves
 were crushed in chilled mortars with a pestle, liquid nitrogen, and a
 pinch of sea sand to disrupt cellular compartmentalization. Enzymes were
 extracted from <em>E</em>. <em>radicans</em>
 tissue with a polyvinylpyrrolidone-phosphate extraction buffer (Mitton et
 al., 1979). The resulting slurry containing crude protein extract was
 absorbed onto 4x6 mm wicks punched from Whatman 3 mm chromatography paper.
 Wicks were stored in microtest plates at -70°C until used for
 electrophoresis. Wicks were placed in horizontal starch gels (10%) and
 electrophoresis was performed.  Nine enzyme stains in
 five buffer systems resolved one monomorphic and 14 putative polymorphic
 loci. Enzymes stained and polymorphic loci (in parentheses) for each
 buffer system were: (1) system 4, malate dehydrogenase (EC 1.1.1.37;
 Mdh-1, Mdh-2), 6-phosphogluconate dehydrogenase (EC 1.1.1.44; 6-Pgd-2) and
 shikimic dehydrogenase (EC 1.1.1.25; Skdh);  (2) system 8-, aspartate
 aminotransferase (EC 2.6.1.1; Aat-1, Aat-2), and fluorescent esterase (EC
 3.1.1-; Fe-2, Fe-3); (3) system 10, triosephosphate isomerase (EC 5.3.1.1;
 Tpi-1, Tpi-2) and UTP-glucose-1-phosphate (EC 2.7.7.9; Ugpp-1); (4) system
 11, isocitrate dehydrogenase (EC 1.1.1.42; Idh), and phosphoglucoisomerase
 (EC 5.3.1.9; Pgi-2);  (5) system 6, fluorescent esterase (EC 3.1.1-;
 Fe-4).  All buffer and stain recipes were adapted from
 Soltis et al. (1983) except Aat (Cheliak & Pitel, 1984) and Ugpp
 (Manchenko, 1994). Buffer system 8- is a modification of buffer system 8
 described by Soltis et al. (1983). Banding patterns of both species were
 consistent with Mendelian inheritance patterns expected for each enzyme
 system (Weeden & Wendel, 1989) and were consistent with disomic
 inheritance expected for diploid individuals.
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