DOI: 10.5061/dryad.5qfttdz8z
title: Epidendrum radicans - genetic data of 4 regional 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: Study sites and sampling Samples were obtained
 from five E. radicans 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.  Arenal
 Slope Sites (AS) - 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.  Tabacón Springs Sites (TS) - 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 E. radicans 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.   Arenal 1968 Site (SE) - At this site
 (10.4705° N and 84.7317° W), because of the more challenging terrain, 116
 samples were collected from the nearest E. radicans 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) were collected from each of four E. radicans
 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. 
 Genotyping 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 E. radicans
 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.