DOI: 10.5061/dryad.18931zd22
title: Chloroplast loci <em>Abies religiosa</em> population from La Malinche National Park

description: Genetic structure of a population can be defined by the resistance of the
 landscape, the distance between populations, or geographic barriers. We
 analyzed the population genetic structure of Abies
 religiosa on a fine spatial scale and examined isolation models
 by resistance, distance, and barrier. We collected vegetative tissue from
 populations located at the altitudinal extremes of the distribution range
 of the species on three slopes of La Malinche National Park (LMNP) (South,
 North, and East) in central Mexico. Genomic DNA was obtained using the
 CTAB 2X method, and eight microsatellite chloroplast loci were amplified.
 The genetic structure was identified based on a Discriminant Analysis of
 Principal Components with cross-validation and a spatial Principal
 Component Analysis using the Gabriel-type connectivity network. The
 isolation hypotheses were evaluated by constructing partial Mantel tests
 using Reciprocal Causal Modeling and Maximum Likelihood Population Effects
 models. A genetic structure of isolation by barrier was identified, and
 two genetic groups were recognized: one including populations of the South
 slope and the other comprising populations of the North and East slopes.
 The greatest genetic exchange between groups was recorded in populations
 located at higher altitudes. It is suggested to promote the connectivity
 between slopes through assisted migration and immediately halt land-use
 changes, as part of the actions to preserve genetic diversity at LMPN.
 This study contributes to the knowledge of the spatial genetic structure
 of species at risk that are components of the Mexican temperate
 forest. 
description: <em>Vegetative Tissue
 Collection</em> We toured three slopes of the LMNP that are
 relatively easy to access (North, East, and South) because of the
 orography and the type of ownership (social) of agricultural plots
 (López-Téllez et al., 2019), aiming to identify the relative abundance of
 the species within its altitudinal range (2400–3600 m a.s.l.; Sáenz-Romero
 et al., 2012). Two collection sites were selected on each slope, which
 were assumed to be distinct populations located at the upper and lower
 limits of its altitudinal range, with a total of six populations
 throughout the LMNP (2 populations × 3 slopes) (Fig. 1). We established a
 central point in each population, from which two transects were drawn in
 opposite orientations. Along each transect, vegetative material was
 collected from 20 individuals separated by at least 30 m (Fig. 1). The
 geographic location of each individual was recorded; then, young undamaged
 needles were collected, transported, and stored in 1.5 mL Eppendorf tubes
 with Tris-EDTA pH 8.0 (Sigma-Aldrich) buffer. 
 <em>Laboratory</em>
 Tissue samples were first
 ground with liquid nitrogen (Doyle, 1990); afterward, genomic DNA was
 extracted using the CTAB 2X method. The extractions were visualized in 1%
 agarose electrophoresis to determine their viability. Unsuccessful samples
 were purified using Wizard ® SV Minicolums (Promega) columns and Wash
 Buffer 2 (Qiagen) following the vendor-standardized procedure, although
 modifying the concentrations to improve the
 result.  Ten chloroplast microsatellites (cpSSR) designed
 for <em>Pinus thunbergii</em> and <em>P.
 leucodermis</em> were amplified (Vendramin et al., 1996).
 Amplifications were run with a final volume of 14 µl using the Master Mix
 (Taq DNA Polymerase; Qiagen) solution in a T100
 Touch<sup>TM</sup> Thermal Cycler (Bio-Rad). The PCR
 conditions proposed by Vendramin et al. (1996) were modified: initial
 denaturation at 95 ºC for 5 min, then 32 cycles with denaturation at 94 ºC
 for 1 min, alignment at 50 ºC–58 ºC for 1 min (see Table 1), followed by
 extension at 72 ºC for 1 min. The final extension step was at 72 ºC for 8
 min. The PCR product was read by capillary electrophoresis (QIAxcel,
 Qiagen) using the method OM500, with a 10-bp resolution for fragments of
 100–500 bp. Amplicons were considered different when they were &gt;10
 bp (Qiagen, 2008); amplicon size was determined with the ScreenFel
 software (Qiagen v 1.0.2.0; Ambion Inc., Austin TX) provided by the
 QIAxcel system, using the 15 bp/500 bp QX Alignment Marker and the 25–500
 bp QX DNA Marker. The binning of the fragments obtained was carried out
 with the program Allelogram v. 2.2 (Morin et al.,
 2009).
description: <em>Genetic
 Diversity</em> Since the chloroplast has a haploid genome, it
 has paternal inheritance in conifers and is not subject to recombination
 (Neale and Sederoff, 1989; Watano et al., 1996); hence, it can be
 considered as a locus (Rai and Ginwal, 2018). It can also be perceived as
 a circular haploid chromosome in which sequence variation generates
 different alleles within individual non-recombinant loci (Echt et al.,
 1998). Therefore, this study analyzed genetic diversity based on
 haplotypes formed by combining fragments from the eight successfully
 amplified microsatellite loci (cpSSR) (Rai and Ginwal, 2018). Genetic
 diversity was described through the number of observed alleles
 (N<sub>a</sub>), number of effective alleles
 (N<sub>e</sub>), Shannon-Weiner index (I), haplotype diversity
 (h), percentage of polymorphic loci (%P), and genetic distances (Nei,
 1987), using the program GenAlEx v. 6.4 (Peakall and Smouse,
 2006).  <em>Genetic
 Structure</em> The pattern of spatial genetic variation was
 evaluated using a Discriminant Analysis of Principal Components (DAPC)
 with cross-validation (Jombart et al., 2010). Connectivity was determined
 by constructing a spatial Principal Components Analysis (sPCA) (Jombart et
 al., 2008) through a Gabriel-type network. This network was selected
 because it has moderate saturation, which makes networks more informative
 than saturated ones (Dyer and Nason, 2004; Naujokaitis-Lewis et al.,
 2013). All analyses were carried out with the software R v. 4.0.3
 (packages adegenet, akima, poppr, ResistanceGA, and tess3r; R Core Team,
 2020).  To evaluate the hypotheses of isolation, we
 constructed matrices of resistance distances (IBR) and of barrier
 distances (IBB) between pairs of populations, with the program
 Circuitscape v. 4 (McRae and Shah, 2009). To this end, for IBR we
 elaborated a raster from the visual categorization of land use (farming,
 forest, secondary vegetation, urban, bare ground) with the program ArcGIS
 v. 10.4 (ESRI, 2018), from Landsat 5 images (30 m × 30 m resolution)
 downloaded from GloVis (https://glovis.usgs.gov). And for IBB, we obtained an elevation raster of the study area using the Digital Elevation Model downloaded from ASF Data Search Vertex (https://search.asf.alaska.edu/#/; Alos Pasar images, 12.5 m resolution). In the case of the IBD, we used linear geographic distances between population pairs calculated with the program GenAlEx v. 6.4 (Peakall and Smouse, 2006).  Once the matrices of each isolation hypothesis were constructed, partial Mantel tests were performed with the genetic distances (Nei, 1987), using Reciprocal Causal Modeling (RCM) to avoid erroneous conclusions due to the high correlation of alternative models (Cushman et al., 2006; Cushman and Landguth, 2010). In addition, to identify the factors that contribute to gene flow, Maximum Likelihood Population Effects (MLPE) models were performed, these linear random effects models account for the lack of independence between pairwise comparisons (Row et al. 2017; Burgess and Garrick 2020). The analyses were carried out using the software R v. 4.0.3 (packages MuMIn, vegan, and ComMLPE; R Core Team, 2020).
description: # Abies religiosa chloroplast microsatellite loci # Authors Brbara
 Cruz-Salazar (Consejo Nacional de Ciencia y Tecnologa-Universidad Autnoma
 de Tlaxcala, Centro Tlaxcala de Biologa de la Conducta-Estacin Cientfica
 La Malinche, Tlaxcala, Mxico) Maricela Garca-Bautista (El Colegio de la
 Frontera Sur, Laboratorio de Gentica, Carretera Panamericana y Perifrico
 Sur s/n, Barrio de Mara Auxiliadora, San Cristbal de Las Casas, Chiapas,
 Mxico). Lorena Ruiz-Montoya (El Colegio de la Frontera Sur, Departamento
 de Conservacin de la Biodiversidad, Carretera Panamericana y Perifrico Sur
 s/n, Barrio de Mara Auxiliadora, San Cristbal de Las Casas, Chiapas,
 Mxico). Jos Luis Martnez y-Prez (Centro de Investigacin en Gentica y
 Ambiente, Universidad Autnoma de Tlaxcala. Km 10.5 Autopista Tlaxcala-San
 Martn, Ixtacuixtla, Tlaxcala, Mxico). # Description of the data and file
 structure The file includes eight chloroplast microsatellite loci
 (Vendramin et al. 1996) of Abies religiosa obtained with capillar
 electrophoresis. The first column is the individuals. The second column is
 the population, and the third and fourth are the geographical position.
 From the column 5 to 12 are the the haplotypes of each loci for all
 individuals. With this haplotypes, we generate a file with one haplotype
 per individual.