Chloroplast loci <em>Abies religiosa</em> population from La Malinche National Park

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. Methods Vegetative Tissue Collection 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. Laboratory 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 Pinus thunbergii and P. leucodermis 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 TouchTM 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 >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).