Study Area
This study was carried in the region known as the extreme northwest Balsas-Jalisco region in Mexico's Michoacan state. This region is an area with a complex topography due to the Western Sierra Madre, where many climatic conditions can be found in small distances. The annual mean temperature ranges are around 14 to 18°C, an altitude of 1500 to 1700 mamsl, and annual precipitation of 400 to 800 mm.
The agricultural activities in this region are influenced by Chapala lake. Chapala is the biggest lake in Mexico, with an extension of 1100 km2. However, this lake has reduced its extension caused by natural and human events (as dams’ construction). Such events reduced the lake extension leaving new cultivable areas where agricultural areas have been established. Actually, this zone is one of the principal zones for producing berries and avocados in the world.
Collection of Biological Material
The collection of the biological samples used in this study was obtained in the itinerant-local markets of the region named "tianguis". In such markets, the local producer sells their products every week. Six local markets were visited; each market is ubicated in the respective municipality center. The municipalities sampled in this study were Chavinda, Jacona, Tangamandapio, Villamar, Sahuayo, and La Barca (Fig. 1). We used the domesticated husk tomatoe (P. philadelphica) as a reference group.
In each market, local producers were localized and asked for their husk tomatoes' origin. The husk tomatoes were bought from farmers that cultivated and collected their own P. ixocarpa fruits. In La Barca's market, the seller can not identify their husk tomatoes' origin. La Barca is one of the bigger cities of the region, and the principal economic activity is intensive agriculture. However, this municipality was included because this is the east limit of the Cienega-Chapala region.
The samples were washed two times with water and soap and washed three times with distillate water. After, samples separated considering each fruit as and different individual and were lyophilized in a vacuum chamber at − 50°C for 72 h. Lyophilized samples were stored hermetically until their utilization.
Amplification by PCR of Intergenic Chloroplast Regions psbJ – petA and trnL – rpL32
Individual lyophilized samples were ground and used for DNA extraction using the CTAB method (Chen and Ronald 1999). As external groups, four genus species were used P. angulata, P. lagascae, P. peruviana, and P. pruinosa. Such species were obtained from the herbarium of CIIDIR-IPN, Unidad Michoacán (Centro Interdisciplinario de Investigación para el Desarrollo Integral Regional). The quality of extraction was corroborated on 1% agarose gel in 1 % TBA buffer at 76 V for 40 min.
For the amplification of intergenic chloroplast regions, five individual of each population were taken and amplified with a set of primers designed by Shaw et al. (2005). These primers are universal for whatever plant species; besides, chloroplast regions' use establishes in a better way the phylogenetic relations among populations (Shaw et al. 2005). The two amplified regions correspond to psbJ – petA, and trnL – rpL32.
The PCR mix contained 1x of the buffer, 10 mM of dNTPs, 1 µl of DNA, 1.5 mM of MgCl2, 1 U of Taq (Go Taq® Flexi DNA polymerase; Promega), and 10 mM of each primer in a final volume of 15 µL. The amplification conditions were as follows: denaturation at 80°C for 5 min followed by 30 cycles of 95°C for 1 m, 50–65°C (gradient of 0.25°C each 1 s) for 1 min, and 72°C for 4 min, followed by a final extension at 72°C for 5 min. The amplicons were sent to the Macrogen, Korea for sequencing.
Sequence Analyses
Sequence analysis was performed using the software MEGA (Molecular Evolutionary Genetic Analysis) version 7 (Kumar et al. 2016). We confirmed the quality and effective size of the sequences included in the alignment before analysis. The sequences were aligned by each intergenic region, after both alignments were linked, indicating each fragment's start and end site. Each region's alignment included sequences P. angulata, P. lagascae, P. peruviana, and P. pruinosa were used as external groups. A model test for finding the best evolutive model was calculated.
Genetic diversity (π) was quantified within populations based on the number of mutations. Additionally, Fu's Fs' neutrality test was calculated for each population. We evaluated the genetic distance between populations (FST) with a test performed with Arlequin Software v.3 (Excoffier et al. 2005). using 100 000 steps in the Markov chain and 1000 dememorization steps with a significance level of 0.05. To establish whether the genetic distance between accessions is independent of geographic location, we performed a Mantel test with 1000 permutations.
Phylogenetic Reconstruction
Phylogenetic reconstruction of the different populations was made using two approaches. The first method used was constructing a phylogenetic tree using 1000 bootstrap replicates under the maximum likelihood method, gamma distribution, and invariant sites under the evolutive model of Tamura-3 parameters. Additionally, due to genetic differentiation being a continuum, and to draw the possible expansion of P. ixocarpa populations, a haplotype network was estimated. This network was constructed in the PopArt software (Leigh and Bryant 2015) using the TCS method (Clement et al. 2000).
Metabolomic Fingerprinting Using GC-MS
Fifty milligrams of individual samples lyophilized and ground tissue were placed in amber vials (five individuals for each population). Vials were incubated at 80°C for one h. After this time, solid-phase microextraction (SPME) was used, inserting the fiber holder into vials and awaiting 10 minutes for each sample. Samples were injected into a gas chromatograph (Clarus 680, Perkin-Elmer Inc., Waltham, MA, USA), equipped with a phase capillary column: 5% diphenyl 95% dimethylpolysiloxane 30 m long, 0.32 mm i.d., 0.25 µm film thickness, temperature limits between − 60 a 320/350°C (Elite-5 MS, Perkin-Elmer Inc., Waltham, MA, USA). Helium gas was used at a flow rate of 1 mL min− 1, the flow remained constant, and there was an initial wait time of 0.5 min. The column temperature was initially maintained at 50°C for 1 min and then ramped to 250°C at 30°C/min, remaining at this temperature for a further 10 min. The temperature of the injector was 230°C. A mass spectrometer (Clarus SQ8T, Perkin-Elmer Inc., Waltham, MA, USA), with an electron impact ionization source (70 eV) in full scan mode was used. The analysis range was 40–500 m/z. The transfer line and ionization source temperatures were 230 and 250°C, respectively.
The feature detection, retention time correction, and peak alignment of the original chromatograms were made in XCMS Online (https://xcmsonline.scripps.edu) (Tautenhahn et al. 2012). To avoid false positives in the detection of metabolites, we only used metabolites with q-values ≤ of 0.05. A Principal Component Analysis (PCA) was made to select the metabolites with significant participation in the metabolomic differentiation. The annotation of the principal metabolites was made using the NIST library using a cut-off value of 0.8.
The results were represented in a heatmap-bicluster. An ion matrix was constructed using the metabolites with the highest differentiation levels. The heatmap construction was made using the platform Metaboanalyst (www.metaboanalyst.ca) (Chong et al. 2018). For the heatmap, the data were normalized and auto-scaled. The dendrograms used the Minkowski correlation as a distance function and the Ward clustering algorithm; the branches' significance was p ≤ 0.05.