We further amplified the primers with ID “a” for AT3G48380 and AT1G18270 genes (Table 1) in 26 individuals from the nine Opuntia species (Figure 1) and the outgroup. PCR amplification followed conditions on Tables 2, ,3,3, respectively.

Phylogenetic networks analyses aimed to identify found hybridization among sampled Opuntia species in two levels : all the 27 sampled taxa, and at the individual level in O. pilifera and O. tehuacana. Therefore, we divided our data in two sets, one that included all 27 taxa and a second group indicated in Supplementary Table 1 as “hybrid-test,” which included only one random individual per species and one putative hybrid. In total we analyzed nine “hybrid-test” matrices with 10 taxa each. To perform the phylogenetic network analysis, we used as a base maximum likelihood (ML) gene trees from RA×ML v.8.2 (Stamatakis, 2014) with the GTR + Γ model, bootstrapping with the autoMRE option and a search for the best-scoring ML tree after the bootstrap searches to obtain the best tree.

The phylogenetic networks analysis was performed in PhyloNet v.3.6.1 (Wen et al., 2018) using the best ML trees for each data block. First, we tested the non-hybridization hypothesis in the complete data block (27 taxa), using the option Infer_ST_MDC to obtain the species tree under the “Minimize Deep Coalescence” (MDC) criterion; next we used all the available reticulation options for the InferNetwork_MP command to test the possible one, two, and three reticulation scenarios. Afterward, we performed nine individual tests for hybridization using each one of the nine “hybrid-test” matrices. For these tests, we first obtained the species trees (Infer_ST_MDC), and thereafter, we used the InferNetwork_MP with the option -h {putative hybrid} to test each individual hybrid hypothesis. For both analyses, each reticulation inference was replicated 10 times. In each one of the 10 replicates, the network with the lowest number of extra lineages was selected and displayed graphically with Dendroscope v.3.0 (Huson and Scornavacca, 2012). Furthermore, we analyze our complete matrix (without outgroup) in SplitsTree v.4.16.1, with a NeighborNet (Supplementary Figure 1) to identify potential reticulation between lineages with a non-parametric method.

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