Abstract—Pinus subsection Cembroides comprises approximately 15 taxa distributed from the southwestern United States to south central Mexico. Despite previous phylogenetic studies based on morphology, nuclear ribosomal DNA, and plastid DNA, we still lack a robust phylogenetic hypothesis and clear delimitation for the closely-related species within the group. We studied the evolutionary relationships within subsection Cembroides and explored incomplete lineage sorting and reticulation using low-copy number nuclear genes. Concatenation and multispecies coalescent phylogenies were inferred from samples representing all taxa from subsection Cembroides and outgroups corresponding to the closely-related subsections Balfourianae, Nelsoniae, Gerardianae, and Krempfianae. The concatenation and coalescence-based trees mainly agreed with one another in recovering Pinus subsection Cembroides as monophyletic and in recovering similar relationships among species as in previous plastid DNA-based studies. Phylogenetic position and admixture analysis suggest that P. californiarum should be treated as a separate species from P. monophylla. Furthermore, our results support recognizing P. fallax as a species rather than as an infraspecific taxon of P. monophylla or P. edulis. The ASTRAL-III tree was consistent with the presence of very high levels of ILS in the group of pinyon pines with small cones. Analyses that account for both incomplete lineage sorting and reticulation identify some unexpected hybridization scenarios that were not reported in the literature.

Climate change is predicted to affect natural and plantation forests. The responses of conifers to overcome changing environments will depend on their adaptation to local conditions; however, intraspecific adaptive genetic variation is unknown for most gymnosperms. Studying genetic diversity associated with phenotypic variability along environmental gradients will enhance our understanding of adaptation and may reveal genetic pools important for conservation and management. METHODS: We used target enrichment and genome skimming to obtain single nucleotide polymorphisms (SNPs) from 61 individuals of Pinus patula, a pine tree native to Mexico widely used in plantation forestry. We investigated the adaptive genetic variation of two varieties with morphological and distributional differences potentially related to genetic and adaptive divergence. RESULTS: Population structure and haplotype network analyses revealed that genetic diversity between P. patula var. patula and P. patula var. longipedunculata was structured, even within populations of P. patula var. longipedunculata. We observed high genetic diversity, low inbreeding rate, and rapid linkage disequilibrium (LD) decay in the varieties. Based on outlier tests, loci showing signatures of natural selection were detected in geographically distant P. patula var. longipedunculata populations. For both varieties, we found significant correlations between climate-related environmental variation and SNP diversity at loci involved in abiotic stress, cell transport, defense, and cell wall biogenesis, pointing to local adaptation. CONCLUSIONS: Overall, significant intraspecific adaptive genetic variation in P. patula was detected, highlighting the presence of different genetic pools and signs of local adaptation that should be considered in forestry and conservation.

The process of hybridization occurs in approximately 40% of vascular plants, and this exchange of genetic material between non-conspecific individuals occurs unequally among plant lineages, being more frequent in certain groups such as Opuntia (Cactaceae). This genus is known for multiple taxonomic controversies due to widespread polyploidy and probable hybrid origin of several of its species. Southern Mexico species of this genus have been poorly studied despite their great diversity in regions such as the Tehuacán-Cuicatlán Valley which contains around 12% of recognized Mexico’s native Opuntia species. In this work, we focus on testing the hybrid status of two putative hybrids from this region, Opuntia tehuacana and Opuntia pilifera, and estimate if hybridization occurs among sampled southern opuntias using two newly identified nuclear intron markers to construct phylogenetic networks with HyDe and Dsuite and perform invariant analysis under the coalescent model with HyDe and Dsuite. For the test of hybrid origin in O. tehuacana, our results could not recover hybridization as proposed in the literature, but we found introgression into O. tehuacana individuals involving O. decumbens and O. huajuapensis. Regarding O. pilifera, we identified O. decumbens as probable parental species, supported by our analysis, which sustains the previous hybridization hypothesis between Nopalea and Basilares clades. Finally, we suggest new hybridization and introgression cases among southern Mexican species involving O. tehuantepecana and O. depressa as parental species of O. velutina and O. decumbens.

Premise: Accurate species delimitation is essential for evolutionary biology, conservation, and biodiversity management. We studied species delimitation in North American pinyon pines, Pinus subsection Cembroides, a natural group with high levels of incomplete lineage sorting. Methods: We used coalescent-based methods and multivariate analyses of low-copy number nuclear genes and nearly complete high-copy number plastomes generated with the Hyb-Seq method. The three coalescent-based species delimitation methods evaluated were the Generalized Mixed Yule Coalescent (GMYC), Poisson Tree Process (PTP), and Trinomial Distribution of Triplets (Tr2). We also measured admixture in populations with possible introgression. Results: Our results show inconsistencies among GMYC, PTP, and Tr2. The singlelocus based GMYC analysis of plastid DNA recovered a higher number of species (up to 24 entities, including singleton lineages and clusters) than PTP and the multilocus coalescent approach. The PTP analysis identified 10 species whereas Tr2 recovered 13, which agreed closely with taxonomic treatments. Conclusions: We found that PTP and GMYC identified species with low levels of ILS and high morphological divergence (P. maximartinezii, P. pinceana, and P. rzedowskii). However, GMYC method oversplit species by identification of more divergent samples as singletons. Moreover, both PTP and GMYC were incapable of identifying some species that are readily identified morphologically. We suggest that the divergence times between lineages within North American pinyon pines are so disparate that GMYC results are unreliable. Results of the Tr2 method coincided well with previous delimitations based on morphology, DNA, geography, and secondary chemistry.

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