Relationships within the major clades of Cactaceae are relatively well known based on DNA sequence data mostly from the chloroplast genome. Nevertheless, some nodes along the backbone of the phylogeny, and especially generic and species-level relationships, remain poorly resolved and are in need of more informative genetic markers. In this study, we propose a new approach to solve the relationships within Cactaceae, applying a targeted sequence capture pipeline. We designed a custom probe set for Cactaceae using MarkerMiner and complemented it with the Angiosperms353 probe set. We then tested both probe sets against 36 different transcriptomes using Hybpiper preferentially retaining phylogenetically informative loci and reconstructed the relationships using RAxML-NG and Astral. Finally, we tested each probe set through sequencing 96 accessions, representing 88 species across Cactaceae. Our preliminary analyses recovered a well-supported phylogeny across Cactaceae with a near identical topology among major clade relationships as that recovered with plastome data. As expected, however, we found incongruences in relationships when comparing our nuclear probe set results to plastome datasets, especially at the generic level. Our results reveal great potential for the combination of Cactaceae-specific and Angiosperm353 probe set application to improve phylogenetic resolution for Cactaceae and for other studies.

Opuntioideae, composed of roughly 370 species, occur in almost every biome in the Americas, from seasonally dry tropical forests (SDTF) to high-elevation Andean grasslands, American deserts and temperate zones. The interrelationships among the three major clades of Opuntioideae (Cylindropuntieae, Opuntieae and Tephrocacteae) are not well resolved, and thus, the ancestral habitat, biogeographic history and evolution of morphological characters, such as large photosynthetic leaves and flattened stems, are poorly understood. To test their geographic origin and evolution of key morphological characters, we built the largest phylogenomic dataset for Cactaceae to date using 103 plastid genes of 107 taxa of Opuntioideae. The subfamily Opuntioideae likely evolved in South America in a combination of seasonally dry tropical forest (SDTF)/desert habitats. Opuntieae most likely evolved in South America in SDTF and, from there, moved into desert regions, Chaco and temperate/subtropical zones, while Tephrocacteae and Cylindropuntieae evolved in South America in desert regions and moved into SDTF, Chaco and temperate/subtropical zones. Analyses of morphological evolution suggest that, although large leaves are plesiomorphic in Opuntioideae, long-lived, photosynthetically active leaves in Cylindropuntieae and Tephrocacteae are homoplasious and do not represent retained plesiomorphy, as is often assumed. Flattened stems are synapomorphic for Opuntieae, possibly representing adaptation to competition for light resources in SDTF, their most likely ancestral area.