Aim  To test how Pleistocene climatic changes affected diversification of the Crotalus intermedius species complex. Location  Highlands of Mexico and the south‐western United States (Arizona). Methods  We synthesize the matrilineal genealogy based on 2406 base pairs of mitochondrial DNA sequences, fossil‐calibrated molecular dating, reconstruction of ancestral geographic ranges, and climate‐based modelling of species distributions to evaluate the history of female dispersion. Results  The presently fragmented distribution of the C. intermedius group is the result of both Neogene vicariance and Pleistocene pine–oak habitat fragmentation. Most lineages appear to have a Quaternary origin. The Sierra Madre del Sur and northern Sierra Madre Oriental are likely to have been colonized during this time. Species distribution models for the Last Glacial Maximum predict expansions of suitable habitat for taxa in the southern Sierra Madre Occidental and northern Sierra Madre Oriental. Main conclusions  Lineage diversification in the C. intermedius group is a consequence of Pleistocene climate cycling. Distribution models for two sister taxa in the northern and southern Sierra Madre Occidental and northern Sierra Madre Oriental during the Last Glacial Maximum provide evidence for the expansion of pine–oak habitat across the Central Mexican Plateau. Downward displacement and subsequent expansions of highland vegetation across Mexico during cooler glacial cycles may have allowed dispersal between highlands, which resulted in contact between previously isolated taxa and the colonization of new habitats.

With the continued adoption of genome‐scale data in evolutionary biology comes the challenge of adequately harnessing the information to make accurate phylogenetic inferences. Coalescent‐based methods of species tree inference have become common, and concatenation has been shown in simulation to perform well, particularly when levels of incomplete lineage sorting are low. However, simulation conditions are often overly simplistic, leaving empiricists with uncertainty regarding analytical tools. We use a large ultraconserved element data set (>3,000 loci) from rattlesnakes of the Crotalus triseriatus group to delimit lineages and estimate species trees using concatenation and several coalescent‐based methods. Unpartitioned and partitioned maximum likelihood and Bayesian analysis of the concatenated matrix yield a topology identical to coalescent analysis of a subset of the data in bpp. ASTRAL analysis on a subset of the more variable loci also results in a tree consistent with concatenation and bpp, whereas the SVDquartets phylogeny differs at additional nodes. The size of the concatenated matrix has a strong effect on species tree inference using SVDquartets, warranting additional investigation on optimal data characteristics for this method. Species delimitation analyses suggest up to 16 unique lineages may be present within the C. triseriatus group, with divergences occurring during the Neogene and Quaternary. Network analyses suggest hybridization within the group is relatively rare. Altogether, our results reaffirm the Mexican highlands as a biodiversity hotspot and suggest that coalescent‐based species tree inference on data subsets can provide a strongly supported species tree consistent with concatenation of all loci with a large amount of missing data.

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