Journal of Biogeography (J. Biogeogr.) (2013) 40, 1850–1860

ORIGINAL Biogeography of in the ARTICLE Pseudouroctonus minimus complex (Vaejovidae) from south-western North America: implications of ecological specialization for pre-Quaternary diversification Robert W. Bryson Jr1*, Warren E. Savary2 and Lorenzo Prendini3

1Department of Biology and Burke Museum ABSTRACT of Natural History and Culture, University of Aim The aim of this study was to assess the impact of pre-Quaternary tecton- Washington, Seattle, WA, 98195-1800, USA, 2 ics and orogeny relative to that of Pleistocene climate change on diversification Department of Entomology, California Academy of Sciences, San Francisco, CA, within the Pseudouroctonus minimus complex, a group of vaejovid scorpions 94118, USA, 3Division of Invertebrate with stenotopic habitat requirements. Zoology, American Museum of Natural Location South-western North America (United States and Mexico). History, New York, NY, 10024-5192, USA Methods Multilocus sequence data (1899 base pairs from two mitochondrial and two nuclear genes) were generated from 65 samples of scorpions in the minimus complex. Phylogeographical structure within the minimus complex was explored using model-based phylogenetic methods and a general mixed Yule coalescent model to identify independent geographical clusters. A time- calibrated multilocus species tree was reconstructed using a multispecies coales- cent approach. Ancestral areas were estimated at divergence events across the tree using a probabilistic Bayesian approach. Results Extensive geographical structure was evident within two well- supported clades. These clades probably diverged over 25 million years ago (Ma), based on estimated mean divergence dates, followed by 14 divergences in the Miocene (25–5 Ma) and 4 divergences in the Pliocene and Pleistocene (< 5 Ma). The ancestral origin of the minimus complex was reconstructed to be across California and the Mexican Highlands. The Chihuahuan Desert was colonized twice from the Mexican Highlands, and one dispersal event occurred from the Mexican Highlands back to California. Main conclusions Spatial and temporal patterns of evolution in the minimus complex support predictions that stenotopy promoted pre-Quaternary diversifica- tion. Miocene and Pliocene geomorphology, perhaps in concert with climate change, induced allopatric divergence across the heterogeneous landscape of south-western North America. Stenotopic scorpions such as the minimus complex provide a model *Correspondence: Robert W. Bryson Jr, Department of Biology and Burke Museum of for exploring correlations between Earth history and biological diversification. Natural History and Culture, University of Keywords Washington, Box 351800, Seattle, WA 98195- Pseudouroct- 1800, USA. Biogeography, diversification, North America, phylogeography, E-mail: [email protected] onus, speciation, stenotopy.

ments (stenotopy) that exert a consistent and repeated influ- INTRODUCTION ence upon gene flow among populations may influence The amount of genetic structure in a taxon across a land- diversification across space and time (e.g. Cooper et al., scape is often correlated with its ecological requirements and 2011; Derkarabetian et al., 2011; Hamilton et al., 2011; Keith dispersal ability (Avise, 2000). Specialized ecological require- & Hedin, 2012), a prediction of the ‘effect hypothesis of

1850 http://wileyonlinelibrary.com/journal/jbi ª 2013 John Wiley & Sons Ltd doi:10.1111/jbi.12134 Diversification in the Pseudouroctonus minimus complex macroevolution’ (Vrba, 1980). Many scorpions are character- The complex is distributed across three distinct biogeograph- ized by low dispersal ability and stenotopic habitat require- ical regions (Fig. 1). Pseudouroctonus andreas, P. rufulus and ments, increasing their propensity for diversification in P. minimus (including its subspecies P. m. castaneus and association with long-term processes such as geomorphologi- P. m. thompsoni) occur in the Channel Islands and coastal cal development and climatic cycles (Prendini, 2001, 2005). chaparral of southern California and northern Baja Califor- Stenotopic scorpions with specialized habitat requirements nia. Several hundred kilometres to the east, P. apacheanus may disperse only a few metres a year (Polis et al., 1985), and P. chicano are distributed across the mesic mixed pine- and local populations may diverge allopatrically on small oak woodlands of the Sierra Madre Occidental and the asso- spatial scales when there is decreased gene flow across barri- ciated sky-island outliers in southern Arizona and adjacent ers of unsuitable habitat. Consequently, stenotopic New Mexico, extending southwards into Chihuahua and taxa tend to be more species-rich and range-restricted than Sonora. Pseudouroctonus apacheanus is also found in western eurytopic scorpion taxa with generalized ecological require- Texas on several disjunct mountains and along patches of ments (Prendini, 2001). mesic rocky upland habitat within the Chihuahuan Desert. This study examined the Pseudouroctonus minimus com- The troglophilous P. savvasi inhabits two isolated caves at plex, a monophyletic group of stenotopic, rupicolous scorpi- the eastern edge of the Chihuahuan Desert. ons in the family Vaejovidae Thorell, 1876. The minimus In this study, species of the minimus complex were pre- complex – here defined as comprising the species Pseudou- dicted to retain genetic traces of regional history over rela- roctonus andreas (Gertsch and Soleglad, 1972), P. apacheanus tively small geographical distances because of their (Gertsch and Soleglad, 1972), P. chicano (Gertsch and Sole- stenotopic habitat requirements. Much of the modern land- glad, 1972), P. minimus (Kraepelin, 1911), P. rufulus (Ger- scape of south-western North America evolved prior to the tsch and Soleglad, 1972), P. savvasi Francke, 2009 and Quaternary (Wilson & Pitts, 2010). The cyclical climate possibly P. cazieri (Gertsch and Soleglad, 1972) – is restricted change that characterized the Pleistocene dramatically trans- to humid, rocky habitats throughout south-western North formed biotic communities across south-western North America from southern California and northern Baja Califor- America (Webb & Betancourt, 1990), but may have had little nia across southern Arizona, New Mexico and western Texas, effect on the diversification of scorpions in the minimus and southwards into the Mexican states of Sonora, Chihua- complex closely associated with abiotic rocky habitats. hua and Coahuila (Sissom 2000; Francke & Savary, 2006). Despite the presence of ephemeral woodland corridors across

California N

P. m. thompsoni P. m. minimus P. apacheanus Pinaleño P. m. castaneus P. andreas Catalina Guadalupe Santa Rita Chiricahua Quinlan P. rufulus Peloncillo P. apacheanus Gulf of California Atascosa Davis Grutas Independence Mexican Highlands Chisos P. cazieri P. savvasi P. chicano Chihuahuan

0–750 m Desert 750–1500 m 1500–2250 m 2250–3000 m

0 250 500 750 1000 km

Figure 1 Map of south-western North America plotting collection localities (black circles) of genetic samples of vaejovid scorpions of the Pseudouroctonus minimus complex. Putative species and subspecies are indicated, as well as the geographical localities of P. apacheanus mentioned in the text. The locality of P. cazieri, a possible member of the minimus complex missing from the study, is indicated with a white circle. Approximate boundaries between the three main biogeographical regions inhabited by these scorpions are indicated with dashed lines.

Journal of Biogeography 40, 1850–1860 1851 ª 2013 John Wiley & Sons Ltd R. W. Bryson Jr et al. much of south-western North America during cooler Pleisto- The gametic phase of the variants was determined computa- cene glacial periods (McCormack et al., 2008; Bryson et al., tionally using phase 2.1.1 (Stephens & Donnelly, 2003). Five 2011), the dispersal of scorpions in the minimus complex separate runs of 400 iterations each were conducted for each was probably limited to corridors containing rocky habitat. nuclear data set, and results with a probability threshold of Accordingly, the pre-Quaternary tectonics and orogeny that 0.7 or greater were accepted. All polymorphic sites with a created and altered rocky terrain across the south-western probability < 0.7 were coded in both alleles with the appro- North American landscape may have had a greater impact priate IUPAC ambiguity code. InDelligent 1.2 (Dmitriev & on diversification than Pleistocene climate change in these Rakitov, 2008) was used to resolve insertion/deletion events stenotopic scorpions. between homologous nuclear alleles in ITS2. Sequence These expectations were tested using a molecular phyloge- alignments for individual gene regions were performed with netic approach. First, the phylogeographical structure within mafft 6 (Katoh et al., 2002; Katoh & Toh, 2008) using the minimus complex was explored. Second, a time- default settings, the ‘20PAM/k = 2’ scoring matrix for nucle- calibrated multilocus species tree was reconstructed, and otide sequences, the Q-INS-i algorithm for 16S and 28S data, ancestral areas were estimated at divergence events across the and the E-INS-i algorithm for ITS2 data. The ITS2 gene tree. The resulting patterns of diversification are discussed in region was highly variable with a large number of indels the context of the geomorphological evolution of south-wes- among the various sequences, so ambiguously aligned regions tern North America. were eliminated using Gblocks 0.91b (Talavera & Castresan- a, 2007). A partitioned homogeneity test was conducted in paup* 4.0b10 (Swofford, 2002) with 1000 heuristic replicates, MATERIALS AND METHODS to test for conflicting phylogenetic signals between the mito- chondrial and nuclear genes used in the species tree analyses. Genetic data

DNA sequence data were generated from 65 samples of scor- Phylogeographical estimation pions in the minimus complex collected throughout the known range (Fig. 1 and Appendix S1 in Supporting Infor- The full mtDNA data set (n = 67) was analysed to examine mation). The composition of the minimus complex was geographical structure and delineate geographically cohesive inferred from multilocus and morphological data for samples lineages within the minimus complex. Geographical structure representing all genera and most species in the family Vaejo- was inferred using Bayesian inference and maximum-likeli- vidae (http://www.vaejovidae.com/, in preparation). All hood phylogenetic methods. Bayesian inference analyses were described species known to belong to the monophyletic min- conducted with MrBayes 3.2.1 (Ronquist et al., 2012). imus complex were included (P. andreas, P. apacheanus, MrModeltest 2.1 (Nylander, 2004) was used to select best- P. chicano, P. minimus, P. rufulus and P. savvasi), as well as fit models of evolution, based on the Akaike information cri- both subspecies of P. minimus (P. m. castaneus and terion (Akaike, 1973), for the COI and 16S gene regions. P. m. thompsoni). Pseudouroctonus cazieri, from the Baja Cal- Analyses were conducted with three heated (tempera- ifornia Peninsula, may also belong to the minimus complex, ture = 0.05) and one cold Markov chain, sampling every 100 but its placement has not been confirmed, as samples could generations for 4 million generations. Adjustment of the not be obtained for molecular analysis. Pseudouroctonus redd- heated chain temperature from the default value of 0.2 to elli and P. williamsi were included as outgroups (Stockwell, 0.05 resulted in higher harmonic mean log-likelihoods and 1989). better convergence and mixing. Parameters were unlinked Genomic DNA was extracted from leg muscle tissue. Frag- across partitions, and the gamma-shaped rate variation was ments of mitochondrial DNA were sequenced from a pro- set to variable. Output parameters were visualized using tein-coding gene (cytochrome c oxidase subunit I, COI) and Tracer 1.5 (Rambaut & Drummond, 2007) to ascertain sta- a ribosomal gene (16S rDNA, 16S) using scorpion-specific tionarity and convergence. All samples obtained during the primers (Appendix S2). Two nuclear genes, including frag- first million (25%) generations were discarded as burn-in. ments of the 28S rDNA (28S) and the internal transcribed Maximum likelihood analyses were conducted using RAxML spacer region (ITS2) between the 5.8S and 28S rDNAs, were 7.2.6 (Stamatakis, 2006) under the GTRGAMMA model, sequenced for a subset of samples (n = 44) that were used in with 1000 nonparametric bootstrap replicates to assess nodal species tree analyses (see below). Primer sequences for support. nuclear genes were obtained from Tully et al. (2006) for 28S A general mixed Yule coalescent (GMYC) model was used and from Ji et al. (2003) for ITS2. The laboratory protocols to delineate geographically distinct clusters of samples used to generate the sequence data are provided in Prendini (henceforth ‘lineages’), implemented in the R command-line et al. (2003, 2005) and Bryson & Riddle (2012). package (R Development Core Team, 2011) splits (Pons Heterozygous sites were identified in nuclear segments et al., 2006). The GMYC model identifies independent evolu- when two different nucleotides were observed at the same tionary clusters by detecting a threshold value at the transi- position in electropherograms of both strands, with the tion from interspecific to intraspecific branching patterns, weaker peak reaching at least 50% of the strongest signal. and provides an objective means of delimiting the ‘species’

1852 Journal of Biogeography 40, 1850–1860 ª 2013 John Wiley & Sons Ltd Diversification in the Pseudouroctonus minimus complex required for species tree reconstructions (see below). An ultr- analysis (BBM) as implemented in rasp 2.0b (Yu et al., ametric tree for use in splits was generated with beast 1.7.4 2011). This program determines the probability of an ances- (Drummond et al., 2012). Separate models of evolution were tral range at a node by averaging over a posterior set of trees, used for the COI and 16S gene regions. Analyses were run thereby accounting for phylogenetic uncertainty. A total of for 40 million generations, with samples retained every 1000 40,000 post-burn-in trees were loaded from the *beast anal- generations, using a Yule tree prior. Results were displayed yses into rasp. Each sample from the phylogeny was assigned in Tracer to confirm acceptable mixing and likelihood sta- to one of three broad biogeographical regions (Fig. 1; Udvar- tionarity, appropriate burn-in, and adequate effective sample dy, 1975): (1) the coastal chaparral of southern California sizes above 200 (Drummond et al., 2007) for all estimated (including the Channel Islands) and north-western Baja Cali- parameters. After discarding the first 4 million generations fornia (hereafter referred to as ‘California’); (2) the Sierra (10%) as burn-in, the parameter values of the samples from Madre Occidental and associated sky islands (‘Mexican High- the posterior distribution were summarized on the maxi- lands’); and (3) upland areas within the Chihuahuan Desert mum clade credibility tree using TreeAnnotator 1.7.4 (‘Chihuahuan Desert’). The probabilities for nodes in the (Drummond et al., 2012). Analyses using single- and multi- phylogeny with posterior probability > 0.50 were estimated. ple-threshold models were performed with splits. The The number of areas was set to three, a F81 + G model was multiple-threshold model tested whether allowing the used, and analyses were conducted for 1 million generations species-coalescent transition to vary across the tree signifi- using 10 chains, sampling every 100 generations. Hypotheti- cantly improved the fit to the model (Monaghan et al., 2009). cal outgroups assigned to the phylogeny by the BBM algo- rithm prior to the analyses were given a ‘wide’ distribution across all three biogeographical regions. The first 25% of Species tree and divergence date estimation generations were discarded as burn-in. A time-calibrated species tree was reconstructed for the mini- mus complex from the multilocus data set using *beast RESULTS (Heled & Drummond, 2010; Drummond et al., 2012), a part of the beast package. One or two exemplar samples Genetic data (n = 44) were selected from each geographically delimited mtDNA lineage identified by the GMYC analysis, and out- The complete mtDNA data set contained 321 parsimony- groups were excluded. These GMYC-delineated lineages sat- informative sites (COI: 211 in 756 bp; 16S: 110 in 397 bp). isfy the operational requirements of ‘species’ for the *beast The reduced mtDNA data sets for the species tree analysis analyses (Heled & Drummond, 2010). Best-fit models of evo- contained 200 (COI) and 100 (16S) parsimony-informative lution were selected using MrModeltest, with a Yule speci- sites, respectively. The nuclear gene loci exhibited much less ation prior and relaxed uncorrelated lognormal clocks variation than the mtDNA (parsimony-informative sites: 28S, applied for each gene tree. In analyses of the COI and 16S 7 in 519 bp; ITS2, 46 in 227 bp). Complete sequence data mtDNA data, which represent a single locus, trees were could not be obtained for two samples: P. andreas Mission linked but substitution and clock models were unlinked. The Trails CA (ITS2) and P. apacheanus Vallecito SON (COI and clock was calibrated using scorpion-specific mutation rates of ITS2). The 28S gene contained three heterozygous sites, all À 5 9 10 3 substitutions/site/Myr for 16S (Gantenbein & Larg- confidently resolved. The ITS2 gene contained eight hetero- À iader, 2003) and of 7 9 10 3 substitutions/site/Myr for COI zygous sites. Two of those sites in one individual (P. apache- (Gantenbein et al., 2005). Clock rates for the 28S and ITS2 genes anus Pinaleno~ AZ 1) were not resolved above the 0.7 were estimated relative to the mtDNA rates. This ‘scorpion acceptance threshold. The partitioned homogeneity test clock’ was calculated from buthid scorpions, distantly related to revealed no significant conflict between the mitochondrial the vaejovids of the minimus complex, as no other mutation and nuclear genes in the reduced data set (P = 0.96). rates have been estimated for scorpions to date. Analyses were GTR+I+G models of sequence evolution were selected for run for 8 9 107 generations, with samples retained every 1000 both genes in the mtDNA tree data set, and GTR+I+G(COI, generations. Results were displayed in Tracer to confirm 16S), GTR+I(28S) and GTR+G (ITS2) models for the spe- acceptable mixing and likelihood stationarity, appropriate burn- cies tree data sets. All aligned sequences were deposited in in, and adequate effective sample sizes. The first 10% of genera- the Dryad repository: doi:10.5061/dryad.q58r0. tions were discarded as burn-in, and parameter estimates were summarized on the maximum clade credibility tree using Phylogeographical estimation TreeAnnotator. This burn-in and visualization procedure was repeated for each of the three gene trees co-estimated by *beast. Extensive geographical structure in the minimus complex was evident within two well-supported major clades (Fig. 2). One clade (referred to as the ‘NW clade’) comprised Ancestral area reconstruction P. andreas, P. minimus, and its subspecies. The second clade The ancestral range at each divergence event was recon- (‘SE clade’) comprised P. apacheanus, P. chicano, P. rufulus structed using Bayesian binary Markov chain Monte Carlo and P. savvasi.

Journal of Biogeography 40, 1850–1860 1853 ª 2013 John Wiley & Sons Ltd R. W. Bryson Jr et al.

reddelli (a) williamsi m. minimus Catalina Is CA minimus m. thompsoni Santa Cruz Is CA 3 m. thompsoni Santa Cruz Is CA 2 thompsoni m. thompsoni Santa Cruz Is CA 1 m. castaneus Oceanside CA 1 m. castaneus Carlsbad CA castaneus m. castaneus Oceanside CA 2 andreas Banner Canyon CA andreas E andreas Mission Trails CA andreas Penasquitos CA andreas W andreas Rosarito BC 1 andreas S Pseudouroctonus minimus complex andreas Rosarito BC 2 apacheanus Quinlan AZ 3 apacheanus Quinlan AZ 1 apacheanus Quinlan AZ 4 Quinlan apacheanus Quinlan AZ 2 apacheanus Atascosa AZ 2 apacheanus Atascosa AZ 1 Atascosa apacheanus Santa Rita AZ 3 apacheanus Santa Rita AZ 1 Santa Rita apacheanus Santa Rita AZ 2 savvasi Azufrosa COAH savvasi S savvasi Casa Blanca COAH savvasi N chicano SE del Nido CHIH chicano NE chicano del Nido CHIH 2 chicano del Nido CHIH 3 chicano NW (b) NW clade minimus chicano del Nido CHIH 1 thompsoni chicano Horquetudo SON chicano W chicano Vallecito SON .85 / 70 castaneus chicano Balleza CHIH andreas E chicano Guachochi CHIH chicano S andreas W chicano Guasarachic CHIH .63 / 57 andreas S rufulus Punta Banda BC rufulus apacheanus Grutas CHIH Grutas Quinlan apacheanus Peloncillo NM 1 .52 / 90 Peloncillo apacheanus Peloncillo NM 2 Atascosa apacheanus Chiricahua AZ 4 Santa Rita apacheanus Chiricahua AZ 1 Chiricahua SE clade apacheanus Chiricahua AZ 3 1.0 / 61 chicano W apacheanus Chiricahua AZ 2 chicano S apacheanus Pinaleño AZ 4 .97 / 56 Grutas apacheanus Pinaleño AZ 2 rufulus Pinaleño savvasi N apacheanus Pinaleño AZ 3 .76 / <50 savvasi S apacheanus Pinaleño AZ 1 chicano NE .93 / <50 apacheanus Catalina AZ 1 chicano NW apacheanus Catalina AZ 3 Catalina Peloncillo apacheanus Catalina AZ 4 apacheanus Catalina AZ 2 Chiricahua apacheanus Guadalupe TX 2 .61 / <50 apacheanus Guadalupe TX 1 Guadalupe apacheanus Guadalupe TX 3 Pinaleño apacheanus Chisos TX 4 apacheanus Chisos TX 1 Catalina apacheanus Chisos TX 5 Chisos apacheanus Chisos TX 2 Guadalupe apacheanus Chisos TX 3 apacheanus Davis TX 4 apacheanus Davis TX 2 .98 / <50 Chisos apacheanus Davis TX 3 Davis apacheanus Davis TX 1 Davis apacheanus Independence TX 1 apacheanus Independence TX 4 apacheanus Independence TX 5 Independence Independence apacheanus Independence TX 2 apacheanus Independence TX 3

Figure 2 Geographical structure within North American vaejovid scorpions of the Pseudouroctonus minimus complex inferred from phylogenetic analyses of 1153 base pairs of mitochondrial DNA. (a) Ultrametric tree generated with beast. General mixed Yule coalescent (GMYC) groups delimited from the single-threshold model are designated by grey branches. Samples in bold were used in multilocus species tree reconstructions. No nodal support values are shown for this tree because the focus is on GMYC cluster delimitation. (b) Maternal genealogy based on mixed-model Bayesian inference (tree shown) and maximum likelihood analyses. All major nodes that received ≥ 0.95 Bayesian posterior probability and ≥ 70% bootstrap support are depicted with black dots. Support at all other nodes is denoted by the Bayesian posterior probability followed by maximum likelihood bootstrap values. All lineages identified by the GMYC analysis received ≥ 0.95 posterior probability and ≥ 70% bootstrap support. GMYC groups are collapsed for clarity.

The single-threshold and multiple-threshold GMYC mod- douroctonus andreas, P. apacheanus, P. chicano and P. savvasi els were not significantly different from each other each contained several geographically delimited lineages (v2 = 1.9109, d.f. = 9, P = 0.9927), so results from the sin- (including singletons, hereafter referred to as ‘lineages’ for gle-threshold model were used to infer geographically delim- convenience). Pseudouroctonus andreas comprised three lin- ited lineages within the minimus complex (Fig. 2a). eages, one on the eastern and one on the western slopes of Independent evolutionary clusters estimated by the GMYC the Peninsular Range in California, and another west of the model were largely concordant with geography. Individuals Peninsular Range in north-western Baja California. The from isolated mountain ranges and regions in close geo- widespread P. apacheanus comprised 15 distinct geographi- graphical proximity were monophyletic with the exception of cally delimited lineages. Eleven of these were grouped within P. m. thompsoni. Although the GMYC model split this taxon three regional clades. The westernmost clade included three into two groups, it was treated as one lineage because of its lineages from the Quinlan, Santa Rita and Atascosa moun- restricted distribution in the northern Channel Islands. Pseu- tain ranges (Fig. 1), to the north-east of which was a clade

1854 Journal of Biogeography 40, 1850–1860 ª 2013 John Wiley & Sons Ltd Diversification in the Pseudouroctonus minimus complex comprising four lineages from the Catalina, Pinaleno,~ Chiri- phyly of the NW and SE clades was strongly supported in cahua and Peloncillo Mountains. The Chiricahua lineage the species tree reconstruction (Fig. 3). The two clades prob- included four samples from the Chiricahua Mountains and ably diverged prior to 25 Ma, during the Palaeogene (mean one from the Peloncillo Mountains. Further east, a third estimated date 37.9 Ma, 95% posterior credibility inter- regional clade included four Texas lineages from Indepen- val = 47.5–28.9 Ma). A subsequent burst of diversification in dence Creek and the Guadalupe, Davis and Chisos Moun- the SE clade occurred c. 14–21 Ma. During this 7-Myr inter- tains. The last remaining lineage of P. apacheanus comprised val, an estimated six divergence events occurred (based on one sample from a cave in north-western Chihuahua (‘Gru- mean divergence date estimates). A further eight divergences tas’). Pseudouroctonus chicano formed two lineages in the occurred within the SE clade during a second burst of diver- Sierra del Nido, Chihuahua, one at higher elevations in the sification c. 13–8 Ma, followed by four final divergences, one west and the other (represented by one sample) at a lower near the Miocene/Pliocene boundary c. 4 Ma, and three elevation in the east. Two other lineages of P. chicano were during the Pleistocene. Estimated divergences within the found, in south-eastern Sonora and further south in the NW clade all occurred during the Miocene from c. 15 to Sierra Madre Occidental, respectively. Both samples of the 6 Ma. troglophile P. savvasi, collected from isolated caves, were also genetically distinct. Ancestral area reconstruction Pairwise sequence divergences between sister lineages, cal- culated with mega 5.05 (Tamura et al., 2011) using a maxi- The rasp analysis (Fig. 4) supported a widespread ancestral mum composite likelihood correction, were relatively high. origin of the minimus complex across California and the Pairwise divergences between deeply divergent lineages ran- Mexican Highlands. The marginal probability for this basal ged from 8.2% (P. chicano from the eastern and western node reconstruction (P = 53%) was much higher than that slopes of the Sierra del Nido) to 8.3% (P. m. minimus and for alternative geographical areas (California + Mexican P. m. thompsoni), and those between more closely related Highlands + Chihuahuan Desert, P = 22%; California, lineages ranged from 2.2% (P. apacheanus from the P = 9%; Mexican Highlands, P = 8%; and three other area Atascosa and Santa Rita mountain ranges) to 3.0% combinations, P < 5% each). One major vicariance event (P. apacheanus from the Davis Mountains and Indepen- and three major dispersal events appear to have occurred. dence Creek). The basal divergence between the NW clade and the SE clade was probably caused by a vicariance event dating to the Eocene. The Chihuahuan Desert appears to have been colo- Species tree and divergence times nized twice from the Mexican Highlands by members of the The topologies of the gene trees were similar and revealed a SE clade (Fig. 4), whereas one dispersal event occurred from consistent lack of support at the base (Appendix S3). Mono- the Mexican Highlands back to California.

Eocene Oligocene Miocene Plio. Pleist.

11.44 minimus NW clade 15.47 thompsoni castaneus 10.8 andreas E Pseudouroctonus minimus complex 8.13 andreas W 37.93 6.85 andreas S Quinlan 12.28 1.85 Atascosa SE clade Santa Rita 21.02 9.02 savvasi N savvasi S 13.09 chicano W 19.23 chicano S Figure 3 Time-calibrated multilocus chicano NE * 11.53 species tree for North American vaejovid 17.99 chicano NW scorpions of the Pseudouroctonus minimus 11.94 rufulus Grutas complex. Bars indicate 95% highest 16.72 Guadalupe posterior densities of divergence dates, with 11.07 Chisos mean estimates in millions of years ago 15.74 Davis 8.59 2.56 (Ma) given at nodes. Bayesian posterior Independence probability support values for nodes are 14.51 4.74 Pinaleño 1.0–0.95 indicated by coded dots. The single node 0.95–0.70 8.11 Catalina with a posterior probability below 0.50 is 0.70–0.50 2.14 Chiricahua marked with an asterisk. Abbreviations: Peloncillo Pleist., Pleistocene; Plio., Pliocene; Quat., 45 40 35 30 25 20 15 10 5 2.6 0 Ma Quaternary. Palaeogene Neogene Quat.

Journal of Biogeography 40, 1850–1860 1855 ª 2013 John Wiley & Sons Ltd R. W. Bryson Jr et al.

1 3

4

Gulf of California 2

E W S N S W S NE NW Figure 4 Dated multilocus phylogeny for North American vaejovid scorpions of the minimusthompsonicastaneusandreasandreas andreas Quinlan AtascosaSantasavvasi Ritasavvasi chicano chicano chicano chicano rufulus GrutasGuadalupeChisosDavisIndependencePinaleñoCatalinaChiricahuaPeloncillo 0 Ma Pseudouroctonus minimus complex, showing Pleistocene ancestral area reconstructions. Pie charts Pliocene 5 indicate the probability of ancestral area for Miocene nodes that received ≥ 0.50 posterior

4 10 probability support. Sample localities are colour-coded to match the three 3 biogeographical regions indicated in Fig. 1. 15 The locality of P. cazieri, a possible member 2 of the minimus complex missing from the 20 NW clade study, is indicated with a white circle. The dashed line and arrows indicate 25 interpretations of four major historical Oligocene SE clade California (C) events that affected diversification. The thin Mexican Highlands (MH) 30 Chihuahuan Desert (CD) solid line indicates the break between C + MH + CD P. andreas (above the line) and P. rufulus C + MH 1 35 (below) at the end of a possible ring Eocene MH + CD <10% probability distribution formed by the minimus 40 complex.

P. andreas in southern California and northern Baja Califor- DISCUSSION nia may be related to the faulting along the Peninsular Range that began in the late Miocene (Axen & Fletcher, 1998). Pre-Quaternary diversification Diversification within the widespread SE clade appears to Scorpions of the minimus complex appear to be ancient have occurred almost entirely within the Neogene period, in inhabitants of south-western North America. Although the the Miocene and Pliocene. Fifteen of the estimated 18 diver- estimated divergence dates are remarkably old and based on gence events (83%) pre-date the Quaternary (Fig. 3). The a molecular clock rate obtained from a distantly related base of the SE clade is characterized by five weakly supported taxon, the dates of many divergence events within the com- nodes in the species tree reconstruction, suggesting a rapid plex correspond well with geological events and to geologi- radiation. This burst of diversification c. 21–14 Ma corre- cally mediated vicariance. Divergence of the NW and SE sponds with regional geomorphological change and climate clades is estimated to have been prior to 28 Ma, during the shifts during the Miocene, 24–12 Ma (Shafiqullah et al., Palaeogene (Figs 3 & 4), coincident with dramatic landscape 1980; Henry & Aranda-Gomez, 2000; Brand & Stump, 2011). deformation in southern California and the development of Marked extensions of the northern Mexican Highlands the San Andreas fault system at c. 30 Ma, following the colli- occurred during this time (Henry & Aranda-Gomez, 2000; sion of the Pacific-Farallon and North American plates Connell et al., 2005; Brand & Stump, 2011), coincident with (Atwater, 1998). The subsequent formation of the Catalina the onset of a wetter climate (Retallack, 2001). The ancestral and Channel Islands, c. 18–12 Ma (Atwater, 1998; Schoenh- area reconstruction suggests that dispersal and colonization err et al., 1999), corresponds well with the basal divergence of the Chihuahuan Desert occurred during this period of the NW clade at c. 15 Ma, and to the subsequent isolation (Fig. 4), perhaps triggered by changing ecosystems associated of the two insular forms, P. m. minimus and P. m. thomp- with the wetter climate (Retallack, 2001). However, at least soni,atc. 11 Ma (Figs 3 & 4). The later diversification of one of these events may have been caused by allopatric frag-

1856 Journal of Biogeography 40, 1850–1860 ª 2013 John Wiley & Sons Ltd Diversification in the Pseudouroctonus minimus complex mentation associated with the mid-Miocene extension of the relatively few samples per mountain range sequenced in this Rio Grande rift (Fig. 4; Henry & Aranda-Gomez, 2000; Con- study. nell et al., 2005). The two sampled localities of troglophilous P. savvasi are The divergence of P. rufulus on the north-western coast of separated by 127 km of xeric habitat (Fig. 1). Dating esti- Baja California from its sister taxon, P. apacheanus in north- mates place the divergence between these samples at c. 9Ma ern Chihuahua, at c. 12 Ma (mean 11.9 Ma, 95% posterior (Fig. 3), suggesting a long period of isolation, probably credibility interval 15.7–8.2 Ma), corresponds well with tec- caused by the first significant expansions of semi-arid and tonic plate movements during this time (Henry & Aranda- arid habitats across south-western North America that Gomez, 2000) and to the estimated development of the started during the late Miocene (Axelrod, 1950, 1979; Gra- proto-Gulf of California, c. 13–12 Ma (Henry & Aranda- ham, 1999). Although P. savvasi possesses no evident troglo- Gomez, 2000; Zhang & Paulssen, 2012). Although the rasp morphies (Francke, 2009), its reliance on a cool humid cave analysis suggested a dispersal event to explain this split, the environment may have prevented subsequent expansions isolation of P. rufulus may be the result of vicariance associ- across the more suitable mesic habitats that extended across ated with the separation of the Baja California Peninsula and much of the Chihuahuan Desert during Pleistocene glacial subsequent rifting along the Pacific plate to the north-west times (Morafka, 1977). The relatively ancient split between (a passive dispersal event). The dates of this rifting are P. chicano populations in the Sierra del Nido might reflect debated (Umhoefer, 2011), but the two most widely accepted divergences between a highland form (del Nido W) and an models span 12.5–6 Ma (Oskin et al., 2001; Fletcher et al., epigean troglophile on the lower-elevation slopes (del Nido 2007), encompassing most of the 95% posterior credibility E). The Sierra del Nido represents an eastern outlier of the interval of the estimated date of divergence. Sierra Madre Occidental flanked by Chihuahuan Desert scrub. Cavernicole forms are found at low elevations to the north-west (P. apacheanus Grutas) and east (P. savvasi)of Habitat specialization these mountains, suggesting that they are or were more Extrinsic habitat features and intrinsic organismal traits syn- widespread and have gone extinct or undetected. ergistically influence the distribution of taxa across land- scapes. Species in the minimus complex are restricted to Fragments of an ancient ring distribution? humid, rocky habitats throughout south-western North America (Sissom, 2000; Francke & Savary, 2006). The dis- Although few areas of the world present the geographical tinct pattern of pre-Quaternary diversification within the conditions necessary for ring speciation (Mayr, 1963), recent complex inferred in this study is probably related to the spe- research suggests that the unique geomorphology of the Baja cialized ecological requirements of these scorpions. With one California Peninsula may have created ring distributions in exception, samples from all mountain ranges in the SE clade several taxa (Mulcahy & Macey, 2009). Based on the inferred form monophyletic groups (Fig. 2a), consistent with the pre- close relationship between two samples from widely disjunct diction that gene flow between populations on adjacent localities (P. apacheanus from Grutas el Sabinal in northern mountain ranges may have been limited during the Pleisto- Chihuahua, and P. rufulus from north-western Baja Califor- cene. The cooler Pleistocene glacial periods and down-slope nia), scorpions of the minimus complex appear to fit this expansions of mixed pine-oak woodlands may have provided pattern of distribution, forming a ring around the Gulf of only one of the habitat requirements that were needed for California (Fig. 4), the terminus of which is situated in dispersal. If suitable rocky habitat was absent, dispersal by northern Baja California where the distributions of the two these stenotopic rupicolous scorpions would have been lim- species come into proximity (Fig. 4). This ring distribution ited. The sole exception was observed in samples of may have been intact until the mid-Miocene separation of P. apacheanus from the Peloncillo and Chiricahua Mountains the Baja California Peninsula from the Mexican mainland (Fig. 2a). One of the two haplotypes from the Peloncillo severed habitat connectivity. Future studies should test this Mountains grouped with haplotypes from the adjacent Chiri- hypothesis with additional samples of P. apacheanus from cahua Mountains. The closest slopes of these mountain Sonora and of P. rufulus from Baja California, and with the ranges are separated by about 10 km of xeric grassland. At inclusion of P. cazieri, a possible member of the minimus the southern end of the Chiricahua Mountains, however, a complex, also from Baja California, which could not be low-elevation lava field, stretching 25 km, almost connects to obtained for the present study. the Peloncillo Mountains. Although perhaps uninhabited by P. apacheanus today, this rocky habitat may have provided a CONCLUSIONS stepping stone during cooler Pleistocene glacial periods, per- mitting gene flow between the two otherwise isolated popu- Many aspects of biogeography seek to explain the distribu- lations of P. apacheanus in these mountains. Future studies tions of species in terms of historical factors and contempo- with larger sample sizes from each mountain range could rary ecology. Stenotopic scorpions such as the minimus better determine whether the inferred pattern of limited gene complex offer a model for exploring correlations between flow between adjacent mountain ranges is a result of the Earth history and biological diversification. Spatial and tem-

Journal of Biogeography 40, 1850–1860 1857 ª 2013 John Wiley & Sons Ltd R. W. Bryson Jr et al. poral patterns of evolution in the minimus complex suggest Axen, G.J. & Fletcher, J.M. (1998) Late Miocene–Pleistocene that stenotopy promoted pre-Quaternary diversification in extensional faulting, northern Gulf of California, Mexico these rupicolous scorpions. The heterogeneous landscape of and Salton Trough, California. International Geology south-western North America provided the backdrop for Review, 40, 217–244. diversification. Miocene and Pliocene geomorphology, per- Brand, P. & Stump, E. (2011) Tertiary extension and fault haps in concert with climate change, appears to have induced block rotation in the transition zone, Cedar Mountains Area, allopatric divergence on a small spatial scale. Arizona v.1.1. Arizona Geological Survey, Tucson, AZ. Bryson, R.W. & Riddle, B.R. (2012) Tracing the origins of widespread highland species: a case of Neogene diversifica- ACKNOWLEDGEMENTS tion across the Mexican sierras in an endemic lizard. Bio- We thank R.F. Ayrey, A.J. Ballesteros, T. Burkhardt, J. Che- logical Journal of the Linnean Society, 105, 382–394. ma, T. Dikow, C. Duran-Barron, O.F. Francke, J. Galvan, Bryson, R.W., Murphy, R.W., Graham, M.R., Lathrop, A. & A. Gluesenkamp, M.R. Graham, C. Hanna, D. Hartman, Lazcano-Villareal, D. (2011) Ephemeral Pleistocene wood- J. Huff, L. Jarvis, J. Krejca, C. Kristensen, C. Lee, K.J. lands connect the dots for highland rattlesnakes of the McWest, R. Mercurio, H. Montano,~ R. Myers, K. Peterson, Crotalus intermedius group. Journal of Biogeography, 38, M. Rubio, C. Savvas, W.D. Sissom, N.W. Smith, M.E. Sole- 2299–2310. glad, P. Sprouse, M. Torocco, D. Ubick and Z.J. Valois for Connell, S.D., Hawley, J.W. & Love, D.W. (2005) Late Cen- assisting with fieldwork and/or providing samples for analy- ozic drainage development in the southeastern basin and sis; L.A. Esposito for COI primer development; O. Delgado, range of New Mexico, southeasternmost Arizona, and wes- M. Mosier and P. Rubi for generating DNA sequence data at tern Texas. New Mexico’s ice ages (ed. by S.G. Lucas, G.S. the American Museum of Natural History (AMNH); the Morgan and K.E. Zeigler), pp. 125–150. New Mexico National Park Service for granting permits to collect in the Museum of Natural History and Science, Albuquerque, Big Bend, Channel Islands and Guadalupe Mountains NM. National Parks; and The Nature Conservancy and the Chan- Cooper, S.J.B., Harvey, M.S., Saint, K.M. & Main, B.Y. dlers for access to the Independence Creek Preserve. Field- (2011) Deep phylogeographic structuring of populations work in Mexico was conducted under permits granted by of the trapdoor spider Moggridgea tingle (Migidae) from SEMARNAT to O.F. Francke, the late F. Mendoza-Quijano, southwestern Australia: evidence for long-term refugia and C. Solis-Rojas. Other support and assistance was pro- within refugia. Molecular Ecology, 20, 3219–3236. vided by R.F. Ayrey, C.A. Brown, O. Delgado, E. Gonzalez, Derkarabetian, S., Ledford, J. & Hedin, M. (2011) Genetic M.R. Graham, J. Hearst, D. Houston, J. Huff, J. Karges, J. diversification without obvious genitalic morphological Klicka, K.J. McWest, R. Mercurio, C. Neill, R. Skiles and divergence in harvestmen (Opiliones, Laniatores, Sclerob- B.T. Smith. Aspects of the work conducted at the AMNH unus robustus) from montane sky islands of western North were funded by National Science Foundation grants DEB America. Molecular Phylogenetics and Evolution, 61, 844– 0413453 and DEB 0228699, and by a grant from the Richard 853. Lounsbery Foundation to L.P. Dmitriev, D.A. & Rakitov, R.A. (2008) Decoding of superim- posed traces produced by direct sequencing of heterozy- gous indels. 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