Evolution of Scaphinotus Petersi (Coleoptera: Carabidae) and the Role of Climate and Geography in the Madrean Sky Islands of Southeastern Arizona, USA

YQRES-03387; No. of pages: 10; 4C: 3, 5 Quaternary Research xxx (2012) xxx–xxx

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Quaternary Research

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Evolution of Scaphinotus petersi (Coleoptera: Carabidae) and the role of climate and geography in the Madrean sky islands of southeastern Arizona, USA

Sara Gran Mitchell ⁎, Karen A. Ober

Department of Biology, College of the Holy Cross, PO Box B, 1 College St., Worcester, MA 01610, USA article info abstract

Article history: Geographically isolated environments such as the conifer forests atop the Madrean “sky islands” in southeastern Received 21 May 2012 Arizona provide natural laboratories for studying factors involved in speciation and origins of biodiversity. Using Available online xxxx molecular and geospatial analyses, we examine beetle population phylogeny, regional climate records, and the Quaternary paleobiogeography of forests to evaluate four hypothetical scenarios regarding the current Keywords: geographic and population genetic patterns of Scaphinotus petersi. Scaphinotus petersi is a large, ﬂightless beetle Arizona that resides in the Madrean conifer forests above ~1900 m asl. Our results do not support the current hypothesis Sky islands Quaternary that S. petersi populations found on seven separate mountain ranges are genetically distinct and separated as Beetles temperatures warmed after the Last Glacial Maximum (LGM). Rather, we show that only some of the ranges Molecular evolution hold genetically distinct populations, and the timing of separation among the populations does not appear to Population divergence coincide with speciﬁc climatic events such as warming trends. In addition, we show that predicted changes to Paleobiogeography the climate of the Madrean sky islands may result in the disappearance of S. petersi from some of the lower ranges Climate by the end of this century. © 2012 University of Washington. Published by Elsevier Inc. All rights reserved.

Introduction first hypothesis, cooler and/or wetter climate conditions in the southwestern USA during the late Pleistocene favored more widely distributed The Madrean “sky islands” region of southeastern Arizona consists of conifer forests, and thus S. petersi habitat, at lower altitude. Divergence isolated forested mountain ranges that rise above a relatively flat prairie, occurred after post-Last Glacial Maximum (LGM) warming caused the scrub, and desert environment. Steep precipitation and temperature lower limit of conifer forests to contract uphill, eventually disconnecting gradients with altitude give rise to a series of ecological successions on forests and thus separating the beetle populations. If true, we would sky islands, and the ecosystems atop these ranges are sufficiently sepa- predict the following results from our genetic and paleobiogeographic rated from other suitable habitats such that the remoteness promotes analyses: 1) Beetle populations on different ranges will be genetically differentiation of organisms that live there. Such environments provide distinct from the others (in other words, each of the populations will natural laboratories for studying the processes involved in speciation be “monophyletic”). 2) Populations on ranges with conifer forests that and origins of biodiversity of endemic organisms (e.g., Brown, 1971; connect with the least amount of lowering or are close together will be Warshall, 1994). One such organism is the flightless, ground-dwelling more genetically similar and more recently separated than populations beetle, Scaphinotus petersi, characterized by Ball (1966).Today,S. petersi on ranges that require more conifer forest lowering to connect or are is found only in the Madrean montane conifer forest ecosystem (here separated by larger distances. 3) Now-separated populations should referred to as “conifer forests”) above ~1900 m elevation. According to have common ancestors during cooler, wetter times such as the Last Ball (1966), beetle populations on several sky island mountain ranges Glacial Maximum (LGM) and divergence should occur after the initiation have distinct morphological characteristics; therefore, Ball concluded of warmer or drier times during the Holocene. 4) Independent analyses that these ranges now host separate S. petersi subspecies. In this study, of conifer forest paleobiogeography and other paleoecological data will we examine S. petersi phylogeny, regional climate records, and the show that forests were connected during the cooler, possibly wetter paleobiogeography of Madrean montane conifer forests to test four hy- conditions thought to exist during the Pleistocene. potheses that could explain current geographic and population genetic A second possible scenario is that beetle dispersal can occur regard- patterns of this endemic beetle. less of habitat connectivity or proximity (no “island” effect), and is thus Ball (1966) hypothesized that all S. petersi subspecies were derived completely decoupled from changes in climate or forest biogeography. from a common ancestral population that once moved freely between In this scenario, we predict the following: 1) Genetic analysis of beetle mountain ranges when forests were more extensive. According to this populations will show evidence of a diversity of lineages (populations may be “polyphyletic” and contain distantly related individuals). ⁎ Corresponding author. Fax: +1 508 793 2696. 2) There may not be any relationship between mountain range proxim- E-mail address: [email protected] (S.G. Mitchell). ity or saddle altitude and the genetic relatedness of beetle populations.

3) Climate conditions were not necessarily favorable for forest connec- conifer forests that occur on the highest mountain ranges in southeastern tivity during the timeframe represented by the phylogenetic tree. 4) Arizona. Evidence for gene flow or dispersal (the movement of one or more individuals from one range to another) can occur during warm and/or dry Study area climate conditions. 5) The genetic data may be consistent with the re-population of ranges that could have lost and then regained their The Madrean sky islands area encompasses a nearly 200,000 km2 forests during the Holocene. area of southeastern Arizona, southwestern New Mexico and north- A third possible scenario is that the S. petersi populations were ern Mexico. This region is notable for its relatively small but high once fully connected and are now isolated, but they have not been (>2000 m) mountain ranges separated by low (b1000 m) topogra- separated long enough to diverge into distinct phylogenetic groups. phy. S. petersi populations are restricted to a subset of ranges: the In this scenario, 1) there would be no population genetic structure Pinal, Pinaleño, Santa Catalina, Rincon, Santa Rita, Chiricahua, and or genetic divergence seen among the mountain ranges and isolated Huachuca Mountains (Ball, 1966;Fig.2).S. petersi has been sighted populations would not fall into distinct monophyletic groups, and but not formally recorded in the Galliuros, Sierra Anchas, and Mogollon 2) there will be no geographic relationship to genetic structure, and Rim (K. Will and G. Ball, pers. comm., 2010). 3) the timing of divergence will not be dependent on climatic conditions. Tectonically, the Madrean sky island region is located in the Basin and A final scenario is a combination of the above. Perhaps some ranges RangeprovinceofNorthAmerica.Themountainrangeshereformedasa are more geographically isolated than others, and thus have distinct, result of the extension and uplift, likely in two stages (Wagner and possibly monophyletic beetle populations. But in this scenario, dispersal Johnson, 2006). The last period of peak uplift likely occurred between may occur between some mountains despite unfavorable climates or a 12 and 6 Ma (Wagner and Johnson, 2006). Due to the geometry of ex- lack of evidence for forest connectivity. In this last scenario, we might tension, the ranges are aligned along two major NW–SE trending ridges. see: 1) A complex phylogenetic tree, with some monophyletic and The ridges are separated by broad expanses, and along ridges, peaks are some polyphyletic groups of beetles on individual ranges. 2) A complex often separated by narrow but deep canyons. The San Pedro River sepa- relationship between mountain range geography and genetic similarity rates the two ridges, and the Gila River separates the northernmost Pinal and divergence times. 3) Climate conditions and their relationship to Mountains from all others to the south. forest connectivity may be irrelevant to the timing of beetle divergence. The steep topographic gradients in the area are associated with gradi- We evaluate these four scenarios by utilizing modern DNA sequenc- ents in temperature and precipitation. These climate gradients in turn ing and analysis tools, a GIS-based analysis of forest paleobiogeography, generate a rich diversity of ecosystems in a relatively small geographic and an analysis of geographic and climatic data. Finally, we use our area, with different ecosystems occupying certain altitudinal ranges results to make predictions on the fate of this endemic species in the (Fig. 3). Sonoran and Chihuahuan deserts dominate at altitudesb1000 m. face of global climate change. Other biomes in the region include grasslands,scrub,chaparral,oak-pine woodlands, montane conifer forest, and spruce-firforest(Halvorsen et al., 2001). S. petersi favors the Madrean Montane Conifer Forest: Background Douglas-fir—Mixed Conifer and Madrean Montane Conifer Forest: pine, as identified in the Arizona Gap vegetation map (Ball, 1966; Halvorsen Description of Scaphinotus petersi et al., 2001). The first of these ecosystems is dominated by Pseudotsuga menziesii (Douglas fir) with some Abies concolor (White fir), Pinus Scaphinotus petersi is a large (1.25 cm) ground beetle (Fig. 1). strobiformis (Mexican white pine), Robinia neomexicana (New Mexico S. petersi, like other Scaphinotus,isflightless, with absent flight locust), Acer gradidentatum (Bigtooth maple), and some other species wings under fused elytra. These beetles are specialist predators of present (Halvorsen et al., 2001). The second is characterized by the pres- land snails, possessing highly modified mouthparts for penetrating ence of Pinus arizonica (Arizona pine), Pinus ponderosa (Ponderosa pine), the snail shell and extracting the soft parts of the prey (LaRochelle, and Pinus engelmanii (Apache pine) (Halvorsen et al., 2001). 1972; Digweed, 1993). In part, it is because of their specialized diet The Quaternary climate history in the southwestern USA is de- of snails that they are confined to the moist conifer forests where duced from a variety of paleoclimate proxies, including pluvial lake the snails live. While the genus Scaphinotus is widely distributed in levels, pollen records from lake cores and packrat middens, ground- North America, S. petersi is endemic and restricted to the Madrean water isotopic composition, and speleothems (e.g., Martin, 1963; Waters, 1989; Van Devender, 1990; Zhu et al, 1998; Allen, 2005; Holmgren et al., 2006; Pigati et al., 2009; Wagner, et al., 2010). The region most likely experienced relatively cool and wet conditions during the Wisconsin glaciation, which began ~115–110 ka, with minimum temperature and maximum effective precipitation conditions occurring during the LGM, between 24 and 21 ka (e.g., Menking et al., 2004; Allen, 2005). Post-LGM climate conditions appear to have been quite variable, with multiple pluvial lake highstands during the deglacial period, 18–10 ka (Waters, 1989); for example, the Younger Dryas at ~12.7–11.5 ka was likely cool and wet (Pigati et al., 2009). Warming and increased aridity continued unevenly through the Holo- cene, with some records indicating peaks in aridity and temperature during the Holocene thermal maximum, ~11–6ka(Zhuetal.,1998; Renssen et al., 2009). Globally, the past 2000 yr has been characterized by a general warming trend in multiple climate proxies and the instru- mental record (Mann et al., 2008), and regional records indicate that generally warm and dry conditions have prevailed in the southwestern US since the middle Holocene (Waters, 1989; Van Devender, 1990; Zhu et al., 1998; Sheppard et al., 1999). Climate models project continued Figure 1. Specimens of Scaphinotus petersi. (A) Scaphinotus petersi catalinae from Mt. Lemmon, Santa Catalina Mts. (B) Scaphinotus petersi corvus from Chiricahua Mts. warming and drying through the end of the century (IPCC, 2007; Scale bar is 1 cm. Seager et al., 2007; Weiss et al., 2009).

Figure 2. Study area. Mountain ranges with signiﬁcant area above 1500 m are shown with triangles. Area above 1300 m shown in light gray, area above 1900 m shown in dark gray. Area mapped as forest on the Arizona GAP map is black. Triangles with dots show S. petersi locations. Open triangles are mountain ranges where S. petersi have not been found. S. petersi subspecies names and their corresponding ranges are listed at left.

Previous work on speciation in the Madrean sky islands (Masta, 2000), and beetles (Smith and Farrell, 2005a,b) also tested hypotheses of divergence times among now-isolated populations. Several studies have focused on the biogeography of species on These studies both concluded that populations diverged before 1 Ma, the Madrean sky islands including plants, arthropods, birds, lizards, much earlier than the proposed post-LGM habitat fragmentation. and mammals (Linhart and Permoli, 1994; McCord, 1994; Sullivan, In contrast, some studies of vertebrates (Sullivan, 1994; Holycross and 1994; Barber, 1999a,b; Slentz et al., 1999; Maddison and McMahon, Douglas, 2007; McCormack et al., 2008) suggest a more recent, post- 2000; Masta, 2000; Boyd, 2002; Downie, 2004; Smith and Farrell, LGM effect on population genetic structure. In addition, studies by 2005a,b; McCormack et al., 2008; Tennessen and Zamudio, 2008). Masta (2000), Boyd (2002),andMcCormack et al. (2008) all reported Most of these studies have shown signiﬁcant morphological variation biogeographic patterns of organisms being more closely related along among populations and/or genetic structure in species. However, a north-south mountain ranges than across the east-west gap in the biogeographic study of a galling insect (Downie, 2004) and a study Madrean sky islands. of squirrels (Lamb et al., 1997) failed to detect any evidence for genetic divergence. Quaternary climate changes have also been implicated Methods in affecting the evolution of local species, but in dissimilar ways. For example, phylogeographic studies in other arthropods such as spiders Beetle collection and DNA sequence data

By constructing a DNA-based phylogenetic tree for S. petersi,we can show the evolutionary relationships between beetles within and among different ranges. Populations on individual mountains may be monophyletic (all specimens are most closely related to the others on their same range) or polyphyletic (at least one specimen on the range is more closely related to populations on other ranges than those on its own). A population may also be paraphyletic, which means that it was the ancestral population for another subspecies. If beetle populations are fully mixed because they are able to disperse freely, or have not had time to separate into subspecies, mountain ranges should have polyphyletic populations. If beetles on a particular range remained separated from all others for a long enough time to develop into a subspecies, that range should have a monophyletic population. This information is derived from DNA sequence data. Figure 3. Generalized cross-section of sky island biomes in the Pinaleño Mountains. S. petersi have only been recorded in the conifer forests that exist between ~1900 We collected DNA sequence data from 54 specimens of S. petersi in and 3000 m. nine localities in seven mountain ranges (Fig. 2), Sphaeroderus lecontei

Please cite this article as: Mitchell, S.G., Ober, K.A., Evolution of Scaphinotus petersi (Coleoptera: Carabidae) and the role of climate and geography in the Madrean sky islands of southeastern Arizona, USA, Quaternary Research (2012), http://dx.doi.org/10.1016/j.yqres.2012.11.001 4 S.G. Mitchell, K.A. Ober / Quaternary Research xxx (2012) xxx–xxx and two additional species of Scaphinotus serve as outgroups (Supple- horizontal distance between beetle-inhabited forested mountain mentary materials). Genomic DNA was extracted following the protocol ranges, and 2) vertical distance of forest lowering required to bring for- outlined in Maddison et al. (1999). PCR reactions were performed using ests on separate ranges into contact with each other. For the first anal- amodification of the procedure described in Maddison et al. (1999). ysis, we simply measured the straight-line distance between the Reactions used a 53–56°C annealing temperature. This procedure was peaks from where our beetle specimens were collected. For the forest used to amplify either a 300 base pair portion or 1200 base pairs of connectivity analysis, we first had to identify the altitude of the modern ND1 and adjacent RNA genes, and either a 500 base pair portion or lower Madrean montane conifer forests. We selected the cells in the 1400 base pairs of COI. Macrogen Inc. (Korea) carried out DNA sequenc- DEM that are identified in Halvorsen et al. (2001) as Madrean Montane ing using an Applied Biosystems ABI 3730 48-capillary DNA analyzer Conifer Forest: Douglas-fir—Mixed Conifer and Madrean Montane with Big Dye Terminator Technology according to the manufacturer's Conifer Forest: Pine, and identified the lower limit as the altitude protocols (Applied Biosystems). DNA sequence data was visualized above which 90% of forest exists today. We then determined how using the SEQUENCHER 3.0 software (Gene Codes Corp.). Sequences much Madrean conifer forest depression is required to connect were easily aligned by eye using MACCLADE 4.06 (Maddison and now-separate areas by finding the highest “saddle” between every Maddison, 2005). Data matrices are available from KAO. combination of peaks, and subtracting these altitudes from the contour defining the lower limit of the Madrean conifer forest. Second, we deter- Age estimates of populations mined the size of the modern beetle habitats by measuring both the area of mapped montane conifer forest and the area >1900 m for ranges with To compare the dates of population divergence and/or changes in and without beetle populations today. Our data sets for these analyses populations due to dispersal events with the timing of climate events included a 30-m grid size digital elevation model (DEM) obtained from in the region, we simultaneously reconstructed the phylogenetic tree the USGS Seamless Data Warehouse and vegetation data from the and estimated the divergence dates using BEAST (Drummond and Arizona Gap Analysis Project Vegetation Map (Halvorsen et al., 2001). Rambaut, 2007). BEAST can be used as a method of reconstructing We used two methods to measure the size of modern beetle phylogenies but is also a framework for testing evolutionary hypoth- habitats. First, we determined the contiguous forest area at and eses while incorporating phylogenetic uncertainty. BEAST uses a above the contour defining the lower limit of Madrean conifer forest Bayesian Markov Chain Monte Carlo analysis of molecular sequences for each of the 16 ranges in the study area by extracting the number (Drummond et al., 2002) to average over all evolutionary trees, so of cells with that value or higher from the DEM. We also used the that each tree is weighted proportional to its posterior probability. Arizona GAP vegetation map from Halvorsen et al. (2001) to measure With this method we inferred time-measured phylogenies using a the area mapped as forest on each range. Using information about relaxed molecular clock for all data combined. We partitioned the where beetles are and are not currently living, we then determined combined data into five subsets based on gene and codon position. the minimum habitat size (both area >1900 m and area of montane We chose unlinked GTR+I+Γ models with four gamma categories, conifer forest) required to support a beetle population. We used the a coalescent extended Bayesian skyline plot for the tree prior, and results of this analysis to estimate the effects of further forest retrac- an uncorrelated lognormal relaxed clock model of rate variation esti- tion due to warming and/or increased aridity on the sustainability of mated for each partition with a normal distribution and a mean for each range's beetle population. each gene based on the rates for each gene from Pons et al. (2010). Using GIS grids of temperature, precipitation, and vegetation, we After an initial period of fine-tuning the operators, two separate determined the climate conditions currently favored by the beetles' MCMC analyses were run for 20 million generations with parameters Madrean conifer forest habitat. We then used a simple spatial model sampled every 1000 generations. Independent runs were combined to determine how changes in temperature and precipitation conditions using LOGCOMBINER1.5.4 (Rambaut and Drummond, 2007), and affect forest distributions in both the past and future. Our model only in- the first 30% of the generations from each run was discarded as dicated where climate conditions were likely favorable for forests based burnin. Convergence of the chains was checked using TRACER 1.4 on average maximum temperature and average precipitation; it did not (Rambaut and Drummond, 2007). The searches achieved adequate show where forests did or did not exist in the past, nor did it account for mixing as assessed by the high effective sampling size (ESS) values complexities in forest succession related to seasonality (e.g., Kupfer et for all parameters of 100 or greater. Bayesian posterior probabilities, al., 2005; Kimball et al., 2010). Furthermore, it assumed that vegetation node ages, and upper and lower bounds of the 95% highest posterior den- is in equilibrium with climate today. These assumptions produced a sity interval for divergence times were calculated using TreeAnnotator model that overestimates forest extent. 1.5.4 and visualized using FIGTREE 1.3.1 (Rambaut, 2010). Given the We generated a continuous spatial data layer of temperature by inferred phylogeny and observed localities of the specimens at the tips first calculating the temperature lapse rate using climate and eleva- of the tree, a parsimony criterion was used to select the reconstruction tion data from 28 local weather stations (http://wrcc.dri.edu/). of ancestral population locality and identify changes from an ancestral These stations range from 701 to 2426 m asl; all climate station re- population locality or dispersal event on the phylogeny for each cords have at least 24 years of data, with the exception of the highest branching point (Farris, 1970; Swofford and Maddison, 1987). station, which only extended from 1965 to 1981. Data from these records showed that the lapse rate for average maximum tempera-

Geographic relationships ture is Tmax =34.333–0.0072z, where Tmax is the average maximum annual temperature and z is altitude in meters (Fig. 4A). We used We used ArcGIS and spatial data sets of topography, vegetation, the average maximum annual temperature in our lapse-rate equation and beetle locations to test hypotheses regarding patterns of beetle because it had a better linear regression (r2 =0.94, pb0.01) than sub-speciation and how forest connectivity changes when the alti- mean annual temperature, average minimum temperature, or tem- tude of the lower limit of the Madrean montane conifer forest is perature extremes. We used this lapse rate and altitudes from the moved up and down. Geospatial analyses included island separation, DEM to calculate the Tmax for every cell in the study area, extrapolat- habitat size, and simple predictive vegetation mapping based on cur- ing and interpolating as necessary (Fig. 4B). We also used the PRISM rent forest extent and climate conditions. precipitation grid (average annual precipitation, Pann, from 1991 to According to the separation-by-forest-retraction hypothesis, pop- 2000, PRISM Climate Group, Oregon State University, http://www. ulations that are geographically “close” should be more closely related prismclimate.org)(Fig. 4C) and vegetation from the Arizona GAP pro- and more recently separated than those that are geographically distant. ject in this analysis (Fig. 4D) (Halvorsen et al., 2001). All grids were For this study, “geographic separation” has two components: 1) resampled to 900 m resolution.

Figure 4. (A) Relationship between average maximum annual temperature (Tmax) and altitude (z) from 45 climate stations in southeastern Arizona (wrcc.dri.edu). (B) Average maximum annual temperature, Tmax. (C) Average annual precipitation for 1991-2000 (PRISM Group, Oregon State University, http://www.prismclimate.org, created 2007). (D) Simpliﬁed vegetation and land cover from Arizona GAP Analysis (Halvorsen et al., 2001).

We then determined the means and standard deviations for tem- increase; IPCC, 2007). Constraining precipitation conditions in the past perature and precipitation for both the entire study area and the was difﬁcult, as most analyses of proxies do not result in quantitative Madrean montane conifer forest. We calculated 95% envelopes for estimates of annual precipitation. For our model, we varied precipita-

Tmax and Pann (μ±2σ). Areas that currently fall within the 95% enve- tion by multiplying the modern average annual precipitation by a factor lopes for both Tmax and Pann have climates compatible with Madrean ranging from 0.80 to 2. These factors are within the estimates for montane conifer forest but do not necessarily host forests today; projected increased aridity (IPCC, 2007) and LGM moisture enhance- areas outside both envelopes are unlikely to support forests. ment in the southwestern US (Menking et al., 2004). Using these climate envelopes and the GIS data sets for temperature and precipitation, we mapped “hospitable forest area” for different cli- Results mate scenarios (Table 3). While spatial and vertical patterns in temperature were possibly different under other climate regimes, for example Phylogeography and estimates of divergence times the lapse rate may have been less steep due to higher humidity during colder times, we assumed a constant lapse rate for temperature (e.g., Results of the Bayesian phylogenetic analysis in BEAST show some temperature increases or decreases by the same amount over the entire monophyletic groups (“clades”) corresponding to mountain ranges and area). Based on paleoclimate estimates and climate projections, we de- are spatially structured with genetic variation at deep and shallow scales. pressed temperature up to 5°C to represent regional LGM conditions Overall, there is a geographic pattern to the evolutionary relationships (Zhu et al., 1998; Anderson et al., 2000). To predict future hospitable among S. petersi populations, with two well-supported major clades forest area, we raised Tmax by 4°C, based on projected multi-model (East and West clades in Fig. 5). We include the Santa Rita population in mean annual surface temperature change in western North America the East Clade despite its geographic location. There is not strong support by the years 2080–2099 (scenario A1B with moderate CO2 emissions for the clades closer to the tips of the evolutionary tree (Fig. 5).

Figure 5. A. Phylogeny of S. petersi dated using a Bayesian relaxed molecular clock in BEAST. Outgroups were removed to show greater detail. Specimen numbers are removed, but the mountain range from which they were collected is indicated at right. A star shows nodes with greater than 95% Bayesian posterior probability. Branches are proportional to time in years. 95% conﬁdence intervals for the ages of major clades in the tree are indicated with gray bars. The node letters indicate population fragmentation between mountain ranges (see Table 1). The separation between the East Clade (upper branch) and West Clade (lower branch) at ~60 ka is the oldest divergence within the species. The bar along the bottom indicates the climate timeline, including the Wisconsin glaciation, LGM = Last Glacial Maximum, YD = Younger Dryas, and CO = Climatic Optimum.

The East Clade of populations from the Santa Rita, Chiricahua, and Clade diverged from the West Clade populations at ~60 ka. However, Pinaleño Mountains are clearly phylogenetically distinct from the within the East Clade, the Chiricahua population and Pinaleño popula- West Clade of S. petersi from the Santa Catalina, Rincon, Huachuca, tion last shared an ancestor with the Santa Rita population at ~8 and and Pinal Mountains. Within the East Clade, the S. p. kathleenae pop- 10 ka respectively. The S. p. petersi population in the Pinals diverged ulation from the Santa Rita Mountains appear to be paraphyletic with from the Santa Catalina Mountain population at ~8 ka, and the Rincon respect to (i.e., the source population of) populations now living on population diverged from the Santa Catalina population ~4 ka. More the Chiricahua and Pinaleño ranges. In the West Clade, the Santa than one dispersal event from the Santa Catalinas to the Huachucas Catalina population (S. p. catalinae) is paraphyletic with respect to may have occurred; one ~12 ka and another ~3 ka (Fig. 5 and Table 1). S. p. petersi from the Pinal, S. p. biedermani from the Rincon and S. p. biedermani from the Huachuca Mountains. S. p. biedermani is Geographic relationships not a monophyletic subspecies because the specimen from the Rincons is a separate lineage from the Huachuca population. The Huachuca Ninety percent of mapped Madrean montane conifer forest lies population is also polyphyletic, as one specimen groups with members above ~1900 m. Distances between peaks range from 34 to 215 km of S. p. catalinae from the Santa Catalina range (Fig. 5). and forest lowering to reach the highest saddles between ranges Population divergence time estimates for mtDNA lineages from BEAST reveal a deep and intricate history of diversiﬁcation (Fig. 5, Table 1). Sev- eral major divergences occurred prior to the LGM, while the majority oc- Table 2 – – Horizontal distance (bold, in km) and height above saddle (italics, in m) separating curred during deglaciation (18 10 ka) or the Holocene (10 0 ka). For sample localities. Distances are between highest mountain summits and height above example, the S. p. kathleenae from the Santa Rita range plus the East saddle is the vertical distance from 1900 m to the highest saddle altitude between peaks.

Table 1 Pinal Pinaleño Chiricahua Santa Catalina Rincon Santa Rita Ages of selected nodes estimated from molecular data in Scaphinotus petersi phylogeny Pinaleño 109 from BEAST analysis. Letters correspond to nodes in Figure 5. 1100 Chiricahua 214 110 Node Split between populations Age in ka 95% C.I. age in ka 1100 560 A East Clade vs West Clade 60 12–214 S. Catalina 93 90 156 B Santa Rita 1 vs Santa Rita 2 31 5–112 1100 690 690 C Santa Rita vs Chiricahua 8 9–34 Rincon 121 81 125 34 D Santa Rita vs Pinaleño 14 2–52 1100 670 670 690 E Catalina vs Huachuca 1 12 1–21 S. Rita 176 144 148 83 95 F Catalina vs Pinal 8 2–27 1100 550 560 690 670 G Catalina vs Rincon 4 0.5–12 Huachuca 215 150 108 125 65 62 H Catalina vs Huachuca 2 3 0.5–9 1100 550 560 690 670 420

area on all but the Pinaleño and Chiricahua Mountains, the two highest ranges in the area.

Discussion

Geographic patterns of evolution

The results show a complex evolutionary history for S. petersi. There is a great deal of phylogeographic structure within the species, with some of the ranges showing genetic and geographic isolation, but others showing evidence of dispersal and genetic mixing. The Pinal, Pinaleño, and Chiricahua populations are now genetically and geographically isolated. The Rincon population may also be genetically distinct, but with only one sample we cannot determine that this population is monophyletic. The Santa Catalina population appears Figure 6. Area above 1900 m (black) and largest contiguous area of montane conifer to be the source for populations in the nearby Rincon as well as the forest on each peak (lined) for each mountain range in the study area. Ranges are sorted more distant Pinal Mountains, and there is evidence of at least two in order of decreasing mapped forest area. Beetle sample localities for this study are relatively recent dispersal events from the Santa Catalinas to the marked with a dot, ranges thought to host S. petersi but lacking samples for in this study are marked with a star. All other ranges have no known S. petersi populations. Huachucas. Based on morphological rather than genetic characteristics, Ball (1966) also indicated that Santa Catalina populations may have given rise to Pinal, Rincon, and Huachuca populations. The bee- range from 420 to 1100 m (Fig. 2, Table 2). Geographically, the Pinal tle population on the Pinal range is distinctive in that it is both mono- Mountains are the most separated from all others by its geographic phyletic and recently diverged from other populations. It is possible location at the north end of the study area and deep valley of the the Pinal range lost its beetles during the early Holocene and was Gila River. The Pinaleño and Chiricahua Mountains are separated repopulated at a single time later, thus explaining why their lineage from each other by >100 km and from the Rincon, Santa Catalina, only goes back to 8 ka and why that range is monophyletic today. Santa Rita, and Huachuca Mountains by the San Pedro River valley. The relationship between geographical distance and genetic At 62 km apart and 420 m of forest lowering required to connect similarity is not simple. The Santa Catalina, Rincon, and Huachuca ranges, the geographically “closest” beetle populations are on the populations are evolutionarily closely related to each other according Santa Rita and Huachuca Mountains. The Santa Catalina Mountains to the genetic analysis. They are also geographically close together. are close in terms of distance to the Rincon (34 km) but the saddle However, the Santa Rita population, while geographically close to between them is very deep (1210 m, or 690 m below the current the Huachuca range, is distantly related to the clade containing it, lower Madrean conifer forest limit). and more closely related to geographically distant populations in The area of mapped forest and area above 1900 m are both good the Pinaleños and Chiricahuas. These results do, however, agree with predictors of beetle presence or absence (Fig. 6). All eight ranges Ball's (1966) original hypothesis of the divergence of S. petersi based with >8 km2 of contiguous forest area according to Halvorsen et al. on morphology. He suggested the Pinaleño, Chiricahua, and Santa Rita (2001) support a beetle population today. No beetles have been for- populations formed a clade. Our molecular results also agree with Ball's mally reported on any ranges with b8km2 of contiguous forest. hypothesis that the Rincon, Huachuca, and Pinal populations are related Area above 1900 m is also a good predictor of beetle presence; all to each other even though the Pinal Mountains are somewhat more ranges with >65 km2 above 1900 m have beetle populations. Only geographically distant from the rest of the clade. one of the eleven ranges with b65 km2 of area above 1900 m has The fact that the Santa Rita and eastern populations are more closely beetles; the Pinal range supports a S. petersi population with only related despite the close topographical connection of the Santa Rita 26 km2 of area >1900 m and 27 km2 of contiguous forest area. with the Huachuca ranges suggests an unexpected phylogeographic The temperature and precipitation conditions supporting the divide. It is possible that an early fragmentation event separated the Madrean montane conifer forest are cooler and wetter than that of the habitat ranges, cutting off the Santa Rita from the West Clade pop- region as a whole. The average and standard deviation of Tmax and ulations. This phylogeographic separation is based on mitochondrial Pann of the whole region are 24.6±2.3°C and 41.8±11.2 cm/yr respec- loci alone and may not reflect patterns seen at other loci. Other evolu- tively. Tmax and Pann of the forested areas are 18.2±2.1°C and 79.6± tionary processes, such as microhabitat factors, competition, predation, 16.8 cm/yr respectively. Therefore, the 1-σ climate envelopes are 16.1 or intrinsic barriers may have limited dispersal from the Santa Ritas to to 20.3°C for Tmax and 62.8 to 96.4 cm/yr for Pann. The area encompassed the Huachucas even when climate, topography, or vegetation favored it. by both the temperature and precipitation envelopes is somewhat more extensive than the mapped forests today, but their geographic distribu- Madrean montane conifer forest connectivity tions are similar (Fig. 7A). We also determined the potential forest extent for two “wet and While our model shows that the altitude of the Madrean montane co- cold” climate scenarios and one “warm and dry” scenario (Table 3, nifer forests may possibly have been depressed to as low as 1200 m asl Figs. 7B,C, and D). The potential forest area under the first wet and during the LGM due to temperature depression and precipitation cold climate scenario, with temperatures 3°C colder and 150% of enhancement, there is no paleobotanical evidence that the area was modern precipitation, is more extensive than under the modern cli- blanketed in widespread Madrean montane conifer forests at any time mate, but does not result in forest connections between mountain during the late Pleistocene or Holocene (Betancourt, 1990; Holmgren ranges. Under the second cold and wet scenario, with temperatures et al., 2006). Paleoecological records from packrat middens in the 5°C colder and 200% of modern precipitation, some, but not all, of Peloncillo Mountains to the east indicate that the occurrence of pinyon the hospitable forest areas connect; the Pinal, Santa Catalina, Santa pine during glacial times was approximately 350 m lower than today Rita, and Rincon Mountains all remain isolated. Conversely, under (Holmgren et al., 2006). However, most of the middens in Holmgren et the warm and dry scenario (4°C warmer and 80% of modern precipi- al.'s, 2006 study are at altitudes between 1300 and 1500 m, and none tation), the model predicts the elimination of forests on all but five of them indicate significant populations of the Madrean montane conifer mountain ranges in the region, and b8km2 of contiguous forest key species such as Pseudotsuga menziesii (Douglas fir), Abies concolor

A) Modern B) Cold and wet scenario 1

33°N 33°N

32°N 32°N

Kilometers 50

111°W 110°W 109°W 111°W 110°W 109°W

C) Cold and wet scenario 2 D) Warm and dry scenario

33°N 33°N

32°N 32°N

111°W 110°W 109°W 111°W 110°W 109°W

Figure 7. Climate envelopes favorable for montane conifer forests for four climate scenarios. Areas with either favorable temperature or precipitation are light gray, areas with favorable temperature and precipitation are dark gray. Other areas are white. The highest peak within each range are indicated by black triangles. A) Modern climate; modern montane conifer forest extent is shown in white hatching. B) 3°C cooler and 150% of modern annual precipitation. C) 5°C cooler and 200% of modern precipitation. D) 4°C warmer and 80% of modern precipitation.

(White ﬁr), or Pinus strobiformis (Mexican white pine) at those altitudes (IPCC, 2007; Pigati et al., 2009; Asmerom et al., 2010; Wagner et al., in the past 36 ka. Thus, the Madrean montane conifer forest regions have 2010). Our climate-envelope model, which increases temperature likely been geographically separated for at least the last 36 ka. Further- by 4°C, indicates that the climate will become unfavorable for wide- more, many of the beetle populations had their last inter-mountain spread montane conifer forests on all ranges except the Pinaleños dispersal events during the Holocene, not the Pleistocene. We can thus and Chiricahuas by the end of this century. Fortunately, the mid- conclude that beetles do not need connected Madrean montane conifer Holocene dispersal patterns we see in the phylogeny indicate that forests to disperse between ranges. beetles may be able to come from other ranges to repopulate if forests However, our results show that it is likely that the montane coni- ever do reappear. fer forests will continue to contract upslope if subjected to increased temperatures and aridity. Today, with the exception of the Pinal Mountains, the beetles exist only on ranges with contiguous forest 2 2 Table 3 area of at least 8 km , with 65 km or more area above 1900 m asl. Climate scenarios for predicting potential forest extent. The “moderate” A1B greenhouse gas scenario from the IPCC leads to a prediction of increased temperatures ranging from 2.5 to 6°C Scenario Temperature difference (°C) % of modern precipitation above early 20th century averages in western North America by the Cold and wet 1 3°C colder than modern 150% year 2099, and several paleoclimate investigations reveal that higher Cold and wet 2 5°C colder than modern 200% temperatures were associated with increased aridity in the past Warm and dry 4°C warmer than modern 80%

Timing of population divergence Supplementary data to this article can be found online at http:// dx.doi.org/10.1016/j.yqres.2012.11.001. The divergence time estimates of highest probability from the molecular data suggest that shared ancestral populations of S. petersi Acknowledgments lived in the late Pleistocene. The ancestral population of the entire species existed at ~60 ka, which was during a glacial period, prior Some outgroup specimens were collected by Elizabeth Jockusch. to its divergence into the East and West clades. This population may We thank Susan Masta and one anonymous reviewer for their helpful have been widespread in southeastern Arizona. The lineages in the comments. The authors also thank the College of the Holy Cross and Santa Ritas and Pinaleños also last shared a common ancestor during the Robert L. Ardizzone Faculty Excellence Fellowship for funding a cool wet time, as did the initial Catalina and Huachuca populations. for this project. The cooler and wetter conditions during these times allowed dispersal despite a high likelihood of non-connectivity of forests at that time. Although populations of S. petersi are isolated and confined to cool References moist forests, they may not need contiguous pine forest to disperse, Allen, B.D., 2005. Ice age lakes in New Mexico. In: Lucas, S.G., Morgan, G.S., Zeigler, K.E. for example, traveling along river corridors at lower elevations during (Eds.), New Mexico's Ice Ages. New Mexico Museum of Natural History and Science warmer times. It is also possible that the beetles were able to move Bulletin No. 28, pp. 107–114. Anderson, R.S., Betancourt, J.L., Mead, J.I., Hevly, R.H., Adam, D.P., 2000. Middle- and through the pinyon-juniper ecosystem that was much more wide- late-Wisconsin paleobotanic and paleoclimatic records from the southern Colorado spread in the past (e.g., Betancourt, 1990), even though they are not Plateau, USA. 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