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Home , Amargosa toad, Black toad, Dixie Valley, Lake Bonneville, Lake Manly, Western toad

University of , Reno

Reconciling Western phylogeography with prehistory

A thesis submitted in partial fulfillment of the

requirements for the degree of Master of Science in

Geography

by

Pete M. Noles

Dr. Jill S. Heaton/Thesis Advisor

December, 2010

THE GRADUATE SCHOOL

We recommend that the thesis

prepared under our supervision by

PETE M. NOLES

entitled

Reconciling phylogeography with Great Basin prehistory

be accepted in partial fulfillment of the

requirements for the degree of

MASTER OF SCIENCE

Jill S. Heaton, Ph.D., Advisor

Scott A. Mensing, Ph.D., Committee Member

Kenneth E. Nussear, Ph.D., Committee Member

C. Richard Tracy, Ph.D., Graduate School Representative

Marsha H. Read, Ph. D., Associate Dean, Graduate School

December, 2010

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ABSTRACT

This study marks the first attempt to study historical processes that may be responsible for the contemporary geographic distribution and phylogeny of Great Basin Western ( boreas spp.). The dynamic aquatic history of the Great Basin was evaluated as a potential model for early toad dispersal into regions which would later become arid and isolated, affecting gene flow and eventually promoting allopatric speciation. This research was accomplished by exploring the spatial and historical relationships among Western toad clades and Great Basin drainages. Toad clades that are distributed over large areas, and are composed of many populations, tend to fall within the confines of regional, riverine drainages. Smaller, more genetically distant clades are generally harbored in small, riverless drainage basins. Dates of estimated evolutionary divergence among Western toad clades varied considerably using rates of molecular substitution that are reasonable for this organism. In addition, aquatic histories contain last known dates of interbasin connectivity that are well within the range of toad evolutionary divergence times reported here. Although the specific dates presented in this study encourage further refinement, this study suggests that the relative ages of Western toad clades are positively related with their geographic isolation. These results suggest that the evolutionary history of the Western toad may have been affected by prehistoric environments dominated by glacial cycles. This information can be used to inform strategies used by wildlife managers to catalog and protect unique biodiversity.

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ACKNOWLEDGEMENTS

I would like to thank my advisor, Jill S. Heaton, and my advisory committee, Scott A.

Mensing, Kenneth E. Nussear, and C. Richard Tracy for their intellectual contributions to this research. The previous field and lab studies that eventually led to this thesis was completed in collaboration with colleagues: Mo Beck, Bobby Espinoza, Robert Fisher,

Matt Forister, John Gray, Bridgette Hagerty, Fran Sandmeier, and Eric Simandle. I would like to especially acknowledge Ken Adams, Brian Horton, Marith Reheis, Donald Sada, and Alan Wallace for participating in discussions that were invaluable to my understanding of Great Basin aquatic histories. The U.S. Fish & Wildlife Service, and the

U.S. Geological Survey provided generous financial support. I would like to thank my friends and family for their support throughout my years in graduate school: my parents,

Rick & Bonnie Noles, my grandparents, Richard & Patricia Noles, and Lily Mathieu, my brother, Garrett Noles, and my dear friends, Tim & Pam Alpers, Amy Barber, Ankur

Goyal, Brigitte Peterson, Seth Taylor, and Stephanie Wakeling. The rural residents of

Nevada also deserve recognition here for ensuring that "school didn't get in the way of my education", particularly: Shirley Harlan, Dave & Bobbie Murphey, Durk Pearson,

Lina Sharp, and Dave Spicer. Finally, I would like thank my High School geometry teacher and friend, Mr. Tom Beveridge, for teaching me the meaning of perseverance.

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Table of Contents INTRODUCTION ...... 1 METHODS ...... 5 Study Area ...... 6 Clade Mapping ...... 7 Watershed Mapping ...... 8 Estimating Divergence Times ...... 8 RESULTS ...... 9 Clade Mapping ...... 9 Watershed Mapping ...... 10 1. The Northwest Basins ...... 10 2. The Lahontan Basin ...... 10 3. The Manly Basin ...... 11 4. The Railroad Basin ...... 11 5. Fish Valley ...... 11  Area of Sterile Basins ...... 12  Closed Isolated Basins ...... 12 Evolutionary Divergence Times ...... 13 Basin Histories ...... 13 The Mono Basin "Switching Yard" ...... 13 Dixie Valley & ...... 15 The San Joaquin Connection ...... 16 Railroad Valley ...... 16 Oregon...... 18 Isolated and Relict Dace Basins ...... 19 DISCUSSION ...... 19 Absolute vs. Relative Dates ...... 19 Important Caveats to Analyses ...... 20 Sampling Recommendations ...... 23 Conclusions ...... 24 REFERENCES ...... 26 FIGURES AND TABLES ...... 32 FIGURE 1: Western toad range in North America...... 32 iv

FIGURE 2: Great Basin Western toad clades...... 34 FIGURE 3: Great Basin morphological regions...... 36 FIGURE 4: Principal pluvial drainage basins...... 38 FIGURE 5: The.Mono Basin switching yard ...... 40 FIGURE 6: Southern expansion of towards Fish Lake Valley ...... 42 FIGURE 7:.Eastern expansion of Lake Lahontan into Dixie Valley...... 44 FIGURE 8: Pre- Mono Basin to San Joaquin drainage link...... 46 FIGURE 9:. corridors between Railroad Valley and the ...... 48 FIGURE 10:.Closed, isolated drainages around an "Area of Sterile Basins" ...... 50 FIGURE 11:.Relative clade ages vs geographic isolation ...... 51 TABLE 1:.Estimated Western toad evolutionary divergence times (slow rate) ...... 53 TABLE 2: Estimated Western toad evolutionary divergence times (fast rate) ...... 54

1

INTRODUCTION

The Western toad species group (Anaxyrus boreas spp.), as currently recognized

(Stebbins, 2003; Frost, 2007), is composed of two broadly distributed subspecies and three localized species. The geographic range of Anaxyrus boreas (Baird and Girard,

1852) extends from the eastern slopes of the Rocky Mountains to the Pacific Ocean, and from northern Baja to and the Yukon (Figure 1). The subspecies A. b. boreas inhabits most of this range (Baird and Girard, 1852), and A. b. halophilus (Baird and Girard, 1852) is distributed in southern California (largely south of the Tehachapi

Mountains) and into the Baja peninsula. The three other species are considered

Pleistocene relicts (Myers, 1942; Karlstrom, 1958; Karlstrom, 1962), and they are scattered along the California-Nevada border. Anaxyrus exsul, is found only in Deep

Springs Valley (Myers, 1942), in the White Mountains. Anaxyrus nelsoni (Stejneger,

1893) is only recognized in , in southwest Nevada (Altig and Dodd, 1987).

Anaxyrus canorus is found only at high elevations in the , and it is reported to be sympatric with A. boreas at the northern tip of its distribution (Karlstrom, 1962;

Morton and Sokoloski, 1978; Davidson and Fellers, 2005).

Historical classifications of toads have recognized species groups based upon morphological similarity. However, recent genetic studies have identified highly divergent toad lineages not yet recognized by (e.g. Jaeger et al., 2005), including the Western toad species group on a contenitnal scale (Goebel et al., 2009). The phylogeographic relationships of all members of this group, as recognized (Stebbins,

2003; Frost, 2007), were examined by Tracy et al. (in prep) to determine if there were 2 differentiated populations on a more localized scale. Three new species designations were recommended in that analysis, and genetic data on the Pleistocene relict species, A. canorus, suggested that this species could be polyphyletic. All of these populations have very restricted geographic ranges, and their locations fall in central Nevada, specifically,

Dixie Valley, Hot Creek Canyon, and Railroad Valley.

Explanations for this biogeographic pattern of species divergence may hinge on the dispersal ability and physiology of western toads. Movements of western toads are predominantly terrestrial (Bull, 2006), however their dispersal routes often parallel stream networks (Carpenter, 1954). Recent telemetry studies show, that when given the opportunity, individuals of this species will exploit streams as dispersal conduits between overwintering hibernacula, breeding pools, and summer growth sites (Adams et al., 2005). Under these circumstances adults have been documented moving in excess of

13 kilometers in less than six weeks in mesic environments (Schmetterling and Young,

2008). This species is a philopatric generalist (Tracy, 1968), and breeds in diverse locations spanning from roadside ditches to large reservoirs (Stebbins, 1951). More specifically, western toads prefer to breed in wetlands with good sun exposure (Hossack et al., 2006) and tend to oviposit in slow moving to standing water (Jones, 2004). Similar reports claim that low densities of emergent vegetation is a requisite for breeding site selection (Jones, 2004), and that excessive vegetative growth is correlated with declines in toad abundance (Skelly et al., 1999; Rannap et al., 2007).

Populations in these groups often are found in moist areas along major rivers and their tributaries (e.g. the Humboldt River in the North, and the Mojave, Owens, and Amargosa 3

Rivers in the south). These observations per se are necessary but insufficient for explaining why clusters have formed in these areas, because although there are major drainages in the south, none are presently connected. This sets the stage to ask if presently independent watersheds were connected in the past, and if so, have they been isolated long enough to facilitate differentiation among aquatic species.

Fortunately, scientists from many disciplines have contributed to our understanding of the natural history of the Great Basin for nearly a century (for a general overview see

Grayson, 1993); for a comprehensive review see (Hershler et al., 2002). Reconstruction of hydrology and climate has revealed that aquatic habitat in the Great Basin has been dramatically reduced and fragmented by natural processes since the onset of the

Quaternary Period. This process of fragmentation has provided ample opportunity for local biota to become genetically isolated via vicariance and extirpation. This may have been particularly true for aquatic invertebrates (e.g., Hershler and Sada, 2002), fishes

(e.g., Hubbs and Miller, 1948), plants (e.g., Stutz and Sanderson, 1983), and small mammals (e.g., Brown, 1971).

Hubbs and Miller (1948) proposed that modern aquatic species distributions within the

Great Basin could result, in part, by ephemeral aquatic migration routes formed during periods during the Pleistocene. They argued for rapid evolution of fish in now-isolated drainage basins assuming that, in most cases, the last aquatic connections between the fish-bearing basins were during the last pluvial period, now known to have ended about 12,000 years ago (KYA). However, this scenario offered by Hubbs and

Miller (1948) has been challenged recently (Smith et al., 2002) as misrepresenting the 4 much slower rates of evolution observed in the fossil record during the past several million years. As a result, less credence has been placed in rapid evolution, and modern aquatic species distributions are more commonly attributed to inheritance from widespread Tertiary populations followed by extinctions and intermittent population- mixing driven by glacial cycles during the Pleistocene (e.g. Minckley et al., 1986).

The possibility that modern species distributions could be partially produced by aquatic migration corridors older than late Pleistocene, but younger than Tertiary, in age has been given little consideration in biogeographic studies because intermediate-aged migration routes are largely unknown. However, new evidence indicates that connections existed among several large in the Great Basin during the late and early

Pleistocene, but have been severed for the past one-half million years or more (Reheis et al., 2002a). This provides researchers with alternative hypotheses concerning rates of evolution for the region’s aquatic biota.

Accounting for these ancient dispersal windows, in conjunction with what is known about the western toad’s dispersal behavior, physiology, and extant distribution in the region, could reveal conditions that are both necessary and sufficient for producing the phylogeographic configuration for this species group. To facilitate the task of assessing ancient dispersal connections, divergence times were estimated for known Western toad clades, which were then compared to dates when last known aquatic corridors are known to have desiccated. This thesis marks the first attempt to relate these ancient dispersal corridors with the Western toad's dispersal behavior, distribution, and evolutionary history in the Great Basin. 5

Information gained from this study would be of immense importance to conservation efforts because although much work has been done on past distributions of plants, mammals, and fish in the Great Basin, little attention has been given to reptiles and . This is unfortunate because the discipline of conservation biology emerged specifically to understand, quantify, and ameliorate the current biodiversity crisis (Soulé

1986), and amphibians are playing an increasingly prominent role in this issue (Beebee and Griffiths 2005). Since most members of the Western toad species group are critically imperiled in all or parts of their geographic ranges, any insight gained here could help catalog and protect native biodiversity.

METHODS Exploring the possibility that Great Basin Western toad diversity may be partially explainable by dynamic Pleistocene landscapes was a four step process that involved:

1) Plotting clades in geographic space;

2) Delineating principal late Cenozoic drainages;

3) Estimating evolutionary histories; and

4) Reviewing drainage histories.

The first two processes dealt strictly with location, and ask the question: Do the clades form a pattern in physical space, and if so, do they seem to correspond with regional drainages? The second two processes address evolutionary and drainage histories, posing the question: If there seems to be a relationship between toad clades and drainage basins, do evolutionary and aquatic histories support the possibility of late Cenozoic dispersal, isolation, and allopatric speciation? 6

Study Area

The hydrologic definition of the Great Basin (Fremont, 1845; King, 1986) was chosen as the regional study area (black outline in Figures 1 and 2) given the Western toad's physiological dependence on water and the distribution of known populations. This definition represents about 165,000 square miles, ranging from the crest of the Sierra

Nevada and southern Cascades in the west, to the crest of the in the east.

It is bounded in the north by the and in the south by the

Drainages. Thus, the Great Basin includes most of Nevada and , and also encompasses portions of eastern California, southeast Oregon, and southern .

Precipitation that falls within the Great Basin never reaches the ocean through surface drainages. Rather, water is internally drained and eventually culminates in places as aptly named as the Carson Sink. Since Great Basin sinks contain no outlets, saline lakes or playas are the principal physical features on valley floors. The Great Basin also includes several river systems and tributaries, including the Humboldt River (which has many tributaries including the Reese River, the Quiun River, the Mary’s River, and others), the

Walker River (including its tributaries such as the East and West Walker Rivers), the

White River, The (and an abundance of tributaries pouring off the Sierra

Nevada), the , and (see Grayson, 1993, for a review).

The Sierra Nevada mountain range in Californa cools and precipitates the westerly flow of Pacific moisture, placing the Great Basin in an arid . The area has a dry steppe climate classification as a result. Playas form rather than lakes in most cases 7 because evaporation potentials exceed precipitation rates (Trimble, 1989). Average annual temperatures vary from 40˚ F in the northeast to 65˚ F in the south (Western

Regional Climate Center, 2010). Average seasonal temperatures vary considerably with elevation, latitude, and topography. Due to severe surface heating in the day followed by sharp cooling at night, the average range between high and low daily temperatures is about 30˚ F. Extreme regional temperatures region span from 120˚ F in the summer to 50˚

F below zero in the winter(Western Regional Climate Center, 2010). The Great Basin has a winter dominated precipitation regime, with some areas receiving as little as 0.23 inches of summer rain in the south and as much as 300 inches of snow in portions of the eastern Sierra Nevada (Western Regional Climate Center, 2010).

Clade Mapping

Toad population locations were mapped using results from Tracy et al. (in prep), which delineated clusters of genetically and morphologically distinct populations that span all known sites of occurrence of Western toads in the Great Basin. The resulting dataset includes precise locations and clade designations for each population. These data were converted to a spatially explicit point file (i.e. shapefile), assigned an appropriate coordinate system, and projected into geographic space for display in a geographic information system (GIS) using ArcGIS 9.3. Population points were assigned a symbology that indicates clade designations from Tracy et al. The resulting data were plotted over a digital elevation model of the Western , state boundaries, and an outline of the study area (Figure 2). 8

Watershed Mapping

Watershed delineation began by mapping morphological regions (Figure 3) as described by Sigler and Sigler (1987) using basin boundaries derived from high-resolution elevation data obtained during NASA Shuttle Radar Topography Missions (SRTMs; i.e.

USGS HydroSHEDS vector layers). Modifications were made to accommodate updates for the boundary of the Lake Lahontan (Reheis, 1999b; Reheis, 1999a). To place more emphasis on features of biological interest, the ―Central Basin‖ (Sigler and

Sigler, 1987) was modified to emphasize an area of ―sterile basins‖; a term given by

Hubbs and Miller (1948) to indicate the absence of fish in the area. Railroad Valley, which is rich in endemic aquatics (Hershler and Sada, 2002; Hubbs et al., 1974;

Polhemus and Polhemus, 2002), and other drainage basins nestled between the ancient lakes Lahontan and Bonneville that known to harbor Relict Dace (Relictus solitarius)

(Smith et al., 2002; Hubbs and Miller, 1948; Hubbs et al., 1974) were also emphasized.

Estimating Divergence Times

Tracy et al. (in prep) extracted DNA samples from tissues collected from 36 western toad populations assembled from several sources. Distinct haplotypes were identified by sequencing cytochrome-b and control regions of the mitochondrial genome (Tracy et al., in prep). Evolutionary divergence times were then calculated for all pairs of the identified haplotypes. Because a unique molecular clock has not been developed for the Western toad species group, it was assumed that the rate of DNA sequence evolution among lineages was within the range of rates described for Nearctic toads around the world. 9

Since molecular substitution rates vary from 0.69% (Macey et al., 1998) to 1.644%

(Stöck et al., 2006) change per lineage per million years, Great Basin Western toad lineages were assumed to fall within this range. The open-source MEGA4 software package (Tamura et al., 2007) was used to produce pairwise genetic distances, with standard errors, for the clades presented in Figure 2 using both a slow (0.69%) and a fast

(1.644%) rate of substitution. These data were imported into a spreadsheet where divergence envelopes were calculated as:

RESULTS

Clade Mapping

Two clades, each composed of multiple populations, appear in the northern and southern latitudes of the Great Basin (the blue Lahontan and red Mojave [represented by both

California and Amargosa clades in Tables 1 and 2] markers seen in Figure 2). A small clade (likely a fragment of a larger clade extending north of the study area) composed of two populations lies in the northwestern corner of the study area (yellow markers straddling Oregon/Nevada border in Figure 2). Two distinct populations (seemingly polyphyletic) of A. canorus are located along the crest of the Sierra Nevada, north (olive 10 green markers in Figure 2) and south (light purple markers in Figure 2) of the San

Joaquin River. The traditionally recognized species of Anaxyrus exsul, the , lies in of the White Mountains (southern turquoise marker along

California/Nevada border in Figure 2) , however a new population of this species was identified along the northern tip of the White Mountains (northern turquoise marker along

California/Nevada border in Figure 2). The three unnamed relict species proposed by

Tracy et al. (in prep) lie in central Nevada. (bright green [Dixie Valley], dark purple [Hot

Creek], and orange [Railroad Valley] markers in Figure 2).

Watershed Mapping

Watershed delineation produced these features of interest:

1. The Northwest Basins: spans parts of California, Nevada, and Oregon with the

largest land area in southeast Oregon (yellow area in Figure 4). In the northern

extremity, several drainages terminate in lakes Malheur and Harney. The western

portion contains Paulina Marsh, Silver Lake, Summer Lake, Lake Albert, and the

Chewaucan Marsh. This watershed abuts the northwestern flank of the Lahontan

Basin. The Oregon Western toad clade straddles the boundary between this region

and the Lahontan Basin.

2. The Lahontan Basin: is an area of broad, flat valleys of comparatively lower

elevation (blue area in Figure 4, southeast of the Northwest Basins). It is the

terminal drainage basin of the enormous Pleistocene Lake Lahontan, which

occupied the lowest valleys during pluvial periods. This region is composed of 27 11

sub-basins forming a wedge approximately 150 miles wide in a northeasterly

direction, extending from Lake Tahoe on the California-Nevada border, to Oregon

and Idaho.

3. The Manly Basin: encompasses the and Mojave morphological

regions (brown area in Figure 4, immediately south of the Lahontan Basin).

Drainages in this area terminated in Pleistocene at irregular intervals

(especially during high pluvial periods) throughout the Quaternary period. The

northern portion of this region consists of basins with the highest relief found in

the Great Basin, eventually transitioning into relatively flatter and tectonically

stable landscapes in the south. With the exception of the northernmost catchments

of the Amargosa river, the Manly watershed lies almost entirely within California.

4. The Railroad Basin: (pale olive green region along the periphery of the

Lahontan Basin in Figure 4) is nestled in east-central Nevada, and is both smaller

in area and geographically isolated from the other regional watersheds. This

complex of basins is about 118 miles in length from north-northeast to south-

southwest, and around 70 miles at its maximum width. Railroad was

the sink in this complex of basins. The basin is bound on its northwest flank by

the Lahontan Basin, an area of sterile basins to the southwest, basins harboring

Relict Dace to the northeast, and the Colorado River drainage to the southeast.

5. Fish Lake Valley: Formerly recognized as part of the Death Valley

morphological region (Sigler and Sigler, 1987), there is no evidence that Fish

Lake Valley (orange area along the periphery of the Manly Basin in Figure 4) was

ever hydrologically integrated with aquatic networks in the Manly Basin. Thus, 12

Fish Lake Valley is recognized here as having a unique and independent aquatic

history relative to its neighbors.

 Area of Sterile Basins: Coined the area of "Sterile Basins" by Hubbs and

Miller (1948) due to the absence of native fish species, this group of basins

(thatched red region in Figure 4) lies in the arid rain shadow of the Sierra Nevada

and White Mountains, and is bounded by systems that do support fishes. It is

bounded on the west by the Lahontan and Fish Lake Valley watersheds, the

Railroad Valley watershed to the east, and a group of isolated basins along the

northern and southern periphery.

 Closed Isolated Basins: The remaining basins were constituents of the

morphological region formerly known as the "Central Basin" (Sigler and Sigler,

1987). This area is newly configured here based upon biological, geological, and

hydrological evidence. These basins are found along the periphery of the sterile

basins and Railroad Valley, which contains two proposed new Western toad

species. Dixie Valley and Big Smoky Valley are the largest representatives of this

group. The remaining areas are composed of numerous smaller, neighboring

basins.

The Bonneville Basin (purple region in Figure 3) was another obvious feature of interest, but was not considered in this study because toad sample data were not available for that area. 13

Evolutionary Divergence Times

Dates of divergence indicate that the outgroup species, A punctatus (the Red Spotted toad), split in the distant past relative to all other lineages (all pairwise comparisons for clade 11 in Tables 1 and 2). Dates of divergence vary widely depending on the rate of molecular substitution used. The slowest published rate evolution (0.69% change per million years) produced minimum dates of divergence that generally predate the

Pleistocene (Table 1) Divergence envelope values average between 2 (min) and 6 (max)

MYA using that slow rate of substitution. In stark comparison, the fastest rate (1.644% change per million years) generated dates of divergence well within the Pleistocene, averaging around 1 (min) and 2.5 (max) MYA(Table 2). Regardless of the rate used,

Railroad Valley and Hot Creek clades are consistently older than the other lineages.

Basin Histories

Recent studies have reported geomorphic and stratigraphic evidence for multiple lake high water levels that were higher than those in the late Pleistocene and older than those of late Pleistocene lakes in several basins of the western Great Basin (Reheis and

Morrison, 1997; Reheis, 1999a; Reheis et al., 2002a; Redwine, 2003). Of these, several may have served to facilitate sweepstake dispersal routes for aquatic species and may account for the major divisions among divergence times.

The Mono Basin "Switching Yard"

Nestled along the divide between the northern Lake Lahontan and southern Mojave groups of toads lies the Mono Basin. Research on this basin's Pleistocene history builds upon the cornerstone research from the 19th century when geologists first described the 14 ancient shorelines climbing out of what is now modern-day , towards Mud

Canyon in the extreme northeastern portion of that drainage basin (Russell, 1889). This canyon harbors the east fork of the , which terminates in modern-day

Walker lake. These observations were the first who indirectly hinted at the ancient lake's potential external connections.

Later in the 20th century scientists directly proposed that pluvial lakes Lahontan and

Manly may have been hydrologically contiguous in the past based on the morphology of modern fish populations (Hubbs and Miller, 1948). Decades later a team of researchers unearthed fish fossils in Mud Canyon and the abandoned shorelines of ancient Lake

Russell (now called Mono Lake) that were allied with lineages of fish found in the

Lahontan basin (Miller and Smith, 1981). This evidence served as a smoking-gun that the presently alkaline lake was once freshwater and harbored Lake Lahontan fish.

Subsequent research has yielded evidence that Lake Russell once had an interval during which it had drainage north into the east Walker river (route indicated by red arrow in

Figure 5), a tributary of Lake Lahontan, possibly between 1.6 and 1.3 Ma, and again after

1.3 Ma (Reheis et al., 2002b; Reheis et al., 2002a). However, it was not until the Long

Valley Caldera erupted ca. 760 Ka, causing extreme tectonic activity along the southeastern shore of Lake Russell, that a paleochannel connected Lake Russell with neighboring Lake Adobe (Reheis et al., 2002b; Reheis et al., 2002a; suspected route indicated by northern green arrow in Figure 5). Because the sill into Adobe Valley was lower than the previous outlet, the northern drainage into the Walker River was abandoned. Due to the substantial influx of water from Lake Russell, Lake Adobe spilled 15 into the nearly dry east fork of the Owens River (see Figure 20 of Hubbs & Miller, 1948, for ground level photo of spill incision; green arrow, Figure 5 in this study), which was a

Lake Manly tributary several times during the Quaternary (Phillips, 2008).

These lines of evidence suggest that climates were substantially wetter in the early to mid-Pleistocene than those later in the Quaternary. Moreover, the drainage behavior of

Lake Russell probably permitted the movement of Lake Lahontan aquatic species into southern Mojave drainages. The key date to consider is the eruption of the Long Valley caldera 760 Ka, which served as the switching point between the two watersheds. The dynamics described here may be a plausible mechanism for dispersal, followed by genetic isolation of the western toad complex.

Dixie Valley & Fish Lake Valley

The divergence times reported in this study indicate that the Black Toad began to split from the Lahontan clade around the same time as did the new species suggested by Tracy et al. (In prep) in Dixie Valley. These populations may have been affected by the same hydrologic event. Evidence suggests that the Pleistocene climate around 650 ka was significantly cooler and wetter than it was during the last pluvial (Reheis et al., 2002a).

At that time, Lake Lahontan expanded into previously isolated basins (light orange lake extents seen in Figures 6 [Dixie Valley] and 7 [Fish Lake Valley]), and the area of sterile basins abutting Fish Lake Valley (Figure 7). Since that time, the region has undergone desiccation, causing Lake Lahontan to withdraw from the acquired basins, re-establishing aquatic isolation shortly after 650 KYA (Reheis et al., 2002a), which is a time falling between the divergence times under the two models of divergence (Table 2). 16

The San Joaquin Connection

Prior to the uplift of the Sierra Nevada, the Humboldt River, and what is presently known as the Mono basin may have drained into the Pacific Ocean (Huber and Rinehart, 1967;

Smith et al., 2002). Before approximately 3.5 million years ago (Carlson et al., 2010) pluvial Lake Russell may have formed the headwaters of the (Huber,

1981; Smith et al., 2002). At that time, narrow v-shaped basalt channel permitted drainage through the area presently known as "Dead Man Pass", serving as a dispersal corridor for aquatic species between the Mono basin and the Pacific ocean. (Huber, 1981; potential flow route indicated by yellow arrow in Figure 9). Between 5 and 2 MYA the

Sierra Nevada underwent a significant rate of upheaval that raised the channel perhaps as much as of 3000 ft (Huber, 1981). Any remaining drainage would have been eliminated when volcanic flows dammed this channel around 3.5 million years ago (Carlson et al.,

2010). These events would have changed both barriers and corridors for gene flow among toad populations distributed across the Sierra Nevada. Though this is an area of debate among geologists, there is some indication that the rate of Sierra uplift has been substantially greater in the northern half of the range relative to the southern portion

(Wakabayashi, 2010) which is closer to the tectonically stable Mojave . This combination of rapid, and potentially variable uplift may account for the apparent differentiation among sampled northern and southern populations.

Railroad Valley

The Railroad Valley (RV) and Hot Creek Canyon (HC) populations of toads fall in a watershed (light green Figure 9) rich with endemic fishes (Sigler and Sigler, 1987; Hubbs 17 and Miller, 1948; Smith et al., 2002), insects (Polhemus and Polhemus, 2002), and mollusks (Hershler and Sada, 2002). These two populations of toads are highly divergent from other toad species, and thus, similar to other divergent species in the region, providing evidence that this watershed has been hydrologically fragmented and isolated for a long time, potentially since the late Miocene (Polhemus and Polhemus,2002

Biologists have long hypothesized that the Railroad Valley watershed may have acted as a switching yard between the Lahontan Basin, and the Colorado River, similar to the

Mono Basin switching yard, only much earlier chronologically. Hubbs and Miller (1948) were the first to propose that there was an interval of northern drainage from Railroad

Valley into the Lahontan Basin, through water overflow at Lake Snyder, into the region of Relict Dace basins, based on fish morphology. Little, to no, research has been reported to validate this hypothesis. However, abandoned streambeds adorn the ridge tops between

Little Fish Lake Valley, and the Reese River drainage, indicating that there was at one time an interval of drainage between these basins, probably during the Pleistocene

(Wallace et al., 2008; Wallace pers. comm.). Aquatic insects in the Railroad Valley have their closest sister taxa in Northwest Texas, Baja California, and Northern Mexico

(Polhemus and Polhemus, 2002), suggesting an interval of southern drainage into the

Colorado River. Again, Hubbs and Miller (1948) were the first to propose a hydrologic connection to the south, before the northern drainage into the Lahontan Basin, through a series of low divides between the White River drainage and southeast extent of the

Railroad Valley watershed. However, subsequent geological examination of a small portion of alluvial fill along the eastern border of the Railroad Valley watershed, coined 18 the "Horse Camp Formation" (HCF) (Moores et al., 1968), contains a variety of flora and for date calibration. These species include, gastropods, mollusks (Moores et al.

1968), and mammals (Horton and Schmitt, 1996; Horton and Schmitt, 1998). Dating of these specimens indicate that this formation is Miocene in age (5-20 ma; Horton and

Schmitt, 1998). The Horse Camp Formation marks the lowest spillover point between the

Railroad Valley and White River watersheds. This eastern connection (Figure 10) is shorter, and it is a more direct route than the southeast route described by Hubbs and

Miller (1948). If aquatic species used this route, the development of the Horse Camp

Formation during the Miocene would have severed as a Sweepstakes dispersal route, and may account for this unique endemic biodiversity in this watershed. If further genetic analyses support Miocene divergence for the Hot Creek and Railroad Valley clades, these lines of evidence might explain the high degree of genetic isolation from all other toad populations in the Great Basin.

Oregon

The Oregon watershed has a geologic landscape that is distinct from the neighboring

Lahontan Basin. The geology of Southeastern Oregon has been tectonically stable for the last 20 ma, whereas, the Lahontan Basin has been continually faulting and sinking during the same time interval (Cummings et al., 2000; Beranek et al., 2006). There is little to no evidence in the geologic record for southern drainage into the Lahontan Basin supporting the hypothesis that the Northwest basins drained into the Lahontan Basin, rather this entire area tilts northwards into the Columbia River drainage (Wallace et al., 2008). Thus, the geological and geographical evidence on relationships between toad populations in 19

Southwest Oregon and those in the remnants of the ancient Lake Lahontan is scant and obscure.

Isolated and Relict Dace Basins

Nestled between the Lahontan and Bonneville basins lies a group of valleys (emphasized by the yellow basin outline in Figure 10) that contain Relict Dace (Hubbs and Miller,

1948; Hubbs et al., 1974). This species of dace, which is the only member of its genus, is related to fishes in both the Bonneville and Lahontan basins, but closer to the former.

During the Pliocene this area was a raised plateau (Alan Wallace, personal comm.), which drained into both basins simultaneously, probably becoming isolated from the

Bonneville Basin first. There is evidence that the areas closest to the Lahontan Basin, in and around the vicinity of basins bearing Relict Dace, were hydrologically integrated during the early to mid Pleistocene and served as the headwaters of the Humboldt River

(Reheis et al., 2002a; indicated by the additional Lahontan Basin boundary in Figure 10).

The remaining basins in this group have unknown drainage histories.

DISCUSSION Absolute vs. Relative Dates

Drainage histories and dates of clade divergence do not preclude the possibility that

Western toads moved into presently closed and geographically isolated basins when aquatic corridors were more abundant during the Pleistocene. However, like evolutionary divergence times, there is a considerable amount of error inherent in drainage histories.

But, unlike dates of evolutionary divergence, the errors around geologic dates are rarely discussed in publications. Therefore the absolute dates of basin connectivity and 20 evolutionary divergence are, at this point, too imprecise to make strong inferences.

Relative ages remain informative. If clades are ranked relative to their date of evolutionary divergence and geographic location, older, more differentiated clades are generally found in more isolated basins (Figure 11). Three notable exceptions to this pattern are the two Yosemite clades and the Black toad that group together in the evolutionarily young, but highly geographically isolated region of Figure 11.. A likely explanation for the Yosemite population's deviation from the trend is that their geographic displacement is probably driven more by rapid (Carlson et al., 2010; Huber,

1981) and variable (Wakabayashi, 2010) tectonics than by comparatively slower pluvial processes. The Black toad's location in isolation and divergence space remains unaccountable.

Important Caveats to Analyses

Although the divergence timings suggest pre-Pleistocene isolation events, during a time when extant watersheds were in nascent development or completely absent (Wallace et al., 2008), it would be helpful to quickly review similar developments in ichthyological literature.

Hubbs and Miller (1948) were the first to argue that fish lineages should become more diverse in regions with turbulent aquatic histories, such as was the case for the

Quaternary Great Basin. However, a subsequent analysis indicates that although endemism is high among Great Basin fishes, there is much lower diversity than predicted by the pluvial cycles of isolation and allopatric speciation (Smith et al., 2002). Instead, 21 paleontological and molecular evidence demonstrated that most Great Basin fish lineages pre-dated the Quaternary Period (Smith et al., 2002). This has led to the conclusion that extirpation of small populations was a more dominant process than speciation in dynamic isolated environments. In other words, diversity has been lost to extirpation when/where populations cannot move among drainages during a time scale of thousands of years

(Smith et al., 2002).

However, ensuing advancements in molecular techniques offered improved applications for dating reconstructions, and eventually prompted researchers to rescind these conclusions (Smith and Dowling, 2008). Updated methods have allowed users to abandon the assumption that molecular substitution rates are uniform among taxa. This was not theoretically tenable until the mass-specific metabolic rate theory of ecology

(Gillooly et al., 2001) was applied to rates of molecular substitution (Gillooly et al.,

2005). By using fossil data to estimate the body size of ancestral fish lineages, researchers were able to estimate metabolic rates using an allometric equation (Estabrook et al., 2007). Small-bodied species, and those that inhabit warm waters, such as dace and pupfish, have slightly higher metabolic rates and shorter generation times than larger species in colder waters (Estabrook et al., 2007), leading to higher rates of cell division, mutation, and substitution (Gillooly et al., 2005). This phenomenon was documented between large northern and small southern cyprinid fishes (Estabrook et al., 2007), and was in agreement with Pliocene and Pleistocene climatic histories. These methods could be applied to the western toad complex if ancestral body mass and body temperature were extracted from examining the fossil record and physiological models. 22

An additional date refinement step would involve sequencing segments of the nuclear genome. These additional genes would serve as supplementary calibration points for estimating dates of divergence (Matthew Forister, personal comm.).

Using these additional methods in future studies may or may not refine the dates reported here. The latter case would support the hypothesis that extirpation and extinction were more dominant process than speciation during the last 1.8 million years for the Western toad species complex in the Pleistocene Great Basin. This may be tenable because the tectonics that formed the basin and range province were not strong enough during

Miocene/Pliocene time to form the landscapes seen today (Minckley et al., 1986; Stewart,

1980). Instead, sprawling plateaus and plains represented the region's physiography prior before the onset of the Pleistocene. Remnants of this transition include abandoned river beds strewn along the ridges surrounding Little Fish Lake Valley (Alan Wallace, personal comm.). Under these conditions allopatry and extinction may have been dominant factors as ranges gradually fragmented contiguous wetlands and eliminated aquatic dispersal routes while a warming climate desiccated remaining . However, if dating refinements indicate dates of divergence were in the Pleistocene, this would permit the possibility that toads dispersed into basins after they had already diverged into the species we recognize today, much like their fish counterparts.

A key distinction, in terms of tractable research, between these scenarios is that nearly nothing is known about the pre-Pleistocene geology of the study area. However, if toad clades diverged more recently, a rich literature is available to reconstruct meaningful hydrological connections through Pleistocene time. 23

Thus, the times reported here are likely relative, representing the first step of a multistep process. The estimated dates of evolutionary divergence reported in this thesis vary considerably The key insight these data provide are that the toad lineages have dates of divergence with confidence intervals that sometimes span several million years, but often overlap Pleistocene time. Further research refinements may narrow these envelopes by accounting for heterogeneous rates of evolution.

Sampling Recommendations

Like most wetlands, Great Basin toad habitats are threatened by human activities. , altered hydrology, and water pollution have already destroyed or ruined many isolated wetlands in the Western United States (Tiner 2003). Explosive population growth in Las Vegas has increased the demand for additional water supplies. To accommodate this demand, the Southern Nevada Water Authority (SNWA) hopes to obtain rights for 180,800 of the 330,000 acre-feet per year of ground water generated in

Lincoln, White , and Clark counties (SNWA 2004). A water diversion project of this scale will lead to declines in the water table, spring discharge rates, and wetland expanses, adversely affecting 20 federally listed and 137 endemic species in the region

(Deacon et al., 2007). This makes cataloging the genetic and geographic diversity of endemic amphibians more urgent than ever.

Based on the relationships seen in this study, recommendations can be made to help prioritize sampling strategies used by wildlife managers. Chief among these are the isolated drainages that have yet to be sampled for Western toad occupancy. Dixie Valley, 24

Fish Lake Valley, and Railroad Valley, which are all distributed along the periphery of the "Sterile Basins", harbor highly divergent toad lineages. Therefore it seems likely that the remaining isolated basins, namely Big Smoky Valley and drainages bearing Relict

Dace, could also harbor unique Western toad lineages of high conservation value.

Since large riverine drainages are associated with large Western toad clades, it would be reasonable to suspect that the Bonneville basin also contains a broadly distributed toad lineage that is discernable from those found in the Lahontan and Manly drainages.

Though sampling the Bonneville basin would fill a large knowledge gap, Western toad populations found in that drainage are not expected to be as differentiated, and therefore of more conservation concern, than populations potentially distributed in the isolated drainages around the "Area of Sterile Basins".

Conclusions

The aquatic history of the Great Basin landscape can help account for the Western toad phylogeny described by Tracy et al. (in prep.). Early in the Pleistocene, regional watersheds harbored either networks of interconnected lakes or a single sprawling . During this time, small peripheral basins were captured and integrated by neighboring drainages as burgeoning lakes acquired new landscapes. Then, as the region underwent sustained aridification, wetlands desiccated, stranding aquatic animal populations. These relict populations either persisted and differentiated, or many likely underwent extinction. Thus, this system exhibits classic elements of allopatric speciation, and island biogeography. The evolutionary divergence times reported in this study do not 25 preclude this scenario, but new divergence models based upon better assumptions should be encouraged as more precise hypotheses are considered.

26

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32

FIGURES AND TABLES

33

FIGURE 1: The range of the Western Toad in North America as currently recognized by the International Union for Conservation of Nature (ICUN) (shaded). The hydrologic

Great Basin (solid black outline) is the study area. 34

35

FIGURE 2: Geographic locations within the hydrologic Great Basin of sampled Western toad populations. These populations fall within nine discernable clades (colored circles) as defined by Tracy et al. (in prep). 36

37

FIGURE 3: Morphological regions (King, 1986; Sigler and Sigler, 1987) of the Great

Basin were used as a base layer for drainage delineation. Four of the five principal Great

Basin drainages are seen here. The Mojave and Death Valley regions were combined in this study to reflect pluvial drainage integration instead of landscape morphology. 38

39

FIGURE 4: Principal Pluvial Drainage Basins and Great Basin Western toad clades. The

Lahontan Basin contained Pleistocene Lake Lahontan and its tributaries, most notably the

Humboldt River. The Manly Basin harbored a complex drainage network of lakes that were integrated by the Owens, Mojave, and Amargosa Rivers, which eventually terminated in Lake Manly at various intervals throughout the Pleistocene. The Northwest

Basins (NWB) which straddle the California, Nevada, and Oregon borders are geologically distinct from the neighboring Lahontan Basin. Fish Lake Valley (FLV) abuts the Manly Basin and a riverless region of basins devoid of native fishes (Sterile).

Railroad Valley (RV) is nestled between the Colorado River drainage, the Sterile basins, the Lahontan Basin, and a complex of closed, isolated basins with poorly known aquatic histories. The remaining basins lack interbasin river networks and have unknown aquatic histories. Dixie Valley (DV) and Big Smoky Valley (BSV) are the only single basins in this last category, the remaining areas represent groups of neighboring basins. Large

Western toad clades loosely fall into the large riverine Lahontan and Manly Basins, whereas smaller, more differentiated clades tend to fall in small, isolated, riverless basins. 40

41

FIGURE 5: Potential hydrologic connection between Lahontan and Manly basins via pluvial Lake Russell. Earlier interval of northern drainage into the East Walker River is indicated by red arrow., the northern drainage was abandoned after the Long Valley caldera erupted, at which point Lake Russell may have drained south into the Owens

River (green arrow). Dotted blue line (from Reheis, 1999) represents early to mid

Pleistocene extension of Lahontan Basin. Lake Russell flow routes modified from Reheis et al. (2002b). 42

43

FIGURE 6: Purported early to mid Pleistocene southern expansion of Lake Lahontan towards Fish Lake Valley through basins that are presently devoid of native fishes

(Sterile Basins). Lake expansion and additional Lahontan drainage boundary (blue dotted line) layers are from Reheis (1999). 44

45

FIGURE 7: Eastern expansion of Lake Lahontan into Dixie Valley (center basin harboring Lake Dixie, pink shading). Potential early to mid Pleistocene connectivity points are found at the extreme northern and southern flanks of Dixie Valley. These may have provided aquatic dispersal corridors into Dixie Valley from neighboring Lake

Lahontan. Lake expansion and additional Lahontan drainage boundary (blue dotted line) layers are from Reheis (1999). 46

47

FIGURE 8:Geologic (Huber, 1981) and ichtyological (Smith et al., 2002) evidence suggests that the Mono Basin (which harbored pluvial Lake Russell) may have drained south into the San Joaquin River (indicated by yellow arrow), possibly flowing across the

Diablo Uplift and eventually terminating into the Monterey Fan (Cole and Armentrout,

1979). A combination of tectonic uplift (Huber, 1981; Carlson et al., 2010) and volcanic flows (Huber, 1981) eliminated this corridor approximately 3.5 MYA (Carlson et al.,

2010). 48

49

FIGURE 9: Railroad Valley (central grey shaded valley) is rich with endemic aquatic species, some of which have their closest relatives in Texas, Northern Mexico, and the

Amargosa River (Polhemus and Polhemus, 2002). Early examination of local topography led to the suggestion that this basin may have been integrated with the White River, a tributary of the Colorado River, early in or perhaps predating the Pleistocene, through a route (southern yellow arrow) now punctuated with low divides (Hubbs and Miller,

1948). Later examination of the eastern flank of Railroad Valley has revealed an alluvial formation, Miocene in age, which may have blocked a shorter and more direct river channel into the Colorado River drainage.

50

51

FIGURE 10: Isolated, riverless, and often clumped basins distributed along the periphery of the Sterile (fishless) Basins, Colorado River drainage , and Railroad Valley.

These aquatic histories of these areas is poorly known, however there is some indication that some of these presently isolated basins may have been incorporated (blue dotted line;

Reheis, 1999) into the Lahontan Basin early in the Pleistocene (Reheis, 1999). A group of these basins (yellow drainage outline) harbor Relict Dace which are related to both Lake

Lahontan and Fishes (Hubbs et al., 1974).

52

FIGURE 11: Relative ages of Great Basin Western toad clades (presented in a stepwise fashion here) may be positively related to their degree of geographic isolation. Clades falling in large riverine drainages, such as the Manly and Lahontan basins, which are expected to have the least geographic isolation, harbor young to middle aged lineages.

More isolated regions, such as Railroad Valley, are occupied by older clades. Two outstanding exceptions to this observation are the Black toad and Yosemite toad populations. An explanation for the Yosemite toad's deviation may lie in the variable, tectonically driven nature, of the Sierra Nevada relative to the pluvial driven landscapes at lower elevations. The Black toad's relative age and apparent isolation lacks an explanation, encouraging further research. 53

TABLE 1: Pairwise comparisons of estimated Great Basin Western toad evolutionary divergence times using a slow (0.69% change per million years) substitution rate. The Red Spotted toad was the outgroup taxon. This slow rate produces dates of divergence that typically predate the Pleistocene, precluding the possibility of Pleistocene dispersal and allopatric speciation.

Interval Min (Millions of Years) [0.69% change per million years] 1 2 3 4 5 6 7 8 9 10 California 1 Amargosa 2 0.00 Black Toad 3 0.72 0.58 Yosemite South 4 0.43 0.00 0.43 Oregon 5 2.75 2.90 2.32 2.32 Lahontan 6 2.75 2.61 2.75 2.46 1.16 Yosemite North 7 2.75 2.90 2.61 2.46 1.16 0.29 Dixie 8 3.04 3.04 2.75 2.75 1.16 0.72 0.58 Hotcreek 9 3.33 3.33 3.19 3.04 3.19 3.33 3.33 3.19 Railroad 10 3.19 3.19 3.04 2.75 3.33 3.62 3.77 3.48 1.74 Red Spotted Toad 11 24.64 25.22 24.78 24.35 23.77 25.22 23.77 25.80 25.80 25.94

Interval Max (Millions of Years)[0.69% change per million years] 1 2 3 4 5 6 7 8 9 10 California 1 Amargosa 2 0.58 Black Toad 3 1.88 1.74 Yosemite South 4 1.01 1.16 1.59 Oregon 5 5.07 5.22 4.64 4.64 Lahontan 6 5.65 5.51 5.07 5.36 2.90 Yosemite North 7 5.07 5.22 4.93 4.78 2.90 1.45 Dixie 8 5.94 5.94 5.65 5.65 3.48 1.88 1.74 Hotcreek 9 6.23 6.23 6.09 5.36 5.51 6.23 6.23 6.09 Railroad 10 6.09 6.09 5.94 5.65 6.23 6.52 6.67 6.38 4.06 Red Spotted Toad 11 33.91 34.49 34.06 33.62 33.04 34.49 33.04 35.65 35.65 35.80

54

TABLE 2: Pairwise comparisons of estimated Great Basin Western toad evolutionary divergence times using a fast (1.644% change per million years) substitution rate. The Red Spotted toad was the outgroup taxon. This fast rate produces dates of divergence that generally fall well within the Pleistocene, maintaining the possibility of Pleistocene dispersal followed by speciation.

Interval Min (Millions of Years)[1.644% change per million years] 1 2 3 4 5 6 7 8 9 10 California 1 Amargosa 2 0.00 Black Toad 3 0.30 0.24 Yosemite South 4 0.18 0.00 0.18 Oregon 5 1.16 1.22 0.97 0.97 Lahontan 6 1.16 1.09 1.16 1.03 0.49 Yosemite North 7 1.16 1.22 1.09 1.03 0.49 0.12 Dixie 8 1.28 1.28 1.16 1.16 0.49 0.30 0.24 Hotcreek 9 1.40 1.40 1.34 1.28 1.34 1.40 1.40 1.34 Railroad 10 1.34 1.34 1.28 1.16 1.40 1.52 1.58 1.46 0.73 Red Spotted Toad 11 10.34 10.58 10.40 10.22 9.98 10.58 9.98 10.83 10.83 10.89

Interval Max (Millions of Years)[1.644% change per million years] 1 2 3 4 5 6 7 8 9 10 California 1 Amargosa 2 0.24 Black Toad 3 0.79 0.73 Yosemite South 4 0.43 0.49 0.67 Oregon 5 2.13 2.19 1.95 1.95 Lahontan 6 2.37 2.31 2.13 2.25 1.22 Yosemite North 7 2.13 2.19 2.07 2.01 1.22 0.61 Dixie 8 2.49 2.49 2.37 2.37 1.46 0.79 0.73 Hotcreek 9 2.62 2.62 2.55 2.25 2.31 2.62 2.62 2.55 Railroad 10 2.55 2.55 2.49 2.37 2.62 2.74 2.80 2.68 1.70 Red Spotted Toad 11 14.23 14.48 14.29 14.11 13.87 14.48 13.87 14.96 14.96 15.02