The Lahontan Basin Evolutionary Lineage of Cutthroat Trout Mary M

The Lahontan Basin Evolutionary Lineage of Cutthroat Trout Mary M. Peacock* Department of Biology, University of Nevada 1664 North Virginia Street, Reno, Nevada 89557, USA and Ecology, Evolution and Conservation Biology Interdisciplinary Program University of Nevada 1664 North Virginia Street, Reno, Nevada 89557, USA Helen M. Neville Trout Unlimited 910 West Main Street, Suite 342, Boise, Idaho 83702, USA Amanda J. Finger Department of Animal Science, University of California 1 Shields Avenue, Davis, California 95616, USA

Abstract.—Lahontan Cutthroat Trout (LCT) Oncorhynchus clarkii henshawi and Paiute Cutthroat Trout (PCT) O. c. selernis are found in the Lahontan hydrographic basin of northern Nevada, northeastern California, and southeastern Oregon and together form the Lahontan basin evolutionary lineage of Cutthroat Trout O. clarkii. The Alvord Cutthroat Trout O. c. ssp. native to the Alvord Lake subbasin in the northwestern Lahontan basin was also part of this lineage but went extinct due to Rainbow Trout O. mykiss introgression in the mid-20th century. Both LCT and PCT are federally listed as threatened under the U.S. Endangered Species Act. Given its historic distribution in a single small stream and both phenotypic and genetic distinctiveness, PCT is currently recognized as a separate evolutionarily significant unit (ESU). For LCT, three ESUs are identified based upon meristic, morphological, ecological, and genetic data. These putative LCT ESUs separate lacustrine forms in the western Lahontan basin (Truckee, Carson, and Walker River basins) from largely fluvial forms in the eastern Lahontan basin (Humboldt and Reese River basins) and northwestern Lahontan basin (Quinn River, Coyote Lake, and Summit Lake basins). The more recent recognition of a much longer evolutionary history of Cutthroat Trout and several influential genetic papers identifying previously unrecognized diversity within Cutthroat Trout have prompted a need to re-evaluate the overall taxonomy of this species. Here, we review earlier literature and draw on new information from recent studies to delineate uniquely identifiable evolutionary units within the Lahontan basin lineage of Cutthroat Trout. Though in several cases various anthropogenic

* Corresponding author: [email protected] 1 2 peacock et al. and natural influences have made definitive conclusions difficult, based on this collective information and the goal of conserving potentially important genetic, evolutionary, and life history diversity, we propose recognition of six uniquely identifiable evolutionary units within the Lahontan Cutthroat Trout lineage: (1) Paiute Cutthroat Trout—upper East Carson River; (2) western Lahontan basin— Truckee, Walker, and Carson rivers together with Summit Lake; (3) northwestern Lahontan basin—Quinn River; (4) eastern Lahontan basin—Humboldt and Re- ese rivers; (5) Lake Alvord basin—Virgin-Thousand and Trout Creek drainages; and (6) Coyote Lake basin—Willow and Whitehorse rivers.

Introduction and Background The Lahontan Cutthroat Trout (LCT) Oncorhynchus clarkii henshawi and Paiute Cutthroat Trout (PCT) O. c. selernis were classified as subspecies of Cutthroat Trout O. clarkii in the 20th century (Behnke 1979). They are found in the Lahontan hydrographic basin, which cov- ers most of northern Nevada and extends into northeastern California and southeastern Or- egon. Together with the extinct Alvord Cutthroat Trout form, these three groups comprise the Lahontan basin evolutionary lineage of Cutthroat Trout (Figure 1; Behnke 1979, 1992). The Lahontan basin evolutionary lineage has been separated from the Snake River Cutthroat Trout lineage (see Campbell et al. 2018) for an estimated 2 million years and from the upper Columbia River lineage for 2.8 million years. Paiute Cutthroat Trout was listed as endangered by the U.S. Fish and Wildlife Service (USFWS) on March 11, 1967 under the Endangered Species Preservation Act of 1966 (USFWS 1967). Cutthroat Trout from all major watersheds in the Lahontan hydrographic basin are considered to be LCT, which was listed as endangered on October 13, 1970 under the En- dangered Species Protection Act of 1969 (USFWS 1970). Both PCT and LCT were subsequently reclassified as threatened on July 16, 1975 to facilitate management (USFWS 1975). Given its small distribution in one historic stream, PCT is currently recognized as a single geographic management unit (GMU) or evolutionarily significant unit (ESU; Waples 1995), with no inter- nal genetic subdivision (Saglam et al. 2017). In contrast, the USFWS initially identified three distinct population segments (DPSs) for LCT in the 1995 Recovery Plan (Coffin and Cowan 1995), based upon meristic, morphological, ecological, and genetic data (Hickman and Behnke 1979; Loudenslager and Gall 1980; Gall and Loudenslager 1981; Behnke 1992; Williams et al. 1992). Later, the USFWS redefined these DPSs as GMUs, largely to acknowledge the physical and ecological differences among Cutthroat Trout found in the major watersheds. The western Lahontan basin GMU (Truckee, Carson, and Walker rivers) contains the only large lacustrine and most of the high-elevation alpine habitat, whereas the eastern Lahontan basin GMU (Hum- boldt and Reese rivers) and northwestern Lahontan basin GMU (Quinn River, Coyote Lake, and Summit Lake; Figure 1) are largely lower elevation fluvial habitats with one small terminal lake (Summit Lake, northwestern Lahontan basin GMU). The combination of more recent recognition of a much longer evolutionary history of Cutthroat Trout (Smith et al. 2002; Smith and Stearley 2018, this volume) and several influential papers identifying previously unrecognized genetic diversity within Cutthroat Trout (Metcalf et al. 2012; Loxterman and Keeley 2012) have prompted the re-evaluation of the overall taxonomy of this species and its major lineages. Lake Lahontan, one of the large pluvial lakes found in the interior hydrographic basins of the western United States during the Pleistocene, played an important role in diversification lahontan basin evolutionary lineage of cutthroat trout 3

Figure 1.—The Lahontan hydrographic basin of northern Nevada, northeastern California, and southwestern Oregon, shaded with major rivers and lakes shown (inset U.S. Fish and Wildlife Service Lahontan Cutthroat Trout geographic management units [GMUs]: western Lahontan basin, eastern Lahontan basin, and northwestern Lahontas basin GMUs). within the LCT lineage. The extent of this large lake, which covered most of northwestern Nevada, ebbed and flowed over time with a high stand about 650 thousand years before present (ka; Figure 2; Reheis et al. 2002). At its fullest, Lake Lahontan covered what are now met- ropolitan areas in western Nevada (Reno and Carson City), extending to the town of Battle Mountain approximately 512 km east of present-day Reno (Reheis et al. 2002). The pluvial lake began to recede and reached present levels approximately 8,000–9,000 years before present (BP), leaving Walker and Pyramid Lakes in the western Lahontan basin as the only 4 peacock et al.

Figure 2.—The high stand of pluvial Lake Lahontan in dark gray shading, the Lahontan hydrographic basin in median gray shading, and the hydrographic Great Basin in light gray shading. Geographic Information System shape file from https://pubs.usgs.gov/mf/1999/mf-2323/. remnants of this large endorheic lake (La Rivers 1962; Thompson et al. 1986; Benson and Thompson 1987). Pyramid Lake, which never went dry during the history of the pluvial lake, is believed to have maintained its native fish community over the past ≥50,000 years (until the arrival of western Europeans: see below), whereas Walker Lake has dried completely and refilled multiple times over the past 11,000 years (Behnke 1992). lahontan basin evolutionary lineage of cutthroat trout 5 The Summit Lake basin in northeastern Nevada has a complex hydrologic history, having been connected to both the Alvord and Lahontan basins at different times during the Pleisto- cene (Curry and Melhorn 1990). Summit Lake was formed when a landslide isolated it from the rest of the Lahontan basin 7,840–19,000 BP (Curry and Melhorn 1990). The northeastern Quinn River was inundated periodically throughout the Pleistocene while the Coyote Lakes basin in southeastern Oregon had a smaller pluvial lake into which the Willow and White- horse rivers drained. Coyote Lakes basin was never connected to larger Lake Lahontan but may have been connected to Lake Alvord during the late Pleistocene (Reheis et al. 2002). Lake Alvord in turn, may have been connected to Lake Lahontan sometime earlier in the Pleistocene, potentially providing a way for LCT to colonize these northern basins. Interest- ingly, LCT is the only native fish found both in the Willow–Whitehorse drainage and in Sum- mit Lake (Behnke 1992). The Humboldt River was never completely inundated by pluvial Lake Lahontan and encompasses many multiple-order stream drainages (Horton 2000). Historically, LCT accessed the large migratory corridors of the Humboldt River and its major tributaries through both fluvial migratory life histories and metapopulation dynamics (Dunham et al. 1997; Neville et al. 2006, 2016). As a result of these different aquatic habitats, LCT has two distinct phenotypic forms: lacustrine and fluvial. Behnke (1992), and Trotter and Behnke (2008) have argued for splitting LCT into two subspecies, O. c. henshawi (western Lahon- tan basin populations) and O. c. humboldtensis (eastern and northwestern Lahontan basin LCT populations), reflective of these distinct lacustrine and fluvial life histories. Rangewide anthropogenic impacts to LCT have included water diversions and dams, habitat degradation and fragmentation, temperature barriers, and transplants of nonnative trout, which were introduced into waters of all three currently designated GMUs and represent hybridization, predation, and competition threats (Dunham et al. 1997, 2003; USFWS 2009). Today, natural populations of LCT are gone from 99% of the historic lake habitat, including both Pyramid and Walker lakes (USFWS 2009). The only extant native, naturally reproducing lacustrine populations now persist in just two small lakes: Independence Lake, high in the Truckee River drainage, and Summit Lake, in the northwestern Lahontan basin GMU. Less than 10% of stream populations remain, which are largely isolated in small headwater reaches with low levels of genetic diversity (Peacock and Kirchoff 2007; USFWS 2009; Peacock and Dochtermann 2012).

Uniquely Identifiable Evolutionary Units Uniquely identifiable evolutionary units (UIEUs) are defined under the unified species -con cept of de Quieroz (2007) as separately evolving metapopulation lineages. Here, we review earlier genetic, geological, hydrographic, meristic, morphological, and ecological information and draw from more recent and extensive nuclear genetic data sets generated for samples collected from the majority of extant LCT and PCT populations in order to refine delineation of UIEUs within the LCT evolutionary lineage. For some basins, resolution of exact relationships remains difficult due to a combination of factors: losses of populations (both modern and historic), low genetic diversity, possible influences of historic stocking (of both LCT and other trout species), data sets that focused on only portions of the range (with the exception of Peacock and Kirchoff 2007), and multiple divergence events over relatively short timescales. However, based upon this collective information and the goal 6 peacock et al. of conserving the remaining genetic, evolutionary, and life history diversity, we propose recognition of six UIEUs within the Lahontan Cutthroat Trout lineage: 1. Paiute Cutthroat Trout—upper East Carson River 2. Western Lahontan basin—Truckee, Walker, and Carson rivers together with Summit Lake 3. Northwestern Lahontan basin—Quinn River 4. Eastern Lahontan basin—Humboldt and Reese rivers 5. Lake Alvord basin—Virgin-Thousand and Trout Creek drainages 6. Coyote Lake basin—Willow and Whitehorse rivers

Paiute Cutthroat Trout Uniquely Identifiable Evolutionary Units The modern-day Truckee, Carson, and Walker rivers, which drain the northeastern side of the Sierra Nevada Mountains, flow approximately 195, 210, and 250 km, respectively, into the hydrographic Lahontan basin (see Figure 1). The Truckee River and Walker River watersheds terminate in the endorheic Pyramid and Walker lakes while the Carson River flows into the Carson Sink. Paiute Cutthroat Trout have the most restricted range of all Cutthroat Trout and historically were found in approximately 18 km of habitat in the Silver King Creek drainage, Alpine County, California, between Llewellyn Falls and Silver King Gorge in Silver King Creek (hereafter, lower SKC), a tributary to the east fork of the Carson River (Figure 1; USFWS 2013). Paiute Cutthroat Trout have no spots but are otherwise consistent with western Lahontan basin LCT morphologically and meristically, with similar numbers of lateral-series scales (150–180), vertebrae (60–63), pyloric caeca (50–70), and gill rakers (21–27; Behnke 1992), placing them within the LCT lineage found in the western Lahontan basin. Behnke (1976) believed that PCT were isolated fairly recently from East Fork Carson River LCT and therefore are more closely related to them than any other LCT populations. Despite Behnke’s (1976) interpretation, multiple lines of genetic evidence suggest that PCT were the first group to diverge within the Lahontan lineage. In phylogenetic analyses using microsatellite data and LCT populations grouped by either watershed or basin, PCT are clearly separate from all of the LCT (Peacock and Kirchoff 2007), with corresponding population level pairwiseF ST estimates that are large and highly significant (Table 1; Nielsen and Sage 2002). Recent phylogenetic analyses using restriction site-associated DNA sequencing (RADseq) single nucleotide polymorphism (SNP) data to address specifically the relationship between LCT (rangewide) and PCT show PCT to be basal to all other LCT clades and therefore ancestral to the LCT group in its entirety (Figure 3; Saglam et al. 2017). Single nucleotide polymorphism data also clearly separate PCT from all LCT populations in principle components analysis (PCA) space (Figure 4; Saglam et al. 2017). In addition, PCT have a single unique haplotype in the NADH dehydrogenase gene subunit 2 (ND2) that differs from unique LCT haplotypes by 1 and 2 base pairs (Loxterman and Keeley 2012). Although considered a separate subspecies of Cutthroat Trout by the USFWS, PCT are part of a monophyletic clade that includes the other LCT UIEUs discussed here, with the closest congeners from the Snake River lineage (see Chapter 12) forming a second distinct monophyletic clade (Figure 3; Loxterman and Keeley 2012; Saglam et al. 2017). Collectively, these results clearly justify PCT as a distinct UIEU within the LCT lineage. As with the LCT, PCT have experienced declines due to anthropogenic mediated perturba- tions (Cordes et al. 2004). A variety of nonnative trout were introduced into lower SKC sometime before the 1930s, resulting in the loss of PCT in its native range. Before these lahontan basin evolutionary lineage of cutthroat trout 7 - FRC 0.110 0.176 0.153 0.163 0.151 0.177 0.139 0.132 0.190 0.151 = 0.0001). = p 0.456 0.482 0.483 0.459 0.436 0.458 0.477 0.479 0.475 MHK TIC 0.636 0.386 0.426 0.405 0.358 0.371 0.392 0.392 0.356 0.380 0.403 0.487 0.363 0.443 INC 0.211 0.211 0.545 0.185 0.208 0.152 0.173 0.188 0.194 0.192 0.459

0.637 0.397 0.412 0.378 0.405 0.371 0.357 0.371 0.373 0.388 0.485 0.388 0.232 0.360 0.165 0.552 ABEL SLC 0.411 0.729 0.389 0.453 0.431 0.421 0.393 0.416 0.403 0.450 0.407 0.495 0.458 0.658 0.641 0.511 0.748 0.402 0.472 0.443 0.436 0.408 0.428 0.420 0.468 0.424 0.428 0.477 ByDay SUM 0.509 0.247 0.240 0.223 0.227 0.186 0.200 0.220 0.453 0.308 0.227 0.199 0.210 0.232 PYR 0.211 0.135 0.466 0.252 0.208 0.209 0.218 0.192 0.158 0.213 0.439 0.298 0.178 0.189 0.192 0.194 0.254 0.617 0.335 0.310 0.274 0.289 0.273 0.283 0.251 0.278 0.566 0.395 0.281 0.282 0.274 MUR 0.375 0.308 0.414 0.669 0.546 0.423 0.433 0.078 0.670 0.540 0.396 0.462 0.772 0.442 0.424 0.413 0.422 0.406 0.417 0.367 0.415 0.681 0.526 0.420 0.408 0.658 0.393 MAR estimates for all populations (18 microsatellite loci, 9,300 permutations [FSTAT]; ST F EFC 0.382 0.178 0.152 0.212 0.485 0.496 0.552 0.289 0.272 0.242 0.238 0.237 0.250 0.234 0.276 0.484 0.260

0.341 0.528 0.372 0.306 0.318 0.531 0.505 0.689 0.515 0.518 0.392 0.334 0.517 0.564 0.335 0.309 0.278 0.290 0.257 0.390 0.285 0.269 0.294 0.271 0.294 0.492 0.281 0.265 0.255 0.407 PILOT IND 0.281 0.225 0.395 0.287 0.138 0.190 0.420 0.403 0.445 0.216 0.171 0.168 0.186 0.159 0.176 0.149 0.188 0.380 0.155

BET 0.511 0.288 0.078 0.347 0.546 0.386 0.308 0.316 0.550 0.436 0.536 0.540 0.404 0.226 0.581 0.345 0.331 0.297 0.312 0.165 0.318 0.289 0.325 0.279 0.321 0.292 0.179 0.304 .—Population-level pairwise .—Population-level

Table 1 Table Nonsignificant values are bolded and Abbreviations italicized. are as follows: BET = Bettridge Creek, IND = Independence Lake, PILOT = Pilot Peak, EFC = East Fork Carson, MAR = Marshall Canyon Creek, MUR = Murray Creek, PYR = Contemporary Pyramid, SUM = Sum Mohawk, = MHK Creek, Tierney = TIC Creek, Indian = INC Creek, Abel = ABEL Creek, Slinkard = SLC Creek, By-Day = ByDay Lake, mit = Coyote Creek, LJC = = Little Beaver Jack Creek, Creek, COY FORE = Foreman Creek, GAN = Gance Creek, FRC = Frazer Creek, BEAV NFH = TCREEK North = MRBC Fork = Humboldt Marys River, River, Marys River Basin Creek, EMR WMR West = = East Marys River, WC = Creek, Whitehorse UWH = Upper Creek, Whitehorse = Little LWH Canyon Creek, LINE = Line Creek, Washburn = WASH Creek, T = Cottonwood Creek. WCOT Creek, and Willow IND PILOT EFC MAR MUR PYR SUM ByDay SLC ABEL INC TIC MHK FRC BEAV COY LJC FORE GAN NFH MRBC EMR WMR TCREEK 8 peacock et al. WC FRC 0.357 0.250 0.272 0.304 0.012 0.266 0.327 0.599 0.588 MHK UWH

TIC LWH 0.583 0.613 0.444 0.496 0.443 0.556 0.531 0.662 0.235 0.236 0.212 0.473 0.494 0.020 INC 0.346 0.375 LINE

0.559 0.426 0.436 0.324 0.459 0.338 0.580 0.356 0.422 0.317 0.438 0.320 0.490 0.378 0.503 0.339 0.229 0.500 0.535 ABEL WASH SLC 0.583 0.499 0.440 0.574 0.246 0.250 0.300 0.255 0.357 0.227 0.467 0.525 TCRK 0.602 0.520 0.452 0.600 0.242 0.246 0.306 0.260 0.088 WMR ByDay SUM EMR 0.372 0.268 0.240 0.269 0.239 0.244 0.294 0.261 0.090 0.067

PYR 0.330 0.251 0.237 0.287 0.229 0.232 0.291 0.248 0.067 0.048 0.021 MRBC 0.235 0.233 0.486 0.324 0.307 0.537 NFH 0.114 0.119 0.096 0.105 0.470 0.346 0.354 0.418 0.259 0.258 0.301 0.256 0.361 0.422 MUR GAN 0.078 0.088 0.100 0.093 0.306 0.339 0.330 0.300 0.202 0.217 0.212 0.229 0.197 0.101 MAR EFC 0.108 0.120 0.136 0.103 0.329 0.199 0.126 0.138 FORE

LJ 0.118 0.439 0.433 0.596 0.314 0.312 0.515 0.375 0.309 0.450 0.388 0.306 0.476 0.419 0.395 0.547 0.409 0.378 0.625 0.130 0.130 0.142 0.105 0.336 0.224 0.274 0.225 0.236 0.271 0.236 0.234 0.317 0.309 0.289 0.274 0.273 0.246 0.122 0.147 PILOT

IND COY 0.115 0.061 0.097 0.099 0.123 0.066 0.316 0.184 0.105 0.098

BET 0.119 0.114 0.455 0.304 0.335 0.205 0.394 0.240 0.406 0.246 0.447 0.306 0.448 0.269 0.078 0.132 0.122 0.130 0.102 0.134 0.346 0.190 0.276 0.264 0.270 0.259 0.320 0.310 0.277 0.262 0.079 BEAV .—Continued.

Table 1 Table WASH LINE LWH UWH WC WCOT COY LJ FORE GAN NFH MRBC EMR WMR TCREEK WAC LINE LWH UWH WC WCOT lahontan basin evolutionary lineage of cutthroat trout 9

Figure 3.—Coalescence-based phylogenetic analysis of Cutthroat Trout populations (from Sa- glam et al. 2017). Upper nodal values represent the posterior probability of each node; lower nodal values are divergence times based upon assumed divergence between Cutthroat and Rainbow trouts (2/4/6 million years before present). Fixed divergence times are given in bold; estimated divergence times are in normal font. Rainbow Trout (RBT), Westslope Cutthroat Trout (WCT), Bonneville Cut- throat Trout (BCT), Yellowstone Cutthroat Trout (YCT), Paiute Cutthroat Trout (PCT), and Lahon- tan Cutthroat Trout populations from the Quinn River, Walker River (WLK), Carson River (Cars), Summit Lake (SUM), Truckee River Independence Lake (IND), and Pilot Peak (Pilot; U.S. Fish and Wildlife Service Lahontan National Fish Hatchery broodstock derived from Morrison Creek in the Pilot Peak Mountains, Utah) and various populations from the Humboldt River. stocking events, a fortuitous introduction in 1912 by sheepherders above Llewellyn Falls allowed PCT to persist in pure form upstream of their native distribution. In 1946, 1968, and 1972, these fish were used to establish four self-sustaining out-of-basin populations in Inyo (White Mountains), Mono, and Madera counties in California (Sierra Nevada; Diana and Lane 1978; Behnke 1992; USFWS 2004). Removing nonnative trout was suggested as a conservation strategy (Cordes et al. 2004) and these treatments were completed in 2015 with plans to restore PCT into lower SKC (Table 2).

Lahontan Cutthroat Trout: Western Basin Uniquely Identifiable Evolutionary Units The large lacustrine habitats are a defining feature of the western Lahontan basin, with Walker and Pyramid lakes representing remnants of ancient Lake Lahontan into which the Truckee, 10 peacock et al.

- - (TPM) 0.221 0.106 0.196 0.114 0.03 0.0009 0.0009 0.187 0.779 0.261 0.00004 0.013 0.009 0.004 0.007 0.009 0.0006 0.00007 -values p <0.000 Bottleneck no. 3.17 3.72 7.56 5.11 1.83 4.39 4.00 2.13 4.13 8.17 6.61 1.94 2.28 1.75 1.75 2.67 4.72 2.72 1.89 alleles Average SD o 0.025 0.032 0.031 0.029 0.026 0.023 0.023 0.027 0.018 H

o H 0.592 0.213 0.506 0.455 0.233 0.694 0.310 0.197 0.156 ) heterozygosities (±SD), number of alleles (no. al (±SD), number of alleles ) heterozygosities e SD H e 0.042 0.540 0.025 0.034 0.550 0.024 0.029 0.720 0.022 0.040 0.053 0.048 0.101 0.085 0.079 0.305 0.025 0.049 0.049 0.615 0.025 0.051 0.056 0.310 0.023 0.086 0.213 0.027 0.084 0.059 0.290 0.022 0.043 0.561 0.024 0.057 0.325 0.023 0.052 H

e H 0.521 0.558 0.753 0.610 0.212 0.548 0.460 0.217 0.342 0.727 0.691 0.302 0.332 0.267 0.269 0.326 0.606 0.354 0.198 ) and expected ( ) and expected o H 18 18 18 18 18 18 10 10 10 18 18 18 18 10 10 18 18 18 18 Loci genotyped Population statistics Lahontan Trout Cutthroat

9 23 24 23 22 15 39 35 42 23 23 24 23 30 30 24 24 24 22 size Sample Population Creek Peak Bettridge Lake Pilot Independence East Fork Carson Marshall Canyon Creek Murray Creek Poison Flat Creek Creek Milk Ranch Creek O’Harrell Lake Contemporary Pyramid Summit Creek By-Day Creek Creek Humboldt Slinkard Wolf Mill Creek Creek River Little Creek Abel Indian Creek Reese Tierney Mohawk .—All Lahontan and Paiute Cutthroat trout populations and their watersheds included in the microsatellite genetic analyses pre genetic in the microsatellite and their watersheds included trout populations Cutthroat .—All Lahontan and Paiute Table 2 Table River River sented here together with sample size, loci genotyped, observed ( sented here together program the in conducted (TPM) model mutational two-phase the under bottlenecks genetic for test excess heterozygous of results and leles), BOTTLENECK (Cornuet and Luikart 1996). Watershed Truckee Carson River Summit Lake River Walker Humboldt lahontan basin evolutionary lineage of cutthroat trout 11

(TPM) 0.065 0.68 0.001 0.03 0.289 0.196 0.304 0.09 0.335 0.0009 0.087 0.013 0.006 0.05 0.05 0.173 0.002 0.004 0.004 0.08 -values p Bottleneck no. 6.94 7.06 5.06 7.22 7.72 8.50 7.83 7.11 7.83 6.78 3.00 4.67 2.88 6.3 4.8 5.60 4.20 1.9 2.1 6.56 alleles Average SD o 0.027 0.022 0.023 0.022 0.022 0.023 0.025 0.032 0.251 0.193 0.017 H

o H 0.611 0.681 0.733 0.706 0.712 0.699 0.677 0.432 0.632 0.192 0.161 SD e 0.028 0.033 0.696 0.027 0.030 0.742 0.026 0.021 0.024 0.024 0.713 0.022 0.040 0.024 0.023 0.025 0.028 0.745 0.023 0.052 0.037 0.079 0.170 0.023 0.047 0.572 0.019 0.060 0.563 0.029 0.065 0.494 0.019 0.066 0.500 0.037 0.289 0.198 H

e H 0.700 0.750 0.769 0.733 0.751 0.780 0.756 0.785 0.758 0.773 0.764 0.444 0.661 0.268 0.633 0.596 0.544 0.566 0.230 0.195 18 18 18 18 18 18 18 18 18 18 18 18 18 10 18 18 18 18 10 10 Loci genotyped Population statistics Paiute Trout Cutthroat

7 45 16 16 16 24 24 23 23 24 24 21 22 13 36 34 15 37 10 24 size Sample Rock Creek Population Rock Frazer Creek creek Maggie Creek Creek Beaver Coyote Little Jack Creek North Fork Humboldt River Creek Foreman Creek Gance North Fork Humboldt River Marys River Marys River Basin Creek East Marys River Creek Marys River West T Creek Washburn Creek Creek Line Canyon Creek Creek Whitehorse Crowley Whitehorse Little Creek Upper Creek Willow Cottonwood Cottonwood Creek San Joaquin River .—Continued. Table 2 Table River Watershed Quinn River Willow– Whitehorse Carson River 12 peacock et al.

Figure 4.—Restriction site associated DNA sequencing single nucleotide polymorphism principle components (PC) analysis relationship among Paiute Cutthroat Trout and Lahontan Cutthroat Trout (LCT) populations in the western, northwestern and eastern Lahontan basins. Figure modified from Saglam et al. 2017. Abbreviations are as follows: PCT = Paiute Cutthroat Trout, IND = Independence Lake LCT, Pilot = Pilot Peak LCT, CARS = Carson River LCT, WLK = Walker River LCT, SUM = Summit Lake LCT, QUINN = Quinn River LCT, REESE = Reese River LCT, and Humboldt = Humboldt River LCT.

Carson, and Walker rivers flowed historically. In addition to mesotrophic Pyramid Lake, the Truckee River watershed has several oligotrophic lakes (Lake Tahoe and Donner, Cascade, Independence, and Fallen Leaf lakes) that supported LCT prior to the extirpation of these populations in the mid-20th century due to overharvest and lack of access to riverine spawn- ing habitat (Gerstung 1988). The western Lahontan basin UIEU portion of the LCT range has seen the largest anthropogenic mediated declines, with more than 95% of natural populations now extirpated. Independence Lake in the upper Truckee River watershed maintains a small, naturally reproducing native LCT population (Rissler et al. 2006). Otherwise, the only remaining naturally reproducing populations in the western watersheds are found (1) in By- Day Creek in the Walker River; (2) above barrier falls in the East Fork Carson River; (3) in lahontan basin evolutionary lineage of cutthroat trout 13 Murray Creek and Poison Flat Creek, two tributaries to the East Fork Carson River; and (4) in Raymond Meadows Creek, a tributary to the West Fork Carson River (Table 2). Several additional populations have been re-established from these original sources. Carson River LCT populations have been established in Marshall Canyon Creek and Milk Ranch Creek in the Mokelumne River system in California, and all current Walker basin stream populations were refounded with LCT from By-Day Creek in the 1980s and 1990s. Because there are very few extant populations within the Truckee, Carson, and Walker rivers, it is unlikely that these populations represent the full range of genetic diversity once found in these watersheds, thus limiting our ability to characterize fully these lineages and reconstruct evolutionary history unequivocally. However, multiple lines of evidence including genetic, meristic, and morphological data, together with geography and hydrographic history, support a western Lahontan basin UIEU composed of the Truckee, Carson, and Walker rivers, including Pyramid and Walker lakes and the Summit Lake drainage. Lahontan Cutthroat Trout that were native to the large terminal lakes (Pyramid and Walk- er) and Lake Tahoe grew to enormous sizes (18–28 kg; Behnke 1992) and were considered the largest western North American trout (Behnke 1992). They were also assumed to have adaptive tolerance of the higher temperatures and salinity of the terminal lakes. Lacustrine LCT can be distinguished from fluvial forms found in the Humboldt and Quinn rivers by (1) a uniform distribution of moderately large, roundish spots over the sides of the body and ven- tral surface; (2) a large number of gill rakers facilitating filter feeding of zooplankton in open water environments; and (3) a large number of pyloric caeca, indicating piscivory (Behnke 1992). Cutthroat Trout in these large lakes spawned in tributary habitat, reared in the river en- vironment, and returned to the lake habitats to mature. Analysis of DNA obtained from museum specimens collected by Snyder (1911–1913) and Henshaw (1872–1876) prior to the extirpation of these populations suggests that some gene flow occurred between the large lakes of Tahoe and Pyramid, which are connected by the Truckee River (Peacock and Kirchoff 2007; Peacock et al. 2017). Though extirpated from virtually the entire Truckee River watershed in the 1940s, LCT from this watershed have a unique conservation history. The contemporary LCT population in Pyramid Lake is a hatchery-maintained fishery of mixed stock origin that was planted in Pyramid Lake after the original, native strain was extirpated. This hatchery stock was created with Carson River, Independence Lake, and Summit Lake LCT and so was not thought to represent the genetic characteristics of the historical and unique Pyramid Lake LCT population. However, a LCT population discovered in the 1970s in Morrison Creek in the Pilot Peak Mountains of Utah (out of the native range of LCT) was ultimately identified as western Lahontan basin LCT, using morphological and meristic evidence, and thought likely to represent the original Pyramid Lake lacustrine strain (Hickman and Behnke 1979). The Cutthroat Trout in the previously fishless Morrison Creek were probably transplanted from Lake Tahoe or Pyramid Lake sometime in the late 19th or early 20th century into Mor- rison Creek in the Pilot Peak drainage, Utah, at a time when LCT from both Pyramid Lake and Lake Tahoe were widely transplanted into interior basin waters by the Nevada Wildlife Commission. In the hopes that introduced LCT in Morrison Creek retained some of the genetic legacy of the original Pyramid–Tahoe population, this population was duplicated by a transplant into neighboring, fishless Bettridge Creek in the 1990s, and in 1995, fish from these two Pilot Peak creeks were used to develop the Pilot Peak strain currently in hatchery production by USFWS Lahontan National Fish Hatchery Complex for lacustrine recovery 14 peacock et al. activities in the Tahoe–Truckee–Pyramid basin. Hickman and Behnke’s (1979) conclusion that the wild Pilot Peak fish in Utah were transplanted from Pyramid Lake was later corrobo- rated by research that found genetic similarities between fish collected from this out-of-basin drainage and Tahoe–Truckee–Pyramid museum samples (Figure 5; Peacock and Kirchoff 2007; Peacock et al. 2017). However, the extensive extirpation of LCT in the western river basins makes deciphering the historical relationships among the river basins difficult. For instance, while the Walker and Carson River populations form single distinct genetic groups in a Bayesian genotype clustering analysis (i.e., the red and yellow clusters in Figure 6), as expected, Independence Lake (IND), the only extant representative of the Truckee River basin, does not assign with the Pilot Peak strain, but rather assigns to the same genotype cluster as the Carson River (CARS), Summit Lake (SUM), and contemporary Pyramid Lake (PYR) populations (Figure 6). Not surprisingly, the contemporary Pyramid Lake hatchery-strain clusters genetically with Summit Lake, Carson River, and Independence Lake, as this stock was created with LCT from these waters. The dual genotype clusters (blue and yellow) observed in the Truckee River watershed could be due to several possibilities (see Figure 6): (1) the Truckee River is geographically proximate to the Carson River, and populations in these watersheds may

Figure 5.—A Cavalli–Sforza chord distance and neighbor joining tree (based upon data from seven microsatellite loci, OCH5-17, Peacock et al. 2004) for the extant Lahontan Cutthroat Trout populations in the Truckee River basin (contemporary Pyramid Lake strain, Independence Lake, Heenan Lake, Pilot Peak wild and hatchery-raised) together with the historic samples collected in the Tahoe–Truckee watershed prior to extirpation (Truckee Basin Museum 1872–1913). lahontan basin evolutionary lineage of cutthroat trout 15

Figure 6.—Bayesian genotype clustering results for Lahontan Cutthroat Trout populations samples from each major watershed (18 microsatellite loci; k = 7; LnP(D) = –41,240.6, SD ±169.42, Δk = 4.38). For population names, see Table 1. have exchanged migrants when the pluvial lake connected them historically; (2) neither the Pilot Peak nor the Independence Lake population, which are relatively small and isolated, capture the historical genetic diversity in the Truckee River watershed, or (3) both of the above. The SNP- based phylogenetic tree (see Figure 3) places Independence Lake in a polytomic clade with Pilot Peak, Carson, and Summit LCT (and this clade also includes the Humboldt: for discussion, see below). However, PCA based upon these same SNP markers clearly delineate the river basins from each other (Figure 7; Saglam et al. 2017). The Humboldt River populations are separated from the western Lahontan basin populations by the first PCA axis (Figure 7) while the second axis separates the Summit Lake, Carson River, and Walker River populations from the Truckee River. Summit Lake was previously placed in the northwestern Lahontan basin GMU with the Quinn River and Coyote Lake basin, but analyses presented here suggest that this population is more closely aligned with the western Lahontan basin (see discussion below). More pronounced than the population losses in the Truckee and Carson River watersheds, the Walker River lineage was reduced to a single small remnant population in By-Day Creek, where the habitat consists of approximately 3 km of stream. The By-Day Creek population has very low levels of genetic diversity and an estimated effective population size (Ne ) of 6.7 (95% confidence interval 5.2–8.6; 18 microsatellite loci, Tables 1 and 2) and was used to establish several additional populations within the Walker basin above fish barriers to protect them from nonnative trout. In Bayesian genotype clustering analysis, PCA, and microsatellite- and SNP-based phylogenetic trees, these Walker River populations are distinct from and distal to other western Lahontan basin LCT populations in the Carson and Truckee rivers (Figures 3, 7, and 9). This result could be due to low levels of genetic variation in the By-Day Creek population and populations founded from it and consequently loss of phylogenetic 16 peacock et al.

Figure 7.—Principle components (PC) analysis using single nucleotide polymorphism data from Saglam et al. (2017). Populations: Truckee River (Pilot Peak and Independence Lake), Car- son River (East Fork Carson River, Poison Flat Creek, and Murray Creek), Summit Lake, Walker River (Slinkard Creek), Humboldt River (Abel, Beaver, Foreman, Frazer, and Gance creeks, West Marys River, and the main-stem Marys River), Reese River (Tierney Creek [TIC]), and Quinn River (Line Canyon and Washburn creeks). signal and/or hydrographic history. According to Benson and Thompson (1987), the Walker River was the first watershed to disconnect from pluvial Lake Lahontan during the lake dry down in the late Pleistocene followed by the Truckee, Quinn, and Carson–Humboldt drainages. This hydrographic history could partially explain the tree topologies for both microsatellite and SNP data, which have Walker River LCT diverging prior to populations in the Truckee, Carson, and Humboldt rivers (Figures 3 and 9). However, as discussed above, SNP- based PCAs still show Walker assorting with other western Lahontan basin populations on axis 1 (Figure 7), a pattern likely due to the long history of the pluvial lake and the resulting interconnectedness among the Truckee, Carson, and Walker River watersheds. We include Summit Lake in the western Lahontan basin UIEU due to morphological and genetic analyses as well as the hydrographic history of the Summit Lake basin, which support a western Lahontan basin origin for these Cutthroat Trout. As noted above, Summit Lake is a relatively young lake created by a landslide, 7,840–19,000 BP, which isolated this lahontan basin evolutionary lineage of cutthroat trout 17 subbasin from the rest of the watersheds in the Lahontan basin. Mahogany Creek, the main tributary to Summit Lake (Curry and Melhorn 1990; Figure 8), originally flowed northward into the Alvord basin during the Pleistocene. A tectonic shift and gradual uplift of the allu- vial fan deposition sent Mahogany Creek southward, connecting it with Lahontan basin and pluvial Lake Lahontan (Curry and Melhorn 1990; Reheis et al. 2002). During this period of connection, Cutthroat Trout from pluvial Lake Lahontan would have accessed the creek prior to the landslide formation and subsequent isolation of Summit Lake (Curry and Mel- horn 1990). This scenario is supported by the genetic similarities observed between Summit Lake and LCT populations in the western Lahontan basin. Using protein electrophoretic data for 14 allozyme loci, Loudenslager and Gall (1980) placed Summit Lake LCT in a monophyletic clade with the Carson and Walker basin populations (no Truckee River populations were included in this analysis), which were clearly differentiated from the Humboldt and Reese River populations. In addition, Curry and Melhorn (1990) estimated that the LCT in Summit Lake diverged from other western LCT populations 3,200–17,000 years ago, coinciding with the timing of the landslide and formation of the lake. Microsatellite- based phylogenetic analyses similarly support a western basin origin for Summit Lake Cut- throat Trout when populations are grouped by river basin—notably forming a monophyletic clade with the Carson River with 100% bootstrap support (Figure 9; Peacock and Kirchoff 2007). Further, Bayesian analysis of microsatellite data for individuals from more than 30 different populations (Figure 6; Peacock et al. 2010) and a SNP-based PCA analysis also confirm the Carson–Summit grouping (Figure 7). Finally, Summit Lake LCT have the same mitochondrial DNA (mtDNA) restriction fragment length polymorphism (RFLP) haplotype as Independence Lake LCT (Williams et al. 1992) and a lacustrine morphology similar to Cutthroat Trout from Lake Tahoe and Pyramid and Walker lakes (Behnke 1992). While patterns from nuclear genetic markers remain somewhat unclear, the genetic data as a whole (including Lahontan mtDNA RFLP haplotypes [Williams et al. 1992], geography, and hydrographic history) support the inclusion of LCT in the Summit Lake drainage in the western Lahontan basin UIEU.

Figure 8.—Summit Lake and in-flowing tributaries, Mahogany and Snow creeks. 18 peacock et al.

Figure 9.—A Cavalli–Sforza chord distance and neighbor joining phylogenetic tree (1,000 iterations) of Lahontan Cutthroat Trout populations grouped by river and based upon data from 18 microsatellites.

Northwestern Basin Uniquely Identifiable Evolutionary Unit The Quinn River was fully inundated by pluvial Lake Lahontan at its high stand (650 ka) and again during the late Pleistocene (Benson and Thompson 1987; Reheis et al. 2002). It lost connection with the Lake Lahontan approximately 12,500–11,500 BP, refilled, and then, approximately 8,000 BP, the lake began to recede, leaving the Quinn River basin entirely isolated (Benson 1977; Thompson et al. 1986; Peacock et al. 2010). Perhaps largely as a result of this dynamic history, classification of Cutthroat Trout from the Quinn River stream system in northeastern Nevada and southeastern Oregon has been notoriously ambiguous (Behnke 1992; Peacock and Kirchoff 2004; Trotter and Behnke 2008; Peacock et al. 2010). Gill raker counts suggest a fluvial life history similar to the LCT in the Humboldt River (Behnke 1992) while mtDNA analyses suggest a common origin with western Lahontan basin populations (Williams et al. 1992, 1998). The mtDNA RFLP haplotype identified for the Quinn River is shared by Summit Lake, as well as Independence Lake (Truckee River) and By-Day Creek lahontan basin evolutionary lineage of cutthroat trout 19 (Walker River), the only western Lahontan basin samples included in the analysis (Williams et al. 1992). Smith et al. (2002) classify Quinn River Cutthroat Trout as O. c. henshawi (which Behnke 1992 refers to as the Lahontan subspecies), distinguishing them the from Humboldt River populations (putative O. c. humboldtensis, Behnke 1992), and estimate sequence divergence at 0.1% (approximately 200,000 years) among LCT from Quinn, Willow–Whitehorse, and Humboldt rivers. Microsatellite data suggest that Willow–Whitehorse and Walker River LCT populations diverged first followed by populations in the Carson, Humboldt, Quinn, and Truckee rivers (Figure 9; Peacock and Kirchoff 2007) while SNP data suggest that the Quinn River populations were the first to diverge after PCT (with high bootstrap support) from all other LCT populations, followed by the Walker River and a polytomic clade consisting of the Summit Lake and Truckee, Carson, and Humboldt River populations (Figure 3; Saglam et al. 2017). According to Benson and Thompson (1987), the pattern of pluvial Lake Lahontan dry- down suggests that the Quinn River disconnected from pluvial Lake Lahontan in the late Pleistocene after the Walker and Truckee rivers but before the Carson and Humboldt River drainages. Multivariate ordination using SNP data place the Quinn River as intermediate to the Humboldt River and western Lahontan basins, reflecting the classification difficul- ties regarding whether these LCT originated from the Humboldt River or Lake Lahontan (Figures 4 and 7). Because the Quinn River Cutthroat Trout have similarities with both the Humboldt (gill rakers) and western Lahontan basin (mtDNA haplotypes) Cutthroat Trout, it is chal- lenging to place them definitively in either UIEU. Based upon available geomorphological evidence, Trotter and Behnke (2008) argue that the Humboldt River flowed northward into the upper Quinn River during the late Pleistocene (Benson and Peterman 1995; J. O. Davis, Desert Research Institute, unpublished), which supports the morphological, meristic, and at least some genetic evidence that the Quinn River Cutthroat Trout are derived from the Humboldt River Cutthroat Trout lineage (Peacock et al. 2010). They further suggest that similarities with the western Lahontan basin LCT are a result of either secondary contact during Lake Lahontan high stands and/or widespread stocking of Truckee River LCT during early European settlement (Trotter and Behnke 2008). However, taxonomic classification of Quinn River Cutthroat Trout is further complicated by population losses in this watershed over the 20th and 21st centuries. There are currently only five to eight populations (recent surveys were inconclusive for some streams) found in isolated streams and approximately 120 km of stream habitat out of at least 46 historically occupied streams, which covered 1,598 km of stream habitat in the Quinn River drainage (Coffin and Cowan 1995; USFWS 2009). Despite this, the morphological/meristic and genetic data, coupled with the hydrographic history of the Lahontan basin and the current isolation of the Quinn River, suggest that this lineage may now be on a separate evolutionary trajectory. Though classification of the Quinn River populations remains problematic, we separate it here, ac- knowledging this trajectory with hopes that future analyses using genomic tools may shed light on the evolutionary history of the Quinn River LCT.

Eastern Basin Uniquely Identifiable Evolutionary Unit Lahontan Cutthroat Trout from the Humboldt River drainage have a fluvial life history and do not share the lacustrine morphological characteristics found in the western Lahontan ba- 20 peacock et al. sin lake forms (Behnke 1992; Trotter and Behnke 2008). Based largely upon these morphological differences, Behnke (1992) and Trotter and Behnke (2008) proposed that LCT from the Humboldt River be considered a separate subspecies (O. c. humboldtensis). While we agree that a separate UIEU is warranted for the LCT populations in the Humboldt River watershed, we acknowledge that multiple genetic data sets suggest a complicated history. We attempt to reconstruct that history here and the results suggest a range of possible relationships between the Humboldt and other LCT populations, including those in both the western Lahontan basin and the Quinn River. These various scenarios likely reflect geography together with the hydrographic history of the pluvial lake and the changing hydrologic connections between the Humboldt River, Quinn River, and pluvial Lake Lahontan over time (Loudenslager and Gall 1980; Bartley et al. 1987; Reheis et al. 2002; Loxterman and Keeley 2012). The main-stem Humboldt River, the largest river in the Lahontan hydrographic basin, flows 530 km from east to west and currently terminates in the Humboldt Sink. Prior to the mid-1800s, the Humboldt River drainage encompassed approximately 6,040 km of cold- water habitat (Coffin 1983; USFWS 2009). Historically, the Humboldt River was inundated by Lake Lahontan up to the confluence with the Reese River and remained so until the lake began to recede during the late Pleistocene (see Figure 1; Reheis et al. 2002). Most extant Humboldt River LCT populations are found primarily in watersheds east of the Re- ese–Humboldt confluence. The Reese River is the largest tributary to the Humboldt and flows 291 km into the main-stem Humboldt River and once provided 806 km of historic LCT habitat (USFWS 2009). Today, extant LCT populations in the Reese River drainage are only found in isolated headwaters in southern tributaries. Streams in the Reese River drainage are no longer connected to the main-stem Reese River, and it is likely that they only connected intermittently historically during high-flow periods. As a result, the extant Reese River LCT populations are geographically quite isolated from the rest of the occupied tributaries within the larger Humboldt River drainage. Similarly, populations in the Little Humboldt River, which is the western-most tributary to the Humboldt and as such was inundated by pluvial Lake Lahontan, are also geographically quite distant from the majority of currently occupied Humboldt River tributaries (see Figure 1). Consistent with historical downstream inundation, the Mohawk and Tierney creeks in the Reese River and Abel Creek (ABEL) in the Little Humboldt River assign to the same microsatellite-based Bayesian genotype cluster (Figure 6) while the remainder of the Humboldt River populations, including Indian Creek (also found in the Little Humboldt River), assign to a second distinct genotype cluster. The genotype cluster comprised of the Reese (Mohawk [MHK] and Tierney [TIC]) and Little Humboldt River populations (ABEL) is likely a result of the relative geographic proximity combined with the rise and fall of the pluvial lake and population losses in the intervening tributaries during the 20th century. Geographic distance from other Humboldt River populations combined with inundation by pluvial Lake Lahontan and potential widespread movement of Cutthroat Trout in the lake could also explain why genetic data suggest that some Humboldt River populations—notably those in the Little Humboldt at the western end of the river—form a clade with the Carson and Walker rivers, separate from the other Humboldt River populations (allozyme-based dendogram of genetic similarity; no Quinn River populations were included in this analysis; Bartley et al. 1987) (Note that the Marys River, which drains into the lahontan basin evolutionary lineage of cutthroat trout 21 eastern-most extent of the Humboldt River, is also in this clade: see below for discussion; Figure 1; Bartley and Gall 1993). The similarity between LCT in the Carson River and the western-most tributaries of the Humboldt River makes geographic sense as, historically, the Carson and Humboldt rivers flowed into the same basin/arm of the pluvial lake (Figure 2), and today, the Carson and Humboldt sinks are in close proximity with a hydrological connection in high water years (Figure 1). Consistent with the possibility of intermixing between Humboldt and western Lahontan basin Cutthroat Trout in the large pluvial lake, results from recent PCA using a large SNP data set (Figure 10; S. J. Amish, University of Montana, and colleagues, unpublished) separate some Humboldt River populations from Pilot Peak (putative Truckee River lineage) but show less divergence from the western Lahontan basin Independence Lake LCT. A similar pattern is observed with mtDNA data, where Williams et al. (1992) identify a single Humboldt River haplotype for Cutthroat Trout from seven Humboldt River watersheds not shared by any Cutthroat Trout in the western or northwestern Lahontan basins, but also found a western Lahontan basin mtDNA haplotype shared with two Humboldt River tributaries, Rock Creek and the Marys River (Figure 1; Table 2). Trotter and Behnke (2008) explained these patterns by suggesting that either the Humboldt River mtDNA haplotype arose after the hydrographic separation of the western and northwestern Lahontan basins from the Humboldt River or that the western

Figure 10.—A principle components analysis (PCA) of Lahontan Cutthroat Trout (LCT) populations (based upon 4,644 single nucleotide polymorphisms, Amish and colleagues, unpublished data) showing the divergence along the first axis between the Lahontan National Fish Hatchery Pilot Peak broodstock (PILOT) and the lacustrine life-history types, Summit Lake (SUM) and Independence Lake (IND), and fluvial life history types, Gance Creek (GAN), West Marys Riv- er (WMR), main-stem Marys River (MSMR), Mohawk Creek (MHK), and Willow Whitehorse Creek (WWH). The second axis differentiates WWH from all other LCT populations. The third axis differentiates the MHK in the Reese River from all other LCT populations. 22 peacock et al. Lahontan basin mtDNA haplotype in Humboldt populations is the result of known stocking of LCT from the Truckee River drainages throughout the range in the early 20th century. While it is difficult to exclude the possibility that stocking explains shared haplotypes, it is equally plausible that there was limited gene flow when the Humboldt River was connected to pluvial Lake Lahontan. For instance, although Rock Creek and Marys River flow into the main-stem Humboldt River east of the confluence with the Reese River and therefore were not likely inundated by pluvial Lake Lahontan, Reheis et al. (2002) identify additional basins, including the Rock Creek basin, that were inundated by mid-Pleistocene lakes that later receded, thus extending the pluvial lake and possibly enabling occasional movement of lacustrine fish far into the Humboldt River during this period. The observation of a significant genetic isolation-by-distance pattern among watersheds draining into the Humboldt River suggests movement influenced by a distance gradient as opposed to random stocking influences (microsatellites,R 2 = 0.49, p = 0.000; Table 1; Peacock and Kirchoff 2007). It is possible that we are observing patterns generated from both natural gene flow and human stocking events, complicating reconstruction of historical movement patterns. In addition, the loss of intervening populations over the 20th century has disrupted historical interconnectedness; this, coupled with contemporary population isolation, low levels of genetic diversity, highly significant population and watershed level pairwise FST estimates (microsatellite data, Tables 1 and 3), and evidence of genetic bottlenecks, suggests that genetic drift is potentially complicating straightforward interpretations of the genetic data. Despite these complexities, we believe that when the geographic and hydrographic history of the Lahontan hydrographic basin is combined with the morphological, meristic, and genetic data for LCT populations in the Humboldt River drainage, a separate UIEU for the Humboldt River LCT populations is indicated. Regardless of divergence order of the UIEUs, the data presented here indicate that the Humboldt River is genetically distinct from all other watersheds in the western, eastern, and northeastern Lahontan basins. The molecular clock estimate of mtDNA sequence divergence of approximately 200–260 ka suggests that the Humboldt River, Quinn River, and western Lahontan basin forms have been separated for a significant period of time and that despite the potential for fish to move within the large lake and provide gene flow among the watersheds, divergent lineages may have already been developing in the tributaries when the large lake was extant (Smith et al. 2002; Trotter and Behnke 2008).

Cutthroat Trout in the Alvord and Willow–Whitehorse Basins—Lake Alvord Uniquely Identifiable Evolutionary Unit and Coyote Lake Basin Uniquely Identifiable Evolutionary Unit The Alvord and Willow–Whitehorse hydrographic subbasins are adjoining endorheic basins covering parts of northwestern Nevada and southeastern Oregon (Figure 1; Behnke 1992; Peacock et al. 2010). During the Pleistocene, Lake Alvord covered the entire Alvord basin and the Willow and Whitehorse rivers drained into pluvial Coyote Lake. Both the Alvord and Coyote lakes disappeared completely during the post-Pleistocene dry-down. Reheis et al. (2002) hypothesized a possible overflow from pluvial Lake Lahontan into Lake Alvord sometime during the mid-Pleistocene, whereas geological evidence shows that subsequently, in the late Pleistocene, Lake Alvord and Coyote Lake were connected when lahontan basin evolutionary lineage of cutthroat trout 23 Quinn 0.134 Marys NFH 0.030 0.201 0.105 0.111 0.042 0.124 0.034 Maggie Rock 0.086 0.064 0.177 0.125 0.122 0.150 0.071 0.153 0.150 0.223 0.213 Reese LH 0.079 0.074 0.167 0.154 0.155 = 0.01. p 0.245 0.067 0.154 0.269 0.106 0.195 0.266 0.240 0.343 0.282 0.305 0.393 Walker 0.085 0.129 0.093 0.083 0.146 0.109 Summit 0.116 0.074 0.175 0.123 0.120 0.202 0.166 estimates, all are significant Carson ST F 0.130 0.094 0.272 0.309 0.252 0.133 0.168 0.126 0.171 0.229 0.169 0.142 0.092 0.089 0.092 0.145 0.147 Truckee

.—Watershed level .—Watershed Table 3 Table River River rivers Carson River Lake Carson River Summit Walker Little Humboldt River River Reese Rock Maggie Creek North Fork Humboldt River Marys River Quinn and Willow Whitehorse 24 peacock et al. Lake Alvord overflowed into the Coyote Lake basin (Behnke 1992; Carter et al. 2006). Cut- throat Trout could have colonized the Alvord basin via the mid-Pleistocene Lake Lahon- tan–Lake Alvord connection or from the late-Pleistocene connection with the Summit Lake basin when Mahogany Creek flowed northward into the Alvord basin prior to the formation of Summit Lake (Curry and Melhorn 1990; Behnke 1992). Colonization of the Coyote Lake basin is thought to have occurred via a Pleistocene headwater transfer from the Quinn River drainage (Behnke 1992), but conceivably, Cutthroat Trout could have come from Lake Al- vord. In either case, Cutthroat Trout in Coyote Lake basin have likely been isolated there for thousands of years. The post Pleistocene dry-down of Lake Alvord reduced the distribution of Cutthroat Trout in the Alvord basin to the Virgin-Thousand Creek and Trout Creek drainages (Bartley and Gall 1991; Behnke 1992; Peacock et al. 2010). Rainbow Trout were introduced into Trout Creek in 1929, and by 1934, most of the specimens examined morphologically were deemed hybrids (Behnke 1992). We could find no genetic analyses of Cutthroat Trout from Trout Creek. Rainbow Trout were first planted in 1933 in Virgin Creek, and by 1970, most fish examined appeared to be pure Rainbow Trout (Behnke 1992). Bartley and Gall (1991) genotyped 59 allozyme loci in 104 Cutthroat Trout collected from Virgin Creek in the 1980s to investigate the extent of Rainbow Trout–Cutthroat Trout hybridization, and no pure Cut- throat Trout were found. There has been considerable interest, however, in a possible out-of-basin transplant of Alvord Cutthroat Trout that is still unresolved. Guano Creek, in the Catlow basin in south- east Oregon, is within the natural range of Great Basin Redband Trout O. mykiss newberrii and contained a pure population of Great Basin Redband Trout until LCT were stocked in 1957, 1969, and 1973 by the Oregon Department of Fish and Wildlife (ODFW; ODFW 2005). In the 1960s, hatchery-raised Coastal Rainbow Trout O. m. irideus were regularly planted into Guano Creek. However, in the 1980s, Behnke (1992) found individuals with an Alvord Cutthroat Trout phenotype in Guano Creek, which suggested that Alvord Cut- throat Trout had been planted there sometime in the early 20th century. This has led to a hatchery program using fish from Guano Creek as an attempt a recover the Alvord Cut- throat Trout phenotype (ODFW, personal communication). However, recent SNP analyses of trout from Guano Creek show these fish to have greater than 50% Yellowstone Cutthroat Trout O. c. bouvieri ancestry, less than 5% Rainbow Trout ancestry, and 45% LCT ancestry (Pritchard et al. 2015). Pure Alvord Cutthroat Trout specimens collected by Carl Hubbs are housed at the University of Michigan in the Museum of Zoology and genetic comparisons of these specimens with the Guano Creek Cutthroat Trout and LCT populations will be important to conduct. However, currently we do not have genetic markers to distinguish Alvord Cutthroat Trout from other Cutthroat Trout, so it is unclear if the trout in Guano Creek retain any Alvord Cutthroat Trout genes. As far as we know, there are no remaining pure populations of Alvord Cutthroat Trout, and the subspecies is considered extinct (Behnke 1992). The Willow and Whitehorse rivers (WWH) are found in the Coyote Lake basin in the southeastern Oregon portion of the Lahontan basin and are the only historic habitat for LCT in this watershed (see Figure 1). Behnke (1992) suggests that these Cutthroat Trout are of Quinn River origin, having colonized the basin during a Pleistocene headwater transfer. However, these Cut- throat Trout have a similar number of gill rakers and lateral line scales as seen in Humboldt lahontan basin evolutionary lineage of cutthroat trout 25 River Cutthroat Trout but well-developed basibranchial teeth and numbers of pyloric caeca similar to the LCT from the western Lahontan basin drainages. Given the possible hydrographic connections to both the Quinn River and pluvial Lake Lahontan during the Pleistocene and ongoing questions about the origin of the Quinn River Cutthroat Trout, LCT may have colonized the Willow–Whitehorse basin from multiple source populations. Behnke (1992) suggested that these trout be classified as a separate O. clarkii subspecies, but in 2008, Trotter and Behnke combined Cutthroat Trout from the Humboldt River, Quinn River, and Willow–Whitehorse drainages into a single O. c. humboldtensis subspecies based on an assumed historical interba- sin transfer. Microsatellite, mtDNA sequence, and SNP data, however, consistently show Wil- low–Whitehorse Cutthroat Trout to be diverged from LCT in all currently designated GMUs (Figures 6, 9, 10, and 11; Williams et al. 1998; Peacock and Kirchoff 2007; Peacock et al. 2010; Amish and colleagues, unpublished). In Bayesian genotype clustering analysis, LCT individuals from the Willow–Whitehorse drainage clearly assign back to this drainage (Figure 6). Phyloge- netic analyses based on different population groupings, either all populations grouped under a larger river designation (i.e., Humboldt River) or combined into a larger GMU designation (i.e., all populations within a single GMU) consistently separate the Willow–Whitehorse Cutthroat Trout populations from all other populations. Willow and Whitehouse River populations are also highly distinct in PCA space (SNPs; Saglam et al. 2017; Figure 10). Moreover, Willow–

Figure 11.—A Cavalli–Sforza chord distance and neighbor joining phylogenetic tree of the extant Lahontan Cutthroat Trout populations grouped by U.S. Fish and Wildlife Service-designated geographic management units, with Paiute Cutthroat Trout and Rainbow Trout as the outgroup. 26 peacock et al. Whitehorse Cutthroat Trout have both western Lahontan basin and unique mtDNA RFLP haplotypes (Williams and Shiozawa 1989), suggesting that these populations may have been derived from western Lahontan basin LCT (see above). Sequence divergence ranged from 0.15% to 0.30% from Quinn River and Summit Lake haplotypes (Williams et al. 1992), and recent ND2 sequence data show the Whitehorse River Cutthroat Trout haplotype differing from the Reese and Little Humboldt River haplotypes by a single base pair (Loxterman and Keeley 2012). Re- gardless of origin, Willow–Whitehorse LCT are clearly genetically distinct with data supporting divergence of WWH lineage from other LCT lineages. The designation of these populations as a separate UIEU is warranted.

Conclusions Here we provide evidence to support six UIEUs (one of which may be extinct), fully acknowl- edging that some relationships are difficult to resolve given extensive population losses, bottlenecks, transfers of LCT, stocking, and the different scopes of the studies referenced. Cutthroat Trout have been in what is now the Lahontan basin for at least 10 million years and during that time have diversified into multiple distinct geographic and genetic units. The patterns and timing of divergence have been driven by the changing hydrographic landscape, which was largely dominated by pluvial Lake Lahontan. The high stand of Lake Lahontan occurred mid-Pleisto- cene (approximately 600 ka), which was earlier and the lake larger than previously thought, providing connectivity among the large river basins that drained into it (Reheis et al. 2002). The size and extent of the pluvial lake and thus degree of connectivity among river basins changed repeatedly over time. The last high stand of the lake occurred approximately 22 ka, after which the lake began to recede and subsequently isolate the river basins. The two terminal desert lakes, Pyramid and Walker, are the remnants of the large pluvial lake and until the mid-20th century supported lacustrine Cutthroat Trout populations, a phenotype thought to be preserved in the Pilot Peak hatchery strain. Phylogenetics of Cutthroat Trout will be ongoing as new research sheds light on the complex evolutionary history of the group.

Acknowledgments We would like to thank Matt Mayfield, Trout Unlimited, for Figure 2. We would also like to thank the anonymous reviewers for improving the manuscript and Patrick Trotter for initiat- ing the project.

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