Genetic and morphological evidence suggests cryptic speciation within the Torrent Sculpin, rhotheus, across the Pacific Northwest

by Benjamin Nicholas

A THESIS

submitted to

Oregon State University

Honors College

in partial fulfillment of the requirements for the degree of

Honors Baccalaureate of Science in Fisheries and Wildlife Sciences (Honors Scholar)

Presented May 28, 2019 Commencement June 2019

AN ABSTRACT OF THE THESIS OF

Benjamin Nicholas for the degree of Honors Baccalaureate of Science in Fisheries and Wildlife Sciences presented on May 28, 2019. Title: Genetic and morphological evidence suggests cryptic speciation within the Torrent Sculpin, Cottus rhotheus, across the Pacific Northwest.

Abstract approved:______Brian Sidlauskas

Dwelling on the bottom of large rivers to high alpine streams, freshwater sculpins (genus Cottus) occur in high densities across the Pacific Northwest, with 21 currently recognized species found in the Pacific Northwest. Sculpin fill many ecological roles, with some species serving as important prey sources, and others competing with or predating upon juvenile salmonids. Fully understanding these ecological interactions requires the ability to recognize and diagnose co-occurring species. However, Cottus contains many similar looking species, leading to frequent misidentifications and the persistence of undiscovered species. I used multilocus phylogenetics and multivariate morphometric analysis to investigate the taxonomic status of Torrent Sculpin, Cottus rhotheus across the Pacific Northwest, and determine whether the current concept of C. rhotheus contains multiple cryptic, unrecognized species. Phylogenetic analysis reveals three distinct and geographically restricted clades, with the and Cascade Range acting as allopatric barriers. Morphometric data corroborates the genetic divisions, identifying the presence of three cryptic species under the current species description of C. rhotheus.

Key Words: Sculpin, cryptic speciation, morphology, phylogeny

Corresponding e-mail address: [email protected]

©Copyright by Benjamin Nicholas May 28, 2019

Genetic and morphological evidence suggests cryptic speciation within the Torrent Sculpin, Cottus rhotheus, across the Pacific Northwest

by Benjamin Nicholas

A THESIS

submitted to

Oregon State University

Honors College

in partial fulfillment of the requirements for the degree of

Honors Baccalaureate of Science in Fisheries and Wildlife Sciences (Honors Scholar)

Presented May 28, 2019 Commencement June 2019

Honors Baccalaureate of Science in Fisheries and Wildlife Sciences project of Benjamin Nicholas presented on May 28, 2019.

APPROVED:

______Brian Sidlauskas, Mentor, representing Fisheries and Wildlife Sciences

______Douglas Markle, Committee Member, representing Fisheries and Wildlife Sciences

______Thaddaeus Buser, Committee Member, representing Fisheries and Wildlife Sciences

______Toni Doolen, Dean, Oregon State University Honors College

I understand that my project will become part of the permanent collection of Oregon State University, Honors College. My signature below authorizes release of my project to any reader upon request.

______Benjamin Nicholas, Author

Introduction

Dwelling on the bottom of large rivers to high alpine streams, freshwater sculpins (genus Cottus) occur in high densities across the Pacific Northwest (PNW) of the , with twenty species currently recognized in that region (Fricke et al. 2019). Sculpins fill many ecological roles, with some species serving as important prey sources for larger (Dryer et al. 1965; Scott and Crossman 1979), and others competing with or predating upon juvenile salmonids (Hunter 1959; Bond 1963; Berejikian 1995; Gabler and Amundsen 1999). Fully understanding these ecological interactions requires the ability to recognize and diagnose co-occurring species. However, Cottus contains many similar looking species, leading to frequent misidentification, redescription and discovery of new species (Daniels and Moyle 1984; Nolte et al. 2005; Lemoine et al. 2014). The most comprehensive and up to date phylogeny of Cottus was produced in 2005, which found Cottus to be paraphyletic using three mitochondrial genes (Kinziger et al. 2005). In an attempt to better understand the ‘Cottopsis’ clade within Cottus, identified by Kinziger et al. (2005), Baumsteiger et al. (2012) analyzed two mitochondrial and nine nuclear genes. This study illuminated complex species structure which occur within Cottus, even identifying three cryptic lineages (Baumsteiger et al. 2012). Presently, there is no comprehensive phylogeny of Cottus with nuclear and mitochondrial genes or complete species representation. In an attempt to better understand Cottus across the United States, Michael Young of the United States Forest Service (USFS) has begun undertaking the challenge to create a complete phylogeny of North American Cottus. In his initial analysis of mitochondrial DNA, Young (pers. comm 2016) found a sculpin in the PNW by the name of Cottus rhotheus, common name Torrent Sculpin, which showed deeply genetically divergent and geographically distinct lineages. Cottus rhotheus has an extensive PNW range, primarily occupying the Columbia River Basin and its tributaries, but also occurs in the Fraser River basin and coastal systems in Oregon and (Bond 1963) (Fig. 1). In the initial mtDNA analysis, three distinct geographic groups were identified in Western Washington, Western Oregon, and east of the Cascades stretching to Montana. Historically, this species is known for having variable morphology and morphological characteristics across its range (Bailey and Dimick 1949; Markle and Bond 1996; Richard and Zaroban 2013). With preliminary genetic results suggesting divergent lineages and historical studies documenting variation in morphological characteristics, it appeared likely that several undiscovered cryptic species existed within a C. rhotheus species complex.

Figure 1: A range map of documented collection localities for C. rhotheus. Different color dots represent the different databases which data was retrieved from (FishMap.org 2019). The type locality for C. rhotheus is denoted by the yellow star.

Cottus rhotheus Little information exists on the life history of Cottus rhotheus, and most available information stems from studies completed prior to 1980 (Northcote 1954; Bond 1963; Patten 1971; Thomas 1973). These early studies examined diet most extensively. Cottus rhotheus is a generalist predator with a relatively large gape size in comparison to other Cottus (Northcote 1954). A study in Arrow Lake, found that the juvenile diet (<39mm, standard length (SL)), primarily included crustacean plankton and insect larvae (Northcote 1954). Once C. rhotheus reach roughly 60mm SL become a larger part of their diet and switch to a near piscivorous diet of cyprinids and salmonid fry above 70mm SL (Northcote 1954). A recent diet study by Tabor et al. (2016) tracked diets in the Cedar River in Washington over the course of several months. In January and February, C. rhotheus between 50-99mm SL primarily fed on stoneflies, mayflies, and caddisflies, and earthworms (Tabor et al. 2016). Cottus rhotheus over 100mm SL fed primarily on other sculpins (Tabor et al. 2016). In March through May, fish eggs accounted account for more than 50% of the diet of specimens >75mm SL. In summer months, C. rhotheus <75mm had a mixed diet of insects and other sculpin, while those over 75mm SL consumed mostly sculpin (Tabor et al. 2016). The primary diet of C. rhotheus in summer months was a mix of sculpins and insect larvae for specimens 55-74mm, with specimens larger than 75mm consuming primarily other sculpin (90%) (Tabor et al. 2016). These two studies suggest that C. rhotheus does not specialize on a prey across habitats and are opportunistic to the prey available to them. Both studies did confirm that large C. rhotheus will prefer a piscivorous diet at larger sizes. Bond (1963) predicted that C. rhotheus reaches sexual maturity between 60-69mm, when roughly three years of age. Fecundity measured across its PNW range in three geographic regions (W. Washington, E. Washington, and W. Oregon) revealed the number of ova correlated significantly and positively with specimen size. Among specimens from the Yakima River in E. Washington, the number of ova ranged from 100 ova at 69mm SL to 412 ova at 108mm SL (Patten 1971). In Newaukum Creek in W. Washington, the number of ova ranged from 63 to 488, among specimens ranging from 59mm SL to 100mm SL (Patten 1971). Specimens of C. rhotheus from Oregon were less fecund than their Washington counterparts, with the number of ova ranging from 166 to 298 in specimens ranging from 60mm SL to 119mm SL (Bond 1963). No study has quantified the home range or movement of C. rhotheus, though Bond (1963) assumed similarity to other Cottus species. Many cottids live a benthic and sedentary life, rarely moving 100m from where they hatched (Natsumeda 1998; Natsumeda et al. 2004; Petty and Grossman 2004; Breen et al. 2009). With such little movement in these species, populations of C. rhotheus in likely have little genetic connectivity with those in the Willamette Basin hundreds of miles away. While many species of Cottus do not move from the portion of the stream they inhabit, Thomas (1973) observed repeated upstream spawning migrations between January and April in a Washington population of C. rhotheus. Females laid eggs during the month of April, and then individuals migrated downstream in May and June (Thomas 1973). This however is the only documented case of such migration. In Bond’s (1963) thesis on Cottus distribution in Oregon, he hypothesized that there would be no need to migrate, as the habitat which C. rhotheus occupies in the Willamette Valley had ample cover under boulders for egg laying and juvenile protection once hatched. Simon and Brown (1943) observed an upstream migration in C bairdii semiscaber, with mature fish seeking out headwater springs to mate. The spawning season of C. rhotheus has been documented between April through June at three localities across the PNW (Bond 1963; Carl et al. 1967; Thomas 1973). After spawning, C. rhotheus guards its nest (Bond 1963; McPhail 2007). Bond (1963) notes that male C. rhotheus only abandon their nests when severely disturbed. This behavior is also observed in several other species of Cottus (Simon and Brown 1943; Marconato and Bisazza 1988; Goto 1993; Fiumera et al. 2002; Bateman and Li 2004). Many researchers have noted that morphology varies across this species range, and that putatively diagnostic morphological characteristics are not perfectly reliable (Bailey and Dimick 1949; Markle and Bond 1996; Wallace et al. 2013). For example, in their description of C. hubbsi, Bailey and Dimick (1949) provided a table of morphological characteristics and morphometrics distinguishing C. rhotheus from C. hubbsi. In the table, they include a note that characteristics such as complete body prickling and lateral lines extending to the caudal fin, only apply to the region in which C. rhotheus and C. hubbsi co-occur. In coastal Washington and Oregon, Bailey and Dimick (1949) stated prickling was reduced or absent and lateral lines were usually incomplete. Markle and Bond’s (1996) sculpin identification workshop guide noted that specimens from Eastern Oregon have less distinct dorsal saddles and an incomplete lateral line, despite the fact that prominent saddles and a complete lateral line characterize C. rhotheus across the rest of its range. Body prickling also varies across C. rhotheus’ range (Bailey and Dimick 1949; Markle and Bond 1996). With considerable geographic variation observed in morphological characteristics and the presence of several distinct mitochondrial clades from Michael Young’s data, further examination into the species complex of Cottus rhotheus was warranted. This thesis aims to test for the presence of cryptic species in the Cottus rhotheus species complex. Cryptic species are classically defined as two or more species which have been classified as a single species because they are nearly morphologically indistinguishable (Bickford et al. 2007). Others have furthered this definition by requiring nominal cryptic species to be reproductively isolated (Mayr 1970; Mottern and Heraty 2014). In this thesis, I attempt to resolve the taxonomic uncertainty surrounding the Cottus rhotheus species complex using morphological comparisons and phylogenetic methods.

Methods

Many papers which test for the presence of cryptic species rely solely on genetics, failing to include any morphometric or phenotypic data (for example, Feulner et al. 2006; Baumsteiger et al. 2012; Puckridge et al. 2013; Dawson and Jacobs 2014). Struck et al. (2018) found that a majority of these studies only used one locus in their genetic analyses. In studies which do include phenotypic data, 25% use only one characteristic (Struck et al. 2018). This study uses nuclear and mitochondrial genes and a suite of morphological characteristics and measurements to assess genetic and phenotypic differences in the Cottus rhotheus species complex.

Sampling A total of 416 tissue samples from 182 localities comprised our molecular data set and 148 whole specimens from 41 localities were measured for morphological data (Fig. 2). Tissue samples and morphological vouchers were provided by the U.S. Geological Survey, U.S. Fish and Wildlife Service, Washington Department of Ecology, Oregon Department of Environmental Quality, Wild Fish Conservancy, Washington State Department of Fish and Wildlife and Oregon Department of Fisheries and Wildlife, Pacific Lutheran University, Oregon State University, University of Washington, and University of Michigan (See Appendix Table 1 for complete list, collection acronyms follow (Sabaj 2019)). Additional specimens were collected with electroshocking backpacks and dip nets and euthanized by OSU personnel operating under Care and Use Protocol 5077, with all participants undergoing the proper training to work with live . Tissues and fin clips were preserved on chromatography paper, in coin envelopes, or in vials containing 99% ethanol, whereas morphological vouchers were fixed in 10% formalin and stored in either 50% isopropanol or 70% ethanol. Most major basins were sampled for genetics and morphometrics, with the exception of several coastal Oregon rivers and the population at Harney Basin at Fish Lake. The C. rhotheus found in Harney are thought to have been introduced with trout, as this is the only location they are found in the Harney Basin (Bisson and Bond 1971). Because they are probably not a naturally occurring population and their origin is unknown, I excluded them from the analysis. Specimens were only included in the morphological data set if in good condition with a straight body and no damage to fin rays or to the portions of the body which were required for accurate measurements in the morphometric data set. Recently collected specimens were preferred over older specimens due better body condition, more visible coloration, and an increased likelihood of accurate identification by original collector.

A B Figure 2: Collection localities for the genetic (A) and morphological (B) samples used in this study.

Molecular analysis The molecular sequencing and analysis included in this thesis were conducted by Michael Young and his lab at the USFS’s National Genomics Center for Wildlife and Fish Conservation in Missoula, Montana. Genomic DNA was extracted from tissues using QIAGEN DNeasy Blood and Tissue kit (QIAGEN Inc.), following the manufacturer’s instructions for tissue. Each gene was amplified with a suite of standard and custom-designed primers and sequenced using standard Sanger’s sequencing protocols at the University of Washington (Seattle, WA) or Eurofins Genomics (Louisville, KY). Young will coauthor a version of this thesis that will be submitted for peer review and eventual publication. A total of 416 tissue samples were used in the analysis of cytochrome c oxidase subunit 1 (COI). From COI analysis, haplotypes were identified using the online version of CD-HT (Huang et al. 2010), with haplotypes labeled in default order with no relation to relatives. A maximum-parsimony network for COI haplotypes was constructed using the TCS algorithm in the program PopART (Clement et al. 2000; Leigh and Bryant 2015). To create a multigene phylogeny for C. rhotheus, thirty-two tissue samples were sequenced at three additional genes: cytochrome b (cytb), S7, and rhodopsin. CytB represents an additional mitochondrial locus, S7 is a nuclear intron, and rhodopsin is a protein coding nuclear gene. Eight species of Cottus were used as outgroups for the phylogeny. From the Uranidea clade identified by Kinziger et al. (2005), specimens from the western C. bairdii species complex and C. cognatus where chosen. Four inland species were included (C. confusus, C. beldingii, and C. schitsuumsh, C. leiopomus), along with C. bairdii from near its eastern type locality. C. aleuticus and C. asper represented the Cottopsis clade (Kinziger et al. 2005), which was used to root the phylogeny. Sequences of COI, cytb, and rhodopsin were aligned by eye and no indels or stop codons were detected. Sequences of S7 were aligned using the online version of MAFFT with the G- INS-i algorithm (Katoh et al. 2017). Gaps present in the S7 gene were coded using FastGap (Borchsenius 2009) and the gap-coding algorithm from Simmons and Ochoterena (2000) and added to the S7 sequences. The concatenated sequences from the four genes were then constructed into a NEWICK tree. Partitions were identified using PartionFinder2 and implemented by the CIPRES gateway (Miller et al. 2010; Lanfear et al. 2017). This data was then used in a maximum-likelihood analysis in RAxML 8 (Stamakis 2014) as implemented by raxmlGUI 1.5 (Silvestro and Michalak 2012). All partitions were analyzed using GTRGAMMA as the evolutionary model, as RAxML restricts analyses to a single model. This analysis was then bootstrapped (n=1,000) and support values were assigned to each branch of the best-fitting tree.

Morphology Thirty-eight measurements were taken per specimen among twenty landmarks. Twenty- six measurements among twelve landmarks formed the truss system used in Strauss (1991), with an additional twelve supporting measurements capturing variation in eye size, opercular and preopercular morphology, and other elements known or suspected to vary among sculpin species from Buser et al. (2017)(Fig 3). After preliminary analysis, I removed measurements 13-21, 18- 21, 20-21, 10-21, 1-21, and 21-23 from this study due to the variability in gill flaring, hyoid depression, and cranial lifting after euthanasia in older specimens, which distorted those measurements. Each measurement was taken with a Bluetooth caliper and recorded to the nearest .01mm. Bilateral measurements were taken on the left side of specimens. A table of complete measurements can be found in Appendix Table 1. In addition to body morphometrics, meristic and morphological characteristic data were recorded. Prickle pattern, the presence of head nubbles, lateral line completion, and six fin-ray counts were recorded from a subsample of specimens within each identified geographic clade. On sculpin, nubbles are found on the cranium, usually as circular bumps, unlike prickles which are located posterior to the head and represent modified scales. The thirty-two measurements were then log transformed and corrected for size. To correct for size, the log transformed data was regressed against standard length, using the function ‘Allometric vs Standard’ in PAST (Hammer et al. 2011). Using this program, a principal component analysis (PCA) was run using a covariance matrix of the size corrected measurements. This allows for visualization of the axes which maximally separate the individuals in this study. To determine whether diagnosable morphometric differences separate the major clades revealed by molecular phylogenetic analysis, a canonical variates analysis was performed. A 1 15 20 2 4 3 13

7 6 21 8 10 B

15 20 1 2 17 24 23 13 5 16 22 19

11

10 TL C

Figure 3: (A) Lateral view of Cottus rhotheus (OS 22275) displaying 20 land marks used for morphometrics (B) The 26 measurements of a 12 landmark truss system (Strauss 1991). (D) Eleven supporting measurements (Buser et al. 2017). Photographs by Brian Sidlauskas.

Eastern Oregon Specimens I originally planned to include specimens from Eastern Oregon (Grande Ronde and John Day basins) matching the current morphological concept of Cottus rhotheus. However, due to a government shutdown, project collaborator Michael Young lost a month of work and was not able to sequence these individuals for all genes needed to include them in the final phylogeny. Preliminary mtDNA analyses of specimens from these two basins suggested they are more closely related to C. hubbsi, than to C. rhotheus, despite the morphological similarity to the latter. More work and analyses are currently being conducted to investigate and potentially resolve the phenotypic contradiction of the preliminary genetic results. However, meristic and morphological characteristic data from those specimens were included in the results and discussion herein, as these data are available now.

Results Molecular Analysis The analyses of both COI and cytb showed multiple divergent groups within the C. rhotheus species complex. Two main geographic clades split by the Columbia River and Cascade mountains were identified with mtDNA data, with one clade representing specimens from Western Washington and the other containing all other C. rhotheus specimens. Within the larger W. Washington clade, Puget Sound and Coastal Washington subgroups were identified (Fig. 4a). The other clade containing all other C. rhotheus contained three geographic subgroups: Western Oregon, Central Washington and Palouse, and Idaho, Montana, and Eastern Washington (Fig. 4a). Thirty-two haplotypes were identified from the 416 specimens analyzed for COI data. The haplotype network displays and agrees with the five geographic clades identified in the mtDNA analysis (Fig. 4b). Analysis of the nuclear genes (S7 and rhodopsin) in isolation resolved less phylogenetic structure, due to the slower rate of evolution at those loci relative to the mitochondrion. Analysis of rhodopsin sequences from across the range of C. rhotheus identified a clear Western Washington clade, while the remaining C. rhotheus samples formed a paraphyletic grade and shared similar sequences with C. bairdii and C. hubbsi specimens. S7 showed more divergence and reconstructed three geographically distinct clades corresponding to specimens from Western Washington, those from Western Oregon, and those originating east of the Cascades.

COI data

Sculpins_OSU.xlsx

1.0 2.0 3.0 4.0 5.0

E. Washington/Palouse

Idaho/Montana (Type Locality)

W. Oregon

Puget Sound Coastal Washington A

AO E. Washington- Palouse Puget Sound AB

AZ BE AR AP BF AA AQ BA AY AE AF Idaho/Montana BB BC AK AM AJ

AN AS AD AC AL AG AH AT BD AI AX AW W. Oregon Coastal Washington AV B AU Figure 4: A) Results of COI data displayed across the PNW with COI gene tree (N=416). B) A haplotype network displaying geographic clades identified in COI data. Each bar represents a difference of one base pair. C. aleuticus, NR0815

C. asper, NR6638

C. ricei, NR3733

C. beldingii, NR0382

C. beldingii, NR1332

C. beldingii, NR1302

C. leiopomus, NR3613

C. leiopomus, NR0691

C. confusus

C. schitsuumsh

C. cognatus

C. bairdii/hubbsi complex

C. bairdii, Ohio, NR7489

C. rhotheus, Rock Cr, OR NR3930

C. rhotheus, Sain Cr, OR NR3928

C. rhotheus, Sain Cr, OR NR3924

C. rhotheus, South Santiam R, OR NR1707 1

C. rhotheus, Silver Cr, OR NR3939

C. rhotheus, Mary’s R, OR NR6591

C. rhotheus, Cougar Creek, ID NR1966

Bootstrap support C. rhotheus, MF Mill Cr, WA NR77 0-49 C. rhotheus, Spokane R, WA NR7471 2 C. rhotheus, Beaver Cr, WA NR434 50-74 C. rhotheus, NF Palouse R, ID NR7303 C. rhotheus, Big Sand Cr, ID NR1761

75-89 C. rhotheus, Willapa R, WA NR848 90-100 C. rhotheus, Ellsworth Cr, WA NR1342 C. rhotheus, EF Quinault R, WA NR1017

C. rhotheus, Cook Cr, WA NR867

C. rhotheus, Speelyai Creek trib, WA NR773

C. rhotheus, NF Newaukum R, WA NR790

C. rhotheus, Chehalis R, WA NR5909

2 C. rhotheus, Chehalis R, WA NR5276 C. rhotheus, SF Newaukum R, WA NR4468 3 3 C. rhotheus, SF Newaukum R, WA NR2188 C. rhotheus, Big Cr, WA NR4491

C. rhotheus, NF Calawah R, WA NR882

C. rhotheus, Kalaloch Cr, WA NR892

C. rhotheus, Bogachiel R, WA NR2200 1 C. rhotheus, Chehalis R, WA NR6035 C. rhotheus, Crescent Cr, WA NR4199

C. rhotheus, Crescent Cr, WA NR4197

C. rhotheus, Jim Cr, WA NR2055

C. rhotheus, Skykomish R, WA NR1003

C. rhotheus, Taylor Cr, WA NR4644

0.006 Figure 5: Multilocus phylogeny of C. rhotheus and several outgroups and map with geographic clades identified. Collapsed outgroups represent monophyletic outgroups. The multilocus phylogeny produced from partitioned, concatenated analysis of the two mitochondrial and two nuclear genes place all C. rhotheus into a strongly supported clade (Fig. 5). Within this clade, three geographically circumscribed clades emerge: Western Oregon, Western Washington, and Eastern Washington/Idaho/Montana. The individual gene trees for cytb, COI, and S7 all support this topology, and the rhodopsin data agree on the distinctiveness of the Western Washington clade (those data failing to return any other clear structure within C. rhotheus). The phylogeny’s basal division between specimens from W. Washington and those from W. Oregon and east of the Cascades is strongly supported (90% bootstrap), as is the division between W. Oregon specimens and those from east of the Cascades (100% bootstrap). The Columbia River and Cascade Mountain Range separate these three major clades of C. rhotheus in the multilocus phylogeny as genetic barriers. There is little statistical support for the split between the Coastal Washington and Puget sound clades identified in the mtDNA-only analysis.

Morphometric analysis After size-correction of the original variables, the first four axes resulting from the Principal Component Analysis (PCA) summarized 63.9% of the total variance remaining. PC1 accounted for 30.1% of the total variance, with measurements related to the caudal peduncle region (3-4, 3-6, 4-7, 6-7) and eye size (15-16, 17-24) loading most greatly. PC2 accounted for 13.8% of the total variance, with caudal peduncle length loading negatively and eye size (15-16, 17-24), pectoral fin width (11-22), anal fin insertion to dorsal insertion (3-7), and distance from supraoccipital crest to base of first dorsal spine (20-1) loading greatest. A table of the loadings from the first two axes from all measurements can be found in the Appendix Table 3. A scatterplot of these axes, with specimens categorized by the three main clades identified in phylogenetic analysis reveals that C. rhotheus found in W. Oregon have longer and more slender caudal peduncles than do C. rhotheus found in Western Washington or east of the Cascades (Fig. 6). There is little overlap between specimens from Western Oregon and the two other geographic groups in the morphospace (Fig. 6). Conversely, specimens from Western Washington and from east of the Cascades overlap substantially in the morphospace.

Legend W. Oregon W. Washington Idaho, Montana, E. Washington

Figure 6: Morphospace of principal components one and two. Groupings based on multilocus analysis.

Despite their partial overlap in principal component space, the canonical variates analysis successfully separated the three main subclades of Cottus rhotheus as identified by phylogenetic analysis (Fig 7). On the first canonical axis, measurements related to the caudal peduncle (3-4, 3- 6, 6-7, 4-7) and the length between the supraoccipital crest and insertion of the first dorsal spine (20-1) separated the three clades. On the second canonical axis, anal fin length (7-8), measurements related to the caudal peduncle (3-4, 3-6, 4-6), and distance between the end of the dorsal and anal fin (3-7) maximally separated the three clades of C. rhotheus. Loadings for the canonical variates can be found in Appendix Table 3.

Legend W. Oregon W. Washington Idaho, Montana, E. Washington

Figure 7: Canonical variates scatterplot. Groupings based on multilocus phylogeny data.

Meristics Across the entire sampling range, there was no significant difference in fin ray counts separating the clades identified from phylogenetic analysis. Dorsal spines varied from 7-9 in all groups and dorsal rays ranged from 15-17. Pectoral fin ray counts varied between 15 and 17, with asymmetrical pectoral ray numbers occurring in some specimens. All specimens sampled had 12 caudal rays and 4 pelvic rays.

Geographic variation in other morphological characters Across the Torrent Sculpin’s PNW range, lateral line completion, coloration, and prickle patterns, and nubble presence vary. Specimens within a single basin or even at the same collection locality exhibit substantial variation. Eye and head nubbles were present on nearly all specimens across the sampled localities. There was variation in the amount of nubbles in some specimens but that did not translate to noticeable differences between the three geographic clades. In all clades but W. Oregon, juvenile (<40mm SL) C. rhotheus displayed incomplete lateral lines, ending anterior to the caudal peduncle. In W. Oregon, complete lateral lines were observed in juveniles as small as 20mm SL. No other specimens from the other two C. rhotheus clades had a fully developed lateral line before 40mm SL. Specimens from W. Oregon overwhelmingly had lateral lines that ended on the caudal fin. Across the rest of the sampled range, the lateral line ended near or on the caudal fin in adult specimens, with the exception of specimens from Washington’s Cowlitz River. There, lateral lines were mostly incomplete, ending at a vertical through the insertion of the second dorsal fin in adult and juvenile specimens. In other W. Washington drainages, the size at which lateral lines reached completion differed. In the Chehalis River, specimens possessed completed lateral lines at 70mm SL and larger. In the Puget Sound, specimens >45mm SL possessed lateral lines that ended near or on the caudal fin. East of the Cascades, specimens >45mm SL had lateral lines which ended on the caudal fin or right before, except for in the Upper Palouse River where specimens did not develop a complete lateral line until <55mm SL. In some instances, specimens were observed with asymmetrical lateral line terminations where one side of the lateral line would end on the caudal fin and the other just anterior to that point. Prickle patterns in Cottus rhotheus across the Pacific Northwest were highly variable. In Western Oregon, many specimens possessed prickles dorsal and ventral to the lateral line extending from the pectoral fin to caudal peduncle, but in some cases only exhibited prickles medial to the pectoral fin. Specimens from W. Washington had little or no prickling. In the Puget Sound, light prickling was observed medial to the pectoral fin, as well as several individual prickles near the posterior of the second dorsal fin base. In the Chehalis drainage, prickling medial to the pectoral fin was more pronounced and occupied a larger area medial to the pectoral fin in comparison to those from the Puget Sound. Specimens from the Cowlitz drainage mostly had prickles medial to the pectoral fin, but some specimens did exhibit prickling which extended towards the first dorsal saddle, above the lateral line. Specimens in Washington east of the Cascades from the Yakima and Chewuch rivers exhibited body prickling similar to those from the Willamette, with extensive prickles dorsal and ventral to the lateral line, extending posteriorly towards the caudal peduncle. In Idaho and Montana, variable prickle patches were observed with some specimens only having prickles medial to the pectoral fin, but a majority had prickles extending towards the caudal fin. Some rivers within Idaho had specimens which exhibited prickles dorsal and ventral to the lateral line as they extend towards the caudal peduncle while other localities only had prickles dorsal to the lateral line. Specimens from the Upper Palouse River in Idaho had few or no prickles. When prickles were present, they were only found medial to the pectoral fin. All specimens from the Willamette basin exhibited two well defined, darkly pigmented dorsal saddles that extended below the lateral line. Similar to specimens from W. Oregon, specimens in E. Washington also possessed clearly defined dorsal saddles that extended below the lateral line. In W. Washington, the two dorsal saddles appeared more distinctly in juveniles than in adult specimens. Greater variation was present in saddle definition and completion across adults in W. Washington, with some saddles barely defined below the lateral line or taking a mottled pattern in some instances. Further east in Idaho and Montana, dorsal saddle definition, width, and number varied among specimens. Some specimens possessed two skinny (2 dorsal rays wide), saddles while others had thicker saddles (3-5 dorsal rays wide). In some instances, coloration was present between the two saddles, almost indicative of a third saddle, but not as dark or well defined. In the Upper Palouse River, the dorsal saddles were poorly defined across all specimens, with many specimens possessing more of a mottled pattern. A majority of the specimens showed evidence of a third saddle which was barely visible and usually asymmetric.

Eastern Oregon Specimens Specimens collected from the John Day basin and Grande Ronde basins in Eastern Oregon turned out to possess mitochondrial DNA sequences most closely matching specimens assigned to C. hubbsi (part of the C. bairdii species complex), despite exhibiting morphological traits similar to those of C. rhotheus. In this region, most larger specimens exhibited complete lateral lines ending right before or on the caudal fin, however asymmetry in the lateral-line endpoint existed. In specimens smaller than 30mm SL, lateral lines were incomplete. Prickling varied by specimen size, with smaller specimens exhibiting extensive prickles above and below the lateral line, from the pectoral fin to caudal peduncle. In large specimens, prickling was most prevalent dorsal to the lateral line with a lesser number of prickles ventral to the lateral line extending towards the caudal fin. Prickling medial to the pectoral fin was also most prevalent in larger specimens. Head and eye nubbles were present on all specimens. Dorsal saddles were strongly defined dorsal and ventral to the lateral line in specimens smaller than 60mm SL. Specimens larger than 60mm SL exhibited varying dorsal saddle completion ventral to the lateral line. Similar to C. rhotheus in Idaho, some specimens had evidence of a third saddle between the two prominent dorsal saddles. In many cases, the third saddle only existed on one side of the specimen.

Discussion

How many species actually exist within the present concept of Cottus rhotheus?

Three cryptic species appear to exist within the current concept of Cottus rhotheus. Mitochondrial DNA analysis revealed five clades across C. rhotheus’ Pacific Northwest range, however, the multilocus phylogeny showed strong support for only three geographically circumscribed clades and did not strongly support any subdivisions within the Western Washington clade. Morphological analysis further supported the three clades from the multilocus phylogeny, with the canonical variate analysis showing no morphological overlap among clades, signifying that morphological differences diagnose and separate all three. Under the universal species concept proposed by De Queiroz (2007), the only qualifier for a species is that it is a separately evolving metapopulation. Other species concepts can then be used to support that species’ existence. This study identified three separately evolving metapopulation lineages across C. rhotheus’ traditional range, each of which is supported by multiple species concepts. The three identified clades all differ genetically, are reciprocally monophyletic, are geographically and genetically isolated from one another, and possess distinguishing phenotypic differences. In following the evolutionary species concept, the three geographic clades have maintained their evolutionary identity, separate from the other two lineages (Wiley 1978). Following the second corollary of the evolutionary species concept, all three clades have remained reproductively isolated from one another, as is evident by no shared haplotypes among the clades and well supported clades within a multilocus phylogeny. The third corollary of the evolutionary species concept does not require morphological differentiation between species but can be used to further support the diagnoses of a species. Wiley (1978) states that the true evolutionary lineages may not be able to be perceive by investigators, but this does not disregard their existence, nor do morphological differences always prove separate evolutionary lineages (i.e. sexual dimorphism). Morphological or phenetic differences should be used in combination with geographic distribution to form a hypothesis for independent evolutionarily lineages. In addition to the evolutionary species concept, the phenetic species concept can be used to in place of the third corollary, which explicitly states that separately evolving lineages do exhibit phenetic differences (Wiley 1978). The Columbia River and Cascade Mountain range seem to serve as genetic barriers for C. rhotheus. The greatest evidence for the Columbia River serving as a genetic barrier occurs in Portland Oregon and Vancouver Washington. Specimens were collected on both sides of the Columbia River, less than twenty miles apart, and shared no mtDNA haplotypes and belonged to distinct clades in the multilocus analysis. It is important to note that seventy-seven of eighty specimens from W. Oregon in the genetic analysis were found upstream of Willamette Falls, a natural waterfall twenty miles from where the Willamette River enters the Columbia River. These falls therefore could also be acting as a genetic barrier. Nevertheless, the presence of three specimens in the Western Oregon clade from below the falls suggests the permeability of that barrier, though perhaps only in the direction of downstream descent. Due to the relatively low dispersal ability and small home ranges of many Cottus species, Torrent Sculpin found in Montana and Idaho have a relatively low likelihood of migrating to other basins and breeding with individuals who reside in Western Oregon or Western Washington (Bailey 1952, Greenberg et al. 1987, Breen et al. 2009). While the home range or dispersal abilities of C. rhotheus have never been tested or observed, many other cottids have been observed to be extremely sedentary. Cottus carolinae was found to have one of the smallest home ranges at 47m (Greenberg et al. 1987). Across the Pacific Ocean in Japan, C. pollux was has a home range between 6.8m and 67.9m (Natsumeda 1998). In Michigan, a study found that 84% of C. bairdii specimens moved less than 100m in a year, with the maximum distance traveled being 511 m (Breen et al. 2009). Another study tracking C. bairdii movement in North Carolina found that adults moved only a single meter on average over 150 days (Petty and Grossman 2004). Other studies of C. bairdii have found home ranges ranging between 45.7m and 143.3m (Bailey 1952 and McCleave 1964). Similar movements and home ranges are expected with C. rhotheus (Bond 1963). A sedentary lifestyle may not be the only thing preventing Cottus rhotheus from dispersing to other basins or drainages. C. rhotheus prefers swifter moving water (>3 ft/s) and water temperatures under 23º C (Bond 1963). The lower reaches of rivers are usually characterized by warmer and slower moving water in comparison to the middle and upper reaches (Crushing and Allan 2001). The lower Willamette River in Portland Oregon occasionally exceeds 23º C in July and August, but rarely sees flow rates above 2 ft/s (US Geological Survey 2019). C. rhotheus also prefers cobble and gravel as a substrate, which are more common in the middle and upper portions of rivers (Crushing and Allan 2001). While these conditions may not physically or physiologically prevent C. rhotheus from moving downstream and dispersing, they may deter or discourage them from doing so. Headwater stream transfers are also an unlikely mode of dispersal for C. rhotheus in W. Oregon, as they prefer streams wider than 10 feet and are primarily observed in the middle reaches of rivers (Bond 1963).

Type Locality The type locality of C. rhotheus is located in Spokane Washington falls within the geographic region occupied by the E. Washington clade (Group 2, Fig. 5), including populations in the Upper Palouse, Idaho, and Montana. With agreement between morphological and genetic data, C. rhotheus specimens from Western Oregon, primarily the Willamette Basin, and those from Western Washington likely represent two new, unnamed cryptic species nestled within the Cottus rhotheus species complex. Presently, there are no described species in the synonymy of C. rhotheus which could be elevated to species status. The W. Washington and W. Oregon clades will require a formal species description and new name.

Which morphological characteristics reliably diagnose clades within Cottus rhotheus? Morphometric analysis across Cottus rhotheus’ range revealed differences in caudal peduncle morphology, anal and dorsal fin endpoints, and anal fin length. Cottus rhotheus found in Western Oregon (primarily from the Willamette Basin) exhibit a narrow and elongate caudal peduncle, separating them from the other two geographic clades which possess shorter and wider caudal peduncles. Specimens from Western Washington had greater distances between the supraoccipital crest and base of the first dorsal ray, wide caudal-fin bases, and longer anal-fin bases. Populations east of the Cascades and north of the Columbia River exhibited a greater distance between the anal-fin insertion and dorsal rays, along with shorter heads. Differences between the three clades were statistically found and not obvious to the eye without closer examination of specimens. In addition to the thirty-two measurements taken from body landmarks, observable characteristics such as prickles, eye nubbles, dorsal saddles, and lateral line terminus were recorded to determine if other diagnosable features existed between the three geographic clades. These features are commonly used in papers or identification guides to help identify C. rhotheus and noted to vary in some portions of the range (Bailey and Dimick 1949; Markle and Bond 1996; McPhail 2007; Wallace et al. 2013). Variations of these characters existed among the three clades but also within them in some cases. The variability within clades prevents these characters from being used to reliable diagnose the clades in addition to the observed differences in body morphology. Prickling among and in river drainages sampled varied greatly and could not be used reliably to diagnose clades. Cottus rhotheus is known to possess heavy prickling across its range (Markle and Bond 1996; Wallace et al. 2013), with the exception of those in W. Washington (Bailey and Dimick 1949). Absent or reduced prickling was commonly found in the specimens from W. Washington, however expections did exist where some specimens were extensively prickled. Conversely, localities from W. Washington and east of the Cascades where specimens primarly exhibit full body pricklying had specimens where prickles we absent or greatly reduced. Eye and head nubbles were found not to be a diagnostic characteristics as they were present across all three clades in C. rhotheus’ range. Among the three clades, the only diagnosable difference between lateral line end point was the size at which a specimen had a completed lateral line. The W. Oregon clade appears to develop a lateral line much smaller (>20mm) than those in W. Washington (45-70mm) or east of the Cascades (>45mm). The possession of a complete lateral line onto the caudal fin historically diagnoses C. rhotheus across the Pacific Northwest (Markle and Bond 1996), with an exception noted in Bailey and Dimick (1949) that specimens in W. Washington had lateral lines which ended short of the caudal fin. Nearly every specimen >60mm examined in this thesis had a completed lateral line onto the caudal fin or ending right before. No single clade had lateral lines which always ended on the caudal fin. Lateral line termini were mostly consistent in W. Oregon but did not always end on the caudal fin. Greater variation between lateral line termini existed in W. Washington and east of the Cascades. Dorsal saddle definition and completion were not reliable in diagnosing the proposed clades as they varied within and among clades. The common primary diagnostic used for C. rhotheus when quickly identifying a specimen in the field is to look for the two prominent dorsal saddles. Specimens from W. Oregon had well defined saddles which extended dorsal to the lateral line. This was also observed in those from W. Washington, however other specimens east of the Cascades were much more variable, with varying saddle definition and number in some cases. In some specimens, one side of the body exhibited three saddles, while the other would show only two. Even within the same collection location saddle definition and completeness was drastically variable.

The Uniqueness of the Upper Palouse River Cottus rhotheus The population of C. rhotheus found in the Upper Palouse, which is seemingly isolated from other populations of east of the Cascades, possess morphological characteristics differ substantially from those of other C. rhotheus found east of the Cascades, such as those from near Yakima and Spokane. Those specimens possessed nearly complete or completed lateral lines in adults, extensive prickling towards the caudal fin, and two well defined dorsal saddles, whereas geographically proximate specimens from the Upper Palouse exhibit reduced or nonexistent prickle patterns and two or three poorly defined saddles. Cottus rhotheus in the Palouse River are separated from the Snake and Columbia drainage by 200’ waterfall. In a 1980 survey of the Palouse River, C. rhotheus was the only species found exclusively above the falls in the upstream portions (Maughan et al. 1980). It has been known that this population of C. rhotheus differs in morphological characteristics from other Idaho populations (Bailey and Dimick 1949; Maughan et al. 1980). Their body morphometrics are however is indistinguishable from other C. rhotheus found in Idaho and Eastern Washington. In analysis of mitochondrial DNA, specimens from E. Washington and the Upper Palouse formed their own clade (from the multilocus phylogeny: Beaver Creek NR434, NF Palouse NR7303, Big Sand Cr NR1761), however, were joined with the rest of Idaho and Montana in nuclear analysis. In the haplotype network, Upper Palouse specimens had the greatest separation of base pair substitutions from the other specimens east of the Cascade. Perhaps this unique isolated population has been subjected to the founder effect and unique environmental conditions which have selected for different prickle and saddle patterns from other populations of C. rhotheus found east of the Cascades.

What is causing variation in body morphology, prickling and dorsal saddles Habitat characteristics may play a role in shaping body morphology in sculpin. In a study of C. carolinae, it was found that two river habitats correlated to two different body morphologies (Kerfoot and Schaefer 2006). In rivers with higher flows, sculpin were more streamlined with deeper and skinnier caudal peduncles. Habitat linked phenotypic plasticity likely exists within many Cottus, however the degree to which it is responsible for the morphological differences observed in this thesis is unknown. Across each of the identified clades, multiple localities from different rivers were sampled. Kerfoot and Schaefer (2006) did not include any genetics in their study, which may have shed light on whether or not populations from either are connected or interbreeding. If evidence suggested that C. carolinae freely moved between streams and there were no genetic difference between populations in the two stream habitats, there would be strong evidence that Cottus body morphology is high linked to environmental variables. A common garden experiment including the three clades and several different stream rearing habitats would be required to test the degree of habitat linked phenotypic plasticity in C. rhotheus. Prickling in sculpin has been used before as a character to help distinguished closely related sculpins (Markle and Hill 2000). However, there is evidence of prickle count being related to ecotypes in British Columbia in populations of C. hubbsi and C. rhotheus (McPhail 2007; COSEWIC 2010). Observations among the three clades identified in the multilocus phylogeny showed variation occurring at some localities within the same clade. For example, east of the Cascades, two clades were identified from mtDNA (Fig. 4a) and one in the multilocus phylogeny (Fig. 5). Even between within one of the mtDNA clades variation in prickles was present. It is likely that environmental factors play a role prickle pattern. However, more work needs to be done to assess the variability of prickle counts in Cottus to examine whether variability is truly caused through environmental variables and to what degree. Coloration in sculpin has also been proven to be environmentally controlled in one species of Cottus. In C. aleuticus, it has been documented that their coloration is plastic and varies between different streams in Alaska (Whiteley et al. 2009). In laboratory experiments, C. aleuticus from different rivers were all place in a tank similar substrate and were observed converging towards similar colorations. It was determined that the differentiation in their coloration was mostly due to substrate composition, not genetic differentiation (Whiteley et al. 2009). Substrate composition across habitats within the clades of C. rhotheus could affect saddle definition, completion, or even number. With variation seen within sampling localities, there is evidence that C. rhotheus may exhibit some degree of plasticity with its dorsal saddles. The W. Oregon clade of C. rhotheus exhibited the least amount of variation in dorsal saddles, but the reasons of this cannot be determined, whether it be from consistency of substrate across its range genetically determined. Laboratory experiments would need to be conducted on specimens across the three clades in a variety of different laboratory streams to test their saddle plasticity.

Future Work Future work on this project will formally describe the two cryptic species discovered in our genetic and morphometric analyses. Those species descriptions detail the geographic ranges of the two new species ranges, their morphological attributes, and any available ecological information. This thesis was not able to fully sample the entire range of this species complex genetically or morphologically. If possible, further genetic sampling should focus on the coastal rivers in Oregon which have C. rhotheus as C. rhotheus is thought to be intolerant to salt water (Bond 1963), and may represent further isolated populations. It is unlikely any of the species within the C. rhotheus species complex will warrant conservation status or attention due to the large ranges each of our identified clades occupies. Future work should also further investigate the degree to which local environmental conditions drive morphological variation within Cottus. Testing the degree to which environmental and genetic factors affect prickle extent, lateral line development, coloration, and body morphology could shed light onto how to diagnose and delimit species in other species complexes which exist within Cottus. Additional work also needs to be done concerning the phenotypic Cottus rhotheus from the Grande Ronde and John Day basin. The specimens sampled from these basins were genetically placed into the C. hubbsi/C. bairdii species concept, raising concern about the ability to morphological distinguish these two species apart where they cooccur. Many of the diagnostic characteristics (dorsal saddles, lateral line, and prickles) which have been used to diagnose C. rhotheus from C. hubbsi are extremely variable between basins and sometimes between individuals of the same collection locality. In some instances, several specimens collected in the Grande Ronde had diagnostic characteristics of C. hubbsi on one side of their body, while the other side was diagnosable as C. rhotheus. For example, some specimens exhibited differing endpoints of the lateral line and differing number of saddles on each side. The prickle patterns were also observed as an intermediate between the two, where prickles did not cover the entire fish in adults, nor were they restricted to solely medial to the pectoral fin. All specimens collected from these two basins showed a majority of diagnosable characters identifiable as C. rhotheus, if not all. It is possible that specimens from these basins have retained an ancestral morphology which closely resembles C. rhotheus. Continuing work on this basin should include further genetic analysis and morphological observation to better understand why traditional distinguishing morphological features did not distinguish genetically distinct clades in this geographic region.

Conclusion Morphological and genetic analysis identified the presence of three cryptic species in the Cottus rhotheus species complex. In W. Washington and W. Oregon, two geographic clades differed genetically and morphologically from populations east of the Cascades where the type locality of C. rhotheus is located. They will need to be formally described. The Cascade Mountain Range and Columbia River appear to act as genetic barriers separating these species. This thesis also sheds some light on future areas which should be studied to better understand the genus Cottus, one of the most speciose groups in the PNW. Little information currently exists about their ecology, life history, or population structures in the PNW. More work should also focus on the variability of morphology and morphological characteristics in sculpin, as it will help create better keys for identification and shed light on whether coloration and prickles are characteristics are determined genetically or environmentally. In fisheries management and conservation, sculpin are usually an afterthought due to their seemingly high abundance, large ranges, and inability to drive license sales. A further in depth look into other Cottus and fishes in the PNW may yield similar findings of undiscovered biodiversity nestled in the Pacific Northwest. Detecting cryptic and undiscovered species is important for our understanding of evolution, biogeography and necessary for biodiversity conservation.

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APENDIX

Table 1: Vouchers Specimens used for morphology Number of ID Museum Locality State Latitude Longitude Specimens College of 4 St. Maries River ID CIDA 68,105 Idaho 47.051217 -116.2878 College of Mannering 4 ID CIDA 68,556 Idaho Creek 47.017683 -116.678883 College of 2 Big Creek ID CIDA 68,560 Idaho 47.00165 -116.7563 College of 5 Jerome Creek ID CIDA 68,578 Idaho 46.956667 -116.732917 College of 6 Merry Creek ID CIDA 68,606 Idaho 47.016333 -116.22585 College of 4 Santa Creek ID 47.155533 -116.515883 CIDA 68554 Idaho College of Wolf Lodge 4 ID 47.673933 -116.607033 CIDA 69,169 Idaho Creek College of 2 Spokane River ID 47.7025 -116.978 CIDA 69,411 Idaho College of South Fork Mica 1 ID 47.597783 -116.88685 CIDA 69133 Idaho Creek College of 2 Cougar Creek ID 47.638233 -116.915083 CIDA 69134 Idaho College of North Fork 1 ID 47.111117 -116.444767 CIDA 70,733 Idaho Tyson Creek College of 3 Renfro Creek ID 47.152983 -116.435200 CIDA 81,700 Idaho College of 2 Palouse River ID 46.91525 -116.9511 CIDA 81,989 Idaho College of North Fork Mica 1 ID 47.598038 -116.897111 CIDA 101,497 Idaho Creek Little Oregon State OS 16432 1 Luckiamute OR 44.79771 -123.2944 University River Oregon State 2 Willamette River OR OS 18427 University 44.86317 -123.175506 Oregon State 10 Trout Creek OR OS 5857 University 45.041448 -122.447803 Oregon State 4 Willamette River OR OS 6897 University 44.566959 -123.254837 Oregon State 2 Willamette River OR OS 18428 University 44.95 -123.04 Oregon State 2 Willamette River OR OS 18448 University 44.85 Oregon State 2 Willamette River OR OS 19226 University 43.7067 -122.296 Oregon State 4 Salt Creek OR OS 19237 University 43.6505 -122.215 Oregon State South Santiam OS 19265 1 OR 44.51664 -122.86285 University River Oregon State OS 19266 1 Klaskanine river OR N/A N/A University Oregon State OS 22227 1 Mary’s River OR 44.553 -123.274 University Oregon State OS 22229 3 Mary’s River OR 44.5533 -123.277 University Pacific East Fork Lutheran 8 WA Satstop River PLU F0679 University 47.047915 -123.514215 University of Pleasant Valley 10 MT UMMZ 164956 Michigan Fisher River 48.041241 -115.287852 University of Pleasant Valley 6 MT N/A N/A UMMZ 173937 Michigan Fisher River University of 4 Skookumchuck WA UMMZ 179392 Michigan 46.728133 -122.970598 University of 10 Cowlitz River WA UMMZ 180382 Michigan 46.576304 -121.704014 University of 6 Mill Creek WA UMMZ 180387 Michigan 46.531216 -122.617029 University of 8 Chewuck River WA UW 20167 Washington 48.569335 -120.172415 University of Cloquallum 7 WA UW 111081 Washington Creek 46.9861 -123.3931 University of North Fork 4 WA UW 112388 Washington Porter Creek 46.9889 -123.2258 University of Apple Tree 8 WA UW 112408 Washington Creek 46.8775 -123.2236 University of 4 Deschutes River WA UW 152214 Washington 46.8605 -122.716 University of 2 Deschutes River WA UW 152220 Washington 46.8316 -122.543

Table 2: Measurement Descriptions

Measurement Descriptions 13-5 Total length (Anterior tip of premaxilla to beginning of caudal fin) 1-2 Base of first dorsal spine to the base first dorsal ray. 2-3 From the base of the first dorsal ray to the base of the 2nd to last dorsal ray. The last dorsal ray was not the chosen endpoint because the final dorsal ray is small and reduced and was usually missing or damaged in smaller specimens. 3-4 From the base of the 2nd to last dorsal ray to the base of the first visible procurrent ray 4-6 Width of caudal fin 6-7 Lower caudal fin base to base of last anal ray 8-7 Base of first anal ray to base of last anal ray 8-10 Base of first anal ray to base of pectoral spine 13-15 Anterior tip of premaxilla to middle of the skull, at the top of orbital 15-20 Anterior tip of premaxilla to supraoccipital crest 20-1 Supraoccipital crest to base of first dorsal spine 13-20 Front of premaxilla to supraoccipital crest 20-21 Supraoccipital crest to basihyal-hypohyal junction 20-10 Supraoccipital crest to base of pectoral spine 1-10 Base of first dorsal spine to base of pectoral spine 1-21 Base of first dorsal spine to basihyal-hypohyal junction 2-10 Base of first dorsal ray of second dorsal fin to base of pectoral spine 1-8 Base of first dorsal spine to base of first anal ray 2-8 Base of first dorsal ray to base of first anal ray 2-7 Base of first dorsal ray to last anal ray 3-8 Base of the 2nd to last dorsal ray to base of first anal ray 3-7 Base of the 2nd to last dorsal ray to base of last anal ray 3-6 Base of the 2nd to last dorsal ray to base of lower caudal ray 4-7 Base of upper caudal ray to base of last anal ray 2-5 Base of first dorsal ray to beginning of caudal fin 10-5 Base of pectoral spine to beginning of caudal fin 17-24 Orbital width 15-16 Orbital height 13-19 Anterior tip of premaxilla to tip of largest preopercle spine 13-23 Anterior tip of premaxilla to tip of opercle spine 20-23 Supraoccipital crest to tip of opercle spine 1-22 Base of first dorsal spine to top of pectoral fin base 11-22 Width of pectoral fin base 2-22 Top of pectoral fin base to base of first dorsal ray

Table 3: PCA and Canonical Variate Loadings. Measurements most discriminating are bolded. Measurement PC 1 PC 2 CV1 CV2 13-18 0.031 0.245 0.0028 0.0049 13-20 -0.011 0.064 -0.0001 0.0013 18-20 -0.002 0.058 -0.0009 -0.0018 20-1 0.030 0.416 0.0110 -0.0072 20-10 -0.070 0.183 0.0055 0.0058 1--2 0.025 0.097 0.0058 -0.0048 1--8 -0.020 0.088 0.0020 0.0020 1--10 -0.025 0.149 0.0036 0.0003 2--3 -0.063 0.063 0.0033 0.0010 2--8 -0.032 0.179 0.0042 -0.0008 2--10 -0.089 -0.011 0.0041 -0.0054 2--7 -0.024 0.016 0.0019 -0.0039 3--4 0.618 -0.190 -0.0192 -0.0168 3--6 0.502 -0.103 -0.0131 -0.0163 3--7 -0.051 0.278 0.0045 0.0093 3--8 -0.095 0.067 0.0065 -0.0006 4--6 -0.010 0.201 0.0075 -0.0137 4--7 0.216 -0.038 -0.0086 -0.0055 6--7 0.265 -0.068 -0.0087 -0.0062 7--8 -0.021 0.014 0.0049 -0.0076 8--10 0.063 -0.079 -0.0058 -0.0011 2--5 0.077 -0.048 -0.0031 -0.0034 10--5 0.039 -0.035 -0.0014 -0.0023 17-24 (Eye) 0.395 0.431 -0.0020 -0.0045 15-16 (Eye) 0.210 0.296 -0.0008 -0.0007 13-19 -0.029 0.099 -0.0003 0.0030 13-23 -0.028 0.121 0.0003 0.0030 20-23 -0.024 0.261 0.0022 0.0064 1--22 -0.004 0.094 -0.0019 0.0032 11--22 0.045 0.294 0.0028 0.0049 2--22 0.011 0.070 -0.0001 0.0013 13-23 0.031 0.245 -0.0009 -0.0018 20-23 -0.011 0.064 0.0110 -0.0072 11--22 -0.002 0.058 0.0055 0.0058 2--22 0.030 0.416 0.0058 -0.0048