Evolutionary Applications

Evolutionary Applications ISSN 1752-4571

ORIGINAL ARTICLE

Genes predict long distance migration and large body size in

a migratory fish, Pacific lamprey
Jon E. Hess,1 Christopher C. Caudill,2 Matthew L. Keefer,2 Brian J. McIlraith,3 Mary L. Moser4 and
Shawn R. Narum1
1 Columbia River Inter-Tribal Fish Commission, Hagerman, ID, USA
2 Department of Fish and Wildlife Sciences, College of Natural Resources, University of Idaho, Moscow, ID, USA
3 Columbia River Inter-Tribal Fish Commission, Portland, OR, USA
4 Fish Ecology Division, Northwest Fisheries Science Center, National Marine Fisheries Service, National Oceanic and Atmospheric Administration,
Seattle, WA, USA

Keywords Abstract
anadromous fishes, association study,
hydrosystem, migratory species, translocation. Elucidation of genetic mechanisms underpinning migratory behavior could help
predict how changes in genetic diversity may affect future spatiotemporal distri-
Correspondence bution of a migratory species. This ability would benefit conservation of one such
Jon E. Hess, Columbia River Inter-Tribal Fish declining species, anadromous Pacific lamprey (Entosphenus tridentatus). Non-
Commission, 3059-F National Fish Hatchery
philopatric migration of adult Pacific lamprey has homogenized population-level
Rd, Hagerman, ID 83332, USA.
neutral variation but has maintained adaptive variation that differentiates groups
Tel.: (208)837-9096, ext. 1107;
fax: (208)837-6047; based on geography, run-timing and adult body form. To investigate causes for
e-mail: [email protected] this adaptive divergence, we examined 647 adult lamprey sampled at a fixed loca-
tion on the Columbia River and radiotracked during their subsequent upstream
Received: 23 June 2014 migration. We tested whether genetic variation [94 neutral and adaptive single
Accepted: 17 August 2014 nucleotide polymorphisms (SNPs) previously identified from a genomewide
association study] was associated with phenotypes of migration distance, migra-
doi:10.1111/eva.12203
tion timing, or morphology. Three adaptive markers were strongly associated
with morphology, and one marker also correlated with upstream migration dis-
tance and timing. Genes physically linked with these markers plausibly influence
differences in body size, which is also consistently associated with migration dis-
tance in Pacific lamprey. Pacific lamprey conservation implications include the
potential to predict an individual’s upstream destination based on its genotype.
More broadly, the results suggest a genetic basis for intrapopulation variation in
migration distance in migratory species.

tory species that have unpredictable movement (e.g., do

Introduction
not home to their natal site).
Genetic mechanisms underpinning various aspects of Genetic tools to help predict various aspects of migra-
migratory behavior have been discovered in animals that tion could benefit conservation of one such species, anad-
traverse great distances and display precise homing ability romous Pacific lamprey (Entosphenus tridentatus). Recent
(e.g., timing, sun compass orientation, and propensity to genetic surveys indicate that the species has seemingly
migrate; Zhu et al. 2009; Hecht et al. 2013; O’Malley et al. nonphilopatric migration of adults from ocean feeding
2013). Characterization of particular genetic traits may sites to freshwater spawning sites based on homogenized
increase our ability to predict the spatial and temporal dis- neutral variation across broad geographical regions (Spice
tribution of migratory species and could thus increase et al. 2012; Hess et al. 2013). Yet this species maintains
effectiveness of the management of such species, many of adaptive variation that differentiates groups based on
which play key cultural and economic roles in our society. geography, run-timing, and adult body form (Hess et al.
Effective management is particularly challenging for migra- 2013). Severe declines in abundance of Pacific lamprey

1192 © 2014 The Authors. Evolutionary Applications published by John Wiley & Sons Ltd. This is an open access article under the terms of the Creative
Commons Attribution License, which permits use, distribution and reproduction in any medium, provided
the original work is properly cited.
Hess et al. Pacific lamprey migration genetics

have occurred throughout its range in the Pacific North- types of lamprey at Willamette Falls may be confounded
west of the USA, including the Columbia River Basin by life-history diversity unique to that tributary. Specifi-
(Close et al. 2002), where multiple anthropogenic factors cally, that early-migrating, short-bodied lamprey may be
(e.g., artificial barriers and past extermination practices) primarily composed by lamprey that have overwintered in
likely contributed to the species’ extirpation in various the river, as opposed to those that have recently entered
tributaries of the interior (Close et al. 2009). These freshwater (Clemens et al. 2010; Hess et al. 2014a). There-
declines have prompted trap and haul translocation efforts fore, it is not clear whether association with length or pas-
as a conservation strategy to help recover Pacific lamprey sage timing explained the genetic divergence observed on
abundance and restore its key role in the ecosystem while a larger scale (Columbia River Basin). A more compre-
providing sustainable harvest in the interior Columbia hensive examination of these potential correlations was
River (Close et al. 2009; Ward et al. 2012). The minimal warranted.
neutral population structure may be a factor that aids Bonneville Dam is the first dam anadromous fish
widespread adoption of a translocation-type strategy (i.e., encounter as they migrate from the Pacific Ocean to the
transporting individuals from a source population to a interior of the Columbia River (river km 235), and it pro-
recipient site) because it precludes need for consideration vides an appealing site at which to collect, measure vari-
of one potential risk of this strategy (i.e., disruption of able phenotypic and genetic traits, and track the
population structure; Weeks et al. 2011). Yet, what would movement of Pacific lamprey through the interior Colum-
be the management implications of pronounced adaptive bia River. This site houses an adult fish facility where Paci-
divergence in this basin? What fitness effects (if any) fic salmonids (Oncorhynchus spp) and Pacific lamprey are
would occur if translocations were to alter the frequencies routinely sampled to address dam passage questions (e.g.,
of particular adaptive genetic variants in different regions Moser et al. 2002a,b; Johnson et al. 2012) and to perform
of the basin? How might adaptive gradients influence the genetic monitoring of stock-specific abundance and run-
effectiveness of translocations? Some of these adaptive loci timing (e.g., Hess et al. 2014b). Further, this location coin-
have been observed to display dramatic shifts in minor cides with a steep genetic transition observed for many
allele frequencies (MAFs) between the lower Columbia adaptive loci in Pacific lamprey (Hess et al. 2013, 2014a).
River and interior Columbia River (Hess et al. 2014a), Finally, past work by Keefer et al. (2009, 2013a) has noted
which represent the source and recipient regions for trans- an important interaction between the size of lamprey and
location, respectively. Therefore, the probability for alter- migration behavior, in that relatively large-bodied Pacific
ing adaptive allele frequencies via Pacific lamprey lamprey typically arrive earlier at Bonneville Dam and tra-
translocation is high, creating added incentive to gain an vel further upstream than small-bodied lamprey in the
understanding of the mechanisms driving this adaptive same year. Unlike the Willamette River, an interaction
divergence and how it relates to the biology of this migra- between size and migration timing is less likely to be con-
tory species. founded by effects of overwintering behavior prior to col-
Hess et al. (2013) identified 162 of 4439 quality-filtered lection, which is rare (<5%) among adults collected at
restriction site–associated DNA sequencing (RAD) single Bonneville Dam.
nucleotide polymorphisms (SNPs) as FST outliers. Further, The primary objective of this study was to test for SNPs
significant linkage among pairs of loci made it possible to associated with migration distance, migration timing, and
categorize the adaptive loci into four groups of linked morphology using individual-based data gathered from
loci, and a subset of four SNPs were selected such that Pacific lamprey captured at Bonneville Dam. Any locus
each SNP represented one of the four groups of linked identified as being significantly associated with one or more
loci (Hess et al. 2013). These four SNPs and five other of these classes of predictor variables was then included in
adaptive SNPs were incorporated into a marker panel a multivariate analysis to determine the combination of
optimized for multiple conservation applications, includ- predictor variables that best explained observed genetic
ing characterization of adaptive variation (Hess et al. variation. We also pursued a secondary objective as a way
2014a). Hess et al. (2014a) showed that body lengths of to relate our primary findings to the translocation strategy
Pacific lamprey captured at Willamette Falls (Willamette being used in conservation efforts for this species. Our sec-
River, OR, USA) significantly correlated with the MAF ondary objective was to determine whether a set of Pacific
of two adaptive loci (Etr_5317, Etr_1806), and run-timing lamprey collected at Bonneville Dam for translocation were
of these lamprey is also significantly correlated with one distinguished morphologically and genetically from Pacific
of these same markers (Etr_1806). Further, these two loci lamprey either selected randomly at different points in the
displayed a steep gradient in MAF between the lower migration season, or from Pacific lamprey known to have
Columbia River and interior Columbia River. However, volitionally migrated to monitoring stations near the trans-
observed correlations between these two loci and pheno- location site.

© 2014 The Authors. Evolutionary Applications published by John Wiley & Sons Ltd 7 (2014) 1192–1208 1193
Pacific lamprey migration genetics Hess et al.

the untagged Pacific lamprey of all sizes was used for trans-
Methods
location to the Umatilla River (Close et al. 2009). This
Pacific lamprey collection, tagging, and monitoring study also utilized data from Hess et al. (2014a) who cap-
Adult Pacific lamprey were collected inside fishways at tured 150 adult Pacific lamprey at the entrance to the fish
Bonneville Dam on the Columbia River (46°N, 124°W; ladder at Willamette Falls (Fig. 1): a random sample of 50
Fig. 1) using passive traps that were deployed at night from Pacific lamprey taken from three time periods (2-week
early June through early September, 2008–2010. Details of intervals) at peaks in the multi-modal abundance distribu-
trapping, anesthesia, and surgical methods were described tion observed in 2012 (April–August). Hess et al. (2014a)
by Moser et al. (2002a,b) and Johnson et al. (2012). All fish recorded passage timing in units of statistical week of col-
were anesthetized (60-ppm eugenol solution) measured lection and measured each individual for total body length
(total length and girth at first dorsal insertion to the nearest (mm).
mm), weighed (nearest g), and scanned with a Distell Fat- For Bonneville Dam tagging, a uniquely coded radio
meter (Distell Industries, Ltd., Lothian, Scotland; Crossin transmitter (Model NTC-4-2L, 8 9 18 mm, 2.1 g; Lotek
and Hinch 2005). Lamprey with girth >9 cm were radio- Wireless, Newmarket, ON, Canada) was placed inside the
tagged in approximate proportion to the run in each year body cavity with its trailing antenna threaded through the
and sampled fish were randomly selected from those that body wall using a cannula or catheter. A secondary tag, a
were trapped insomuch as possible. In 2008, a portion of 4 9 32 mm half-duplex passive integrated transponder

British Columbia 70°N

Canada
50°N
N United States
Washington
48°N 0 1500
' "
30°N Km
Mexico
Priest
140°W 120°W 100°W 80°W
Rapids
Latitude

Dam
Yakima R.
Snake R.
Columbia
River
John Day
Ice Harbor
R. kitat

Bonneville Dam
Dam Dam
Klic

46°N
R.

Pacific McNary Umatilla R.

od

r.

Ocean Dam
R.

eC
Ho
ette

mil

Willamette
llam

een

N Falls John Day R. Idaho

Fift
Wi

The
.
sR

Dalles
ute

Dam
44°N
sch
De

Oregon
0 200 400
Km

124°W 122°W Longitude 120°W 118°W 116°W

Figure 1 Map of study area. The fate categories included below Bonneville Dam, Bonneville Dam to John Day Dam (within main stem or secondary
tributary), above John Day Dam, and above Ice Harbor Dam (Snake River). The Umatilla River is the tributary where the collection of individuals used
for translocation were released to supplement the interior Columbia River population. Collection/detection sites at dams and nondams are shown
with X’s and open circles, respectively.

1194 © 2014 The Authors. Evolutionary Applications published by John Wiley & Sons Ltd 7 (2014) 1192–1208
Hess et al. Pacific lamprey migration genetics

(PIT), was inserted into the body cavity of each fish to have underestimated migration distance in some adults.
assess transmitter loss and failure. After tagging, Pacific However, both potential biases (e.g., fate misclassification)
lamprey recovered in a flow-through tank supplied with should act to decrease any observed effects of body size or
river water for at least 2 h. They were transported by truck fate (i.e., increase the probability of a Type II error) rather
to release sites located about 3 km downstream from Bon- than create false differences.
neville Dam, one on each bank. Pacific lamprey were
released in approximately equal proportions and release
Genetic data collection
site assignment was random with respect to individual fish
(release sites alternated between tag groups). All handling Tissues were collected from the dorsal fin of each adult
and tagging protocols were reviewed and approved by the Pacific lamprey and stored in a buffered solution before
University of Idaho Animal Care and Use Committee and DNA extractions were performed with Qiagen DNeasy kits
conducted under state scientific collection and transporta- (Qiagen, Valencia, CA, USA). We used a set of 96 SNP loci
tion permits. to genotype 647 individual adult lamprey that were sam-
Tagged Pacific lamprey was monitored using an extensive pled at Bonneville Dam and radio-tracked during their
array of aerial and underwater radio antennas (e.g., Keefer upstream migration. This set of 96 SNPs (94 of which are
et al. 2013a,b). Underwater antennas were deployed outside highly polymorphic in Pacific lamprey) was developed fol-
fishway openings, inside fishways, and at fish ladder exits at lowing a genomewide association study (Hess et al. 2013),
Bonneville, The Dalles, John Day, and McNary dams on the which used FST outlier tests to determine that 85 and 9 of
lower Columbia River, Priest Rapids Dam on the upper the SNPs were putatively neutral and adaptive, respectively.
Columbia River, and Ice Harbor Dam on the lower Snake For the genomewide association study, a RAD catalog was
River. Underwater antennas had detection ranges up to constructed using a subset of individuals (6–12 total indi-
approximately 10 m. Aerial radio antennas with longer viduals from 3 to 6 collections) and then used to align and
detection ranges (up to ~1 km) were deployed in dam tail- perform genotyping by sequencing on a larger sample of
races and at the mouths of 14 major Columbia River tribu- individuals (N = 518) from 21 collections distributed
taries between the Willamette River confluence and Priest throughout the Pacific Northwest range of Pacific lamprey
Rapids Dam (Fig. 1): Willamette River, Eagle Creek, (Hess et al. 2013). Subsequently, Hess et al. (2014a) dem-
Herman Creek, Fifteenmile Creek, Rock Creek, Wind River, onstrated that this set of 96 SNPs could perform the follow-
Little White Salmon River, White Salmon River, Hood ing three critical applications in Pacific lamprey: (i) species
River, Klickitat River, Deschutes River, John Day River, Yak- identification, (ii) parentage assignment, and (iii) charac-
ima River, and Umatilla River. Additionally, PIT tag detec- terization of adaptive and neutral variation. These 96 SNPs
tion antennas monitored lamprey passage at the main stem were genotyped using TaqMan assays supplied by Applied
dams (Keefer et al. 2009) and at in-stream sites in the Hood Biosystems (Grand Island, NY, USA). Genotypic data were
River, Fifteenmile Creek, and the Deschutes River basin. collected using the Fluidigm EP-1 96.96 system and follow-
A detection history for each tagged lamprey was gener- ing the manufacturer’s suggested protocol, but modified
ated using an automated program that assigned activity slightly by including a sample preamplification step and
codes to time-stamped detections at each radio antenna. increasing the PCR cycles to 50 (Hess et al. 2014a). All 647
Potential ‘noise’ records were excluded using filters that individuals included in this study passed quality-filtering
identified signal collisions. Each history was then reviewed using a missing data threshold of 10% (i.e., <10 missing
by an experienced technician who further identified records SNPs/individual).
that did not have corroborating support from detections at
nearby sites. A final ‘fate’ was assigned to each lamprey
Statistical analyses
using the last plausible radiotelemetry or PIT tag detection
record. A majority of final detections were at Columbia Linkage disequilibrium (LD) and Hardy–Weinberg equilib-
River dams or reservoirs. This was an artifact, at least in rium (HWE) deviations based on FIS were assessed using a
part, of radio transmitter battery life. Most Pacific lamprey subset of the dataset (515 Pacific lamprey), split among
overwinter in freshwater (including reservoirs) and then 3 years 2008–2010. The Markov Chain Monte Carlo
move to tributary spawning sites in the spring (Robinson approximation of Fisher’s exact test was used to test LD for
and Bayer 2005; Clemens et al. 2010), and the elapsed time all pairs of loci (94 SNPs) within each year sample using
between tagging and springtime tributary entry typically GENEPOP v. 4.1 (Raymond and Rousset 1995), with
exceeded the 162-d battery life (5 s burst rate). Thus, we default parameter settings of 10 000 dememorizations, 100
note that the sampled population may have been biased batches, and 5000 iterations. FSTAT v. 2.9.3.2 (Goudet
because not all lampreys could be tagged due to size restric- 2002) was used to calculate FIS for each year sample, and
tions and other logistical constraints and because we may 282 000 randomizations were used to test significance of

© 2014 The Authors. Evolutionary Applications published by John Wiley & Sons Ltd 7 (2014) 1192–1208 1195
Pacific lamprey migration genetics Hess et al.

each value. A Bonferroni correction was applied to signifi- resent a ‘maximum compression’ option because it treats
cance levels (1.77 9 10 4, initial a = 0.05) for both FIS all individuals as a single group. Permutation tests (1000)
and tests of LD to reduce false positives from multiple were used to calculate P-values to determine significant
tests. associations of SNPs with traits. Due to the high power of
This mixed group of fish was used to test HWE devia- this test to identify associations when many SNPs, traits,
tions because previous studies have not identified more and individuals are included, a Bonferroni correction was
than a single randomly mating population above Bonne- applied to alpha levels (0.05, 0.01, and 0.001; e.g.,
ville Dam using neutral markers (Spice et al. 2012; Hess 8.87 9 10 5 = corrected alpha 0.05) to reduce false posi-
et al. 2013). These Pacific lamprey were randomly selected tives and stringently control for Type I errors. The associa-
at an approximately 2% average sample rate across the tion tests using a GLM to test 6 traits were performed on
majority of the run of Pacific lamprey observed at Bonne- the following two types of data samples: (i) total ‘Bonne-
ville Dam during 2008–2010 (Table 1). Most (98%) of the ville’ sample using PC axes and year as covariates, and (ii)
515 Pacific lamprey had final location fates (Table 2); how- the ‘Bonneville’ sample separated into 3-year samples that
ever, not all fate categories were adequately represented in were each analyzed using PC axes as covariates. These two
this random sample, and so we added 62 Pacific lamprey types of data samples were also analyzed using an MLM,
that were nonrandomly selected to obtain a minimum of and a kinship matrix was included as an additional covari-
50 Pacific lamprey in each fate category (Table 2). This ate.
total ‘Bonneville’ sample (N = 577) was used to test There were six traits included in all the association tests
whether genetic variation was associated with migration performed with TASSEL. Missing trait data (0–4.5% of
fate, migration-timing, or morphology predictor variables; data) were imputed by default parameter settings which
however, we also analyzed each of the 3 year samples sepa- uses an average of three nearest neighbor values. Migration
rately to test for the consistency of associations across years. timing was tested in units of the statistical week in which
Finally, an additional set of 70 Pacific lamprey was also Pacific lamprey were collected and sampled at Bonneville
genotyped and was used to address our secondary objective Dam. Morphology predictor variables included Distell Fat-
(the ‘translocation’ sample). These 70 Pacific lamprey were meter readings, girth, length, and weight. For the purposes
used in translocations in 2008 to supplement the interior of association testing, the river reach where a fish was last
portion of the Columbia River Pacific lamprey population detected and its associated distance units were used to rep-
in the Umatilla River (described in Close et al. 2009). resent migration fate. Migration fate consisted of four river
As a first step to identify loci having significant associa- reaches: downstream from Bonneville Dam (235.1 km),
tions with three main categories of predictor variables between Bonneville Dam and John Day Dam (291.0 km),
(migration timing, morphology, and migration fate), we above John Day Dam (346.9 km), or above Ice Harbor
performed univariate analyses using a general linearized Dam (i.e., near mouth of the Snake River, 537.7 km). In all
model (GLM) and a mixed linearized model (MLM) with analyses aside from association testing, fish that were last
TASSEL v. 5.0.8 (Bradbury et al. 2007). The GLM is a fixed detected above Ice Harbor Dam were pooled with those
effects linear model that is utilized in TASSEL to identify detected above John Day Dam to increase sample size of
significant associations between phenotypes and genotypes. this extreme upstream migration fate category.
TASSEL takes population structure into account by using A multivariate analysis (DISTLMforward, McArdle and
membership in underlying populations as covariates in the Anderson 2001) implemented using the program package
model. The MLM is similar to GLM but includes both fixed PRIMER version 6 and the PERMANOVA+ add-on
and random effects (i.e., relationships among individuals) (http://www.primer-e.com; Plymouth Marine Laboratory,
and can account for both population structure and kinship Plymouth, UK) was used to examine the relative contribu-
to improve statistical power (Yu et al. 2006). Principle tion of each predictor variable to explain variation at the
components of the 85 neutral SNPs (first three PC axes) SNP loci identified by the GLM and MLM association tests.
and a kinship matrix (‘scaled IBS’ method; Endelman and Intercorrelation among these variables was examined
Jannink 2012) based on all 94 SNPs were generated in TAS- (based on Pearson’s r) to avoid excessive redundancy of
SEL to represent population structure and cryptic familial predictor variables (|r| > 0.95), and P-values were calcu-
relationships, respectively. The MLM was implemented lated (SAS Institute, Inc. 2000). This multivariate approach
using the ‘P3D’ (Zhang et al. 2010) parameter option to was applied to the total ‘Bonneville’ sample. DISTLMfor-
shorten computation time, and the ‘without compression’ ward was used for modeling the relationship between a
option to retain full dimensionality of the kinship matrix resemblance matrix (i.e., a Euclidean distance matrix of
(each individual belongs to its own separate group). This genotypes for a particular SNP locus) and multiple predic-
latter option for the MLM provides results that are in tor variables. Genotypes at SNP loci were converted to 0, 1,
extreme contrast to the GLM results, which effectively rep- and 2 based on the number of minor alleles present, and

1196 © 2014 The Authors. Evolutionary Applications published by John Wiley & Sons Ltd 7 (2014) 1192–1208
 Hess et al.

Table 1. Summary information on Pacific lamprey sampling at Bonneville Dam.

2008 2009 2010

Genetic Genetic Genetic

Statistical Bonn. Biosample Rate Fate Bonn. Biosample Rate Fate Bonn. Biosample Rate Fate
week count (N) Random (%) Supp. Trans. count (N) Random (%) Supp. count (N) Random (%) Supp.

20 7 – – – – – 8 – – – – 1 – – – –
21 161 – – – – – 170 – – – – 6 – – – –
22 316 4 4 1.3 0 0 640 0 0 0.0 0 2 1 0 0.0 1
23 280 12 6 2.1 0 0 975 23 20 2.1 0 100 11 2 2.0 3
24 198 9 4 2.0 0 0 612 22 12 2.0 2 286 26 6 2.1 6
25 1401 71 28 2.0 2 0 520 28 10 1.9 2 405 17 8 2.0 1
26 1684 174 35(1) 2.1 5 0 487 30 10 2.1 2 389 9 8 2.1 0
27 1680 271 35(1) 2.1 10 0 721 49 14 1.9 3 328 6 6 1.8 0
28 1267 89 25 2.0 5 0 613 25 12 2.0 1 763 7 7 0.9 0
29 1044 165 24(3) 2.3 3 17 1048 39 21 2.0 0 722 23 14 1.9 1
30 1957 140 37 1.9 4 27 881 52 18 2.0 0 687 22 15(1) 2.2 0
31 866 130 17 2.0 4 26 812 7 7 0.9 0 436 11 10(1) 2.3 0
32 967 71 19 2.0 3 0 344 0 0 0.0 0 623 17 13(1) 2.1 0
33 709 62 14 2.0 2 0 254 7 5 2.0 0 439 12 9 2.1 0

© 2014 The Authors. Evolutionary Applications published by John Wiley & Sons Ltd 7 (2014) 1192–1208
34 937 56 18 1.9 0 0 208 11 4 1.9 1 371 8 6(1) 1.6 1
35 345 56 7 2.0 0 0 109 2 2 1.8 0 130 3 3(2) 2.3 0
36 277 – – – – – 105 – – – – 193 – – – –
37 322 – – – – – 47 – – – – 177 – – – –
38 47 – – – – – 48 – – – – 69 – – – –
39 29 – – – – – 20 – – – – 48 – – – –
40 47 – – – – – 13 – – – – 39 – – – –
41 17 – – – – – 3 – – – – 14 – – – –
Total 14558 1310 273 2.0 38 70 8638 295 135 1.6 11 6228 173 107 1.8 13

The tallies of Pacific lamprey ‘Bonn. Count’ per statistical week (week 20 begins by May 9 and week 41 ends by October 12) are provided by the Fish Passage Center (http://www.fpc.org) as observed
by the Corps of Engineers at the Bonneville Dam fish counting window. For each year, we indicate the total number of fish that were tagged (Biosample N) and the subset of fish that were tissue sam-
pled (Genetic) and used for a random sample, for supplementing spatial fate categories ‘Fate supp.’, and for a translocation ‘Trans.’. The weekly sample ‘Rate’ was calculated for the random sample.
The weekly numbers for the random sample also indicate in parentheses the number of fish that did not have fate information.
Pacific lamprey migration genetics

1197
Pacific lamprey migration genetics Hess et al.

Table 2. Sample sizes per spatial category of Pacific lamprey captured at Bonneville Dam and tracked via radiotelemetry.

Collection (N) Genetic analysis (N)

Final detection reach 2008 2009 2010 2008 2009 2010 Total

Below Bonneville 780 208 100 197 92 60 349

Bonneville to John Day (COL) 253 66 38 47 32 27 106
Bonneville to John Day (trib) 20 12 18 20 (19) 12 (6) 18 (10) 50 (35)
Above John Day (COL) 120 10 9 34 (12) 9 (5) 8 (3) 51 (20)
Above John Day (SNR) 8 1 1 8 (7) 1 1 10 (7)
Total 1181 297 166 306 (38) 146 (11) 114 (13) 566 (62)

A total of 62 fish were used to supplement a random sample to obtain a minimum of 50 fish in each major fate category. The number of supple-
mented fish included in each of the cell subtotals are indicated in parenthesis. The final detection category of Bonneville to John Day was further
divided into fish that were last detected in the mainstem Columbia River (COL) or secondary tributary (trib). The Above John Day category could be
further divided by those fish that reached the Snake River (SNR, above Ice Harbor Dam) versus those last detected in the Columbia River (COL).

the distance matrix was generated from the absolute value collections (by statistical week), and included all newly cat-
of the difference between all pairwise comparisons of indi- egorized collections in a factorial correspondence analysis
viduals. The forward selection procedure fits individual (GENETIX v. 4.03, Belkhir et al. 2004) to examine the rela-
environmental predictor variables or sets of predictor vari- tionships among collections.
ables sequentially in the linear model. In our case, we used We estimated proportions of particular categories of
the following covariates and predictor variables which were genotype, length class, and upstream fate to characterize a
the same as in the previous GLM tests: the first three prin- time series (time units in statistical weeks) of randomly
ciple components of the 85 neutral SNPs (‘PC’), year, fate, sampled Pacific lamprey pooled across the 3 years (2008–
statistical week of passage (‘week’), Fatmeter (‘fat’), girth, 2010). These estimated proportions allowed us to portray
length, and weight. We used 9999 permutations of the how seasonal differences in these proportions could poten-
residuals under a reduced model to test the null hypothesis tially affect the composition of Pacific lamprey used for
of no relationship (Anderson 2003). First, marginal tests translocations. Specifically, the lengths and genotypes
were conducted on each predictor variable individually. (based on pairwise FST) of translocated fish that were taken
Next, conditional tests were then performed using a step- during a time period that spanned three statistical weeks
wise forward selection procedure that identifies the most (29–31) in the year 2008, were compared with the follow-
informative predictor variables sequentially while holding ing groups of fish: (i) Pacific lamprey that were randomly
constant the variables already selected. Similar to the GLM sampled during the same three statistical weeks across
association tests, we treated PC and year as covariates by 3 years, (ii) Pacific lamprey that were randomly sampled
forcing their inclusion into the model to focus on the other during an earlier set of statistical weeks (25–27) across
six predictor variables of interest (fate, week, fat, girth, 3 years, and (iii) Pacific lamprey that were categorized into
length, and weight) which were added sequentially. The three major upstream fate categories. Significance testing of
model ‘Akaike’s Information Criterion’ (AIC, Akaike 1973) pairwise FST values was corrected for multiple testing using
was used as the selection criterion in the sequential tests the B–Y method FDR as modified by Narum (2006) as this
because it imposes a penalty for increases in each predictor method controls Type I errors yet provides improved
variable, resulting in the most parsimonious model. power to differentiate populations over Bonferroni correc-
After demonstrating the importance of length and fate tion.
predictor variables, we categorized the Pacific lamprey with Finally, we performed analyses on the 150 Pacific lam-
complete fate and length data (N = 566) into 12 collec- prey collected at Willamette Falls (Hess et al. 2014a) that
tions, each fate category was divided into a short- and had previously been tested for phenotypic associations of
long-bodied collection (above and below the median size the adaptive loci. These fish only had length and passage-
660 mm, Fig. S1), and the ‘Below Bonneville Dam’ cate- timing (units by statistical ‘week’) information, and we
gory was further split by year due to the ample number of pooled these data with the total ‘Bonneville’ sample to per-
samples represented by that fate category. The ‘Between form association testing on this broader scale between the
Bonneville Dam and John Day Dam’ category could also be two regions (sample referred to as ‘Bonn + Willamette’).
further split based on whether fish were last detected in a Two predictor variables were used in TASSEL (length and
secondary tributary within this reach or not. In addition, week) to identify loci with significant associations. Both
we categorized 70 translocation Pacific lamprey into three GLM and MLM were performed, which required using

1198 © 2014 The Authors. Evolutionary Applications published by John Wiley & Sons Ltd 7 (2014) 1192–1208
Hess et al. Pacific lamprey migration genetics

TASSEL to first generate principle coordinates (first three that had not been found to be linked in previous analyses
PC axes based on the 85 neutral SNPs) to represent popula- across a broader geographic range (Hess et al. 2013,
tion structure and a kinship matrix (based on all 94 SNPs) 2014a). Further, we retained all loci for this study, because
to represent familial relationships. The GLM was per- our downstream analyses found differences in their degrees
formed using the following three covariates: PCs, location of association with predictor variables.
(0 = Willamette, 1 = Bonneville), and year. For the MLM, Only one SNP locus (Etr_673) deviated from HWE
we used the same covariates as in the GLM, and addition- (adjusted alpha of 0.05 = 1.77 9 10 4) in terms of large
ally used the kinship matrix as a covariate. We further positive FIS values, which were significant in both 2008 and
examined the significant loci using marginal tests in PRI- 2009 random samples (FIS = 0.289 and 0.353, respectively).
MER (five covariates and predictor variables: PCs, location, This locus was retained for our analyses as HWE deviations
year, length, and week). Finally, we used PRIMER to con- can be an indication of selection; however, none of our
duct multivariate analyses. Similar to the GLM in TASSEL, downstream analyses indicated statistically significant asso-
we treated the PCs, location, and year as covariates by forc- ciations of this locus with the predictor variables. Further,
ing their inclusion in the multivariate model, and we none of our previous analyses (Hess et al. 2013, 2014a)
implemented forward sequential tests in PRIMER to deter- have indicated similar deviations in FIS for other parts of
mine the model with the best fit using the remaining two the Pacific lamprey range.
predictor variables (length and week).
Association testing
Results
The univariate GLM analyses performed in TASSEL identi-
HWE deviations fied three SNP loci (Etr_1806, Etr_4281, and Etr_5317)
Linkage disequilibrium was statistically significant with significant associations (i.e., < Bonferonni adjusted
(adjusted alpha of 0.05 = 1.77 9 10 4) in three cases alpha level of 0.001) with the girth, length, and weight pre-
involving the following two pairs of loci: Etr_384 dictor variables (‘Bonneville’ sample, Table 3). The MLM
9 Etr_1806 in Pacific lamprey from the random 2008 and results were similar to the GLM but appeared to be more
2009 sample, and Etr_1806 9 Etr_4281 in the random conservative, because two of the same SNPs (Etr_1806 and
2009 sample. These three cases involved only adaptive loci Etr_5317) were significantly associated with the same three

Table 3. Test results using GLM and MLM in TASSEL which show the significant associations between SNP loci and six predictor variables.

Predictor variables

Covariates Sample Locus Fate Week Fat Girth Length Weight

Year + PC (+ kinship) Bonneville Etr_1806 *** (*) *** (**) *** (*)
Etr_4281 *** ***
Etr_5317 *** (***) *** (***) *** (***)
PC (+ kinship) 2008 Etr_1806 *** *** (**) *** (*)
Etr_4281 *
Etr_5317 *** (***) *** (***) *** (***)
PC (+ kinship) 2009 Etr_1806 **
Etr_4281 *** *
Etr_5317 *** *
PC (+ kinship) 2010 Etr_1806
Etr_4281
Etr_5317 *** (**) ** *** (*)
Year + Bonn + Etr_1806 NA NA NA *** (*) NA
Location Willamette Etr_2334 NA NA NA ** NA
+ PC (+ kinship) Etr_4281 NA NA NA *** NA
Etr_5317 NA NA NA *** (***) NA

Bonferroni correction was applied to alpha levels (0.05 ‘*’, 0.01 ‘**’, and 0.001 ‘***’), and were used to provide statistical robustness in identifying
significant P-values for associations between traits and loci. P-values are shown for results from the GLM tests, and P-values from MLM tests are in
parentheses. Covariates utilized in GLM tests are indicated and the same covariates were used in MLM with the addition of a kinship matrix (+ kin-
ship). Covariates included principle coordinates (PC) of variation at 85 neutral SNPs as a proxy for population structure. The analyses were performed
on the following three types of samples: the total ‘Bonneville’ sample, the ‘Bonneville’ sample split into 3 year samples using all six trait predictor vari-
ables, and performed on the ‘Bonn + Willamette’ sample excluding four variables with incomplete information (NA).

© 2014 The Authors. Evolutionary Applications published by John Wiley & Sons Ltd 7 (2014) 1192–1208 1199
Pacific lamprey migration genetics Hess et al.

predictor variables (< Bonferonni adjusted alpha level of The marginal tests for the ‘Bonneville’ sample that were
0.05), but Etr_4281 was no longer significantly associated performed as part of the multivariate analysis were largely
with any traits. This difference likely owes to the extra co- consistent with the TASSEL GLM and MLM results. Mor-
variate (kinship matrix) that results in the MLM test avoid- phological predictor variables girth, length, and weight
ing more false positives, at the expense of increasing false were highly significantly associated (P < 0.001) with the
negatives. three loci identified by TASSEL (Table 5). Length was con-
The TASSEL GLM results from the ‘Bonneville’ sample sistently the predictor variable that explained the largest
that was split into three separate year samples showed there portion of genetic variation for each locus. However, there
were some interannual differences in the number of signifi- was one nonmorphological predictor variable that met the
cant associations between 3 SNPs (Etr_1806, Etr_4281, and most stringent alpha threshold of 0.001 (fate), which was
Etr_5317) and three morphological traits (girth, length, observed to have significant association with Etr_1806. Fur-
and weight; Table 3). For two of the year samples (2008 ther, this association with the fate predictor variable repre-
and 2009), all three SNPs were significantly associated with sented the highest percent of variation (2.6%) explained by
at least one morphological trait (length, < Bonferonni any predictor variable aside from the three main morpho-
adjusted alpha level of 0.05); however, in 2010, only logical variables, girth, length, and weight. For the ‘Bonn +
Etr_5317 was found to be significantly associated with Willamette’ sample, four predictor variables were highly
morphological traits. The more conservative MLM results, significantly (P < 0.001) correlated with three loci
demonstrated only 2 SNPs (Etr_1806 and Etr_5317) to be (Etr_1806, Etr_5317, and Etr_4281). For each of these three
significant in 2008 and 1 SNP (Etr_5317) in 2010; in these loci, the four predictor variables (length, location, year, and
cases, all three morphological traits (girth, length, and week) were consistently ranked from highest to lowest,
weight) were involved. The interannual differences (i.e., respectively, based on percent variation explained
number of significant associations) likely owe to the differ- (Table 5). The locus Etr_2334, in contrast, was significantly
ences in sample sizes which were lower with each subse- correlated with a single predictor variable (length,
quent year. In general, the morphological trait associations P < 0.01).
were most robust across year samples and across GLM and The main pattern observed from the multivariate
MLM tests for 2 SNPs (Etr_1806 and Etr_5317). sequential tests on the ‘Bonneville’ sample showed length
The TASSEL GLM analysis of the ‘Bonn + Willamette’ was the primary predictor variable that explained the larg-
sample also identified Etr_1806, Etr_4281, and Etr_5317 as est percent of variation in the loci (range of 7–17%), fol-
loci with significant association with one of the morpho- lowed by a combination of secondary predictor variables
logical predictor variables (< Bonferroni corrected alpha that were selected for the best fitting multivariate model,
0.001 for length, Table 3). Further, there was a fourth although these secondary variables explained <2% of the
locus, Etr_2334, identified to be significantly associated residual variation (Table 6). For example, weight and girth
with length (< Bonferroni corrected alpha 0.01). Similar to were selected as secondary predictor variables for Etr_4281
previous analyses with the ‘Bonneville’ sample, only 2 loci and Etr_5317, respectively; fate and week were secondary
(Etr_1806 and Etr_5317) were significant in the MLM predictor variables chosen for the best fitting multivariate
analyses. Examination of intercorrelation of the six total model for Etr_1806. Finally, the analysis of the ‘Bonn +
predictor variables indicated that many of the morphologi- Willamette’ sample, which was conditioned on location,
cal predictor variables were moderately to highly correlated year, and PC axes, identified length as the only additional
with each other; however, none of them had a significant predictor variable required for the best fitting model in
correlation that was above 0.95 (Table 4), and so all of the sequential tests (Table 6).
variables were retained for the multivariate analyses.
Morphology and fate
Table 4. Inter-correlation of predictor variables, Pearson’s r.
Based on the results from association testing, we deter-
Week Fate Fat Girth Length Weight mined that length was the primary predictor variable that
Week *** *** *** could explain genetic differences among individuals. To
Fate 0.0552 *** *** *** demonstrate this relationship, we examined a factorial cor-
Fat 0.0556 0.0381 *** *** *** respondence analysis (FCA, Fig. 2) of our ‘Bonneville’ sam-
Girth 0.2490 0.1626 0.1993 *** *** ple of Pacific lamprey that was recategorized into groups
Length 0.1945 0.2100 0.1508 0.7351 *** according to their length class (above and below the med-
Weight 0.2506 0.1748 0.2022 0.9029 0.8831
ian of 660 mm, Fig. S1) and fate. This FCA plot clearly
Pearson’s r values and P-values are on bottom and top triangles, respec- shows that groups of the long- versus short-bodied individ-
tively. All P-values <0.05 were also <0.001 alpha level (***). uals segregate to different halves of the plot, regardless of

1200 © 2014 The Authors. Evolutionary Applications published by John Wiley & Sons Ltd 7 (2014) 1192–1208
Hess et al. Pacific lamprey migration genetics

Table 5. Results from marginal tests of predictor variables and genetic variation of adaptive SNPs.

Locus

Etr_1806 Etr_2334 Etr_4281 Etr_5317

Sample Variable %Var P %Var P %Var P %Var P

Bonneville PC 0.1 NA NA 0.2 0.9

Year 0.1 NA NA 0.0 0.4
Week 1.6 ** NA NA 0.0 0.3
Fate 2.6 *** NA NA 0.6 1.2 **
Fat 0.3 NA NA 0.3 0.3
Girth 6.8 *** NA NA 2.2 *** 15.8 ***
Length 12.2 *** NA NA 7.2 *** 17.0 ***
Weight 9.1 *** NA NA 4.1 *** 16.7 ***
Bonn + Willamette Location 9.8 *** 0.0 10.1 *** 34.2 ***
Year 7.0 *** 0.0 7.8 *** 24.8 ***
PC 0.1 0.7 0.1 0.6
Week 2.8 *** 0.0 3.0 *** 6.8 ***
Length 18.8 *** 1.3 ** 14.6 *** 35.5 ***

For each locus, the percent variation (%Var) explained by a particular predictor variable is provided. The (P) values from the marginal test were calcu-
lated from 9999 permutations, and indicated if below the following alpha levels: 0.05 (*), 0.01 (**), and 0.001 (***). The Bonn + Willamette sample
combined data from two locations (Bonneville Dam and Willamette Falls) and fewer predictor variables for morphology were available to test, as com-
pared to the ‘Bonneville’ sample. Only SNP loci that were identified with significant associations in the GLM and MLM tests in TASSEL (Table 3) were
included in marginal tests for each sample, hence the missing data ‘NA’ for Etr_2334 in the ‘Bonneville’ sample.

Table 6. Results from sequential tests of predictor variables and genetic variation at adaptive loci.

Sample Locus Variable AIC SS (trace) Pseudo-F P Prop. Cumul. Res. df

Bonneville Etr_1806 Length 980.9 14.2 78.8 *** 0.121 0.123 570
Fate 984.4 1.0 5.4 * 0.008 0.131 569
Week 984.6 0.4 2.2 0.1358 0.003 0.134 568
Etr_4281 Length 706.6 12.9 44.6 *** 0.072 0.075 570
Weight 707.8 0.9 3.2 0.0742 0.005 0.080 569
Etr_5317 Length 1200.4 14.1 114.1 *** 0.165 0.177 570
Girth 1211.8 1.6 13.4 *** 0.019 0.196 569
Bonn + Willamette Etr_1806 Length 1045.6 19.5 83.1 *** 0.093 0.193 720
Etr_2334 Length 755.6 6.5 18.4 *** 0.025 0.034 720
Etr_4281 Length 828.5 15.4 48.6 *** 0.057 0.159 720
Etr_5317 Length 1261.1 20.3 115.9 *** 0.091 0.437 720

Predictor variables are listed in the order they were selected to for a best fitting, forward sequential multivariate model using AIC as the selection cri-
terion with the software PERMANOVA. Sum of squares (SS), the Pseudo F-value (Pseudo-F), the permutational P-value (P), proportion of variation
(Prop.) and cumulated variation (Cumul.), and degrees of freedom (df) are shown. The Bonn + Willamette sample combined data from two locations
(Bonneville Dam and Willamette Falls), and these sequential tests were conditioned on population structure (principle coordinates ‘PC’), year, and
location to test the two remaining predictor variables, length and statweek. There were a total of six predictor variables analyzed in sequential testing
with the ‘Bonneville’ sample, and sequential tests were conditioned on PC and year covariates (Table 5). The P values were calculated from 9999 per-
mutations, and indicated if below the following alpha levels: 0.05 (*), 0.01 (**), and 0.001 (***).

their upstream fate category. The exception was the group

Seasonal heterogeneity of morphological and genetic traits
of short-bodied Pacific lamprey from the above John Day
in Pacific lamprey
Dam fate (the most upstream fate category), which may
have been influenced by small sample size (N = 8). The The proportion of long-bodied fish generally decreased by
group of translocated fish were also included in this FCA statistical week throughout the season, but had a peak
plot and they clustered closer to the short-bodied collec- around week 31 (Fig. 3). Further, the weekly proportions
tions, despite the fact that these fish were ultimately of the minor allele at the locus Etr_1806 (used to exemplify
released in a tributary upstream from John Day Dam. the adaptive loci) appears to contrast the pattern observed

© 2014 The Authors. Evolutionary Applications published by John Wiley & Sons Ltd 7 (2014) 1192–1208 1201
Pacific lamprey migration genetics Hess et al.

0.10

BJ
0.08

0.06
BB09 AJ
BB10
0.04
BB10
FC2 (10.2%)

0.02

BB08
0.00 BB08
BJt
BJ BB09
–0.02
T29
T30
AJ
–0.04

T31
–0.06
BJt

–0.08
–0.10 –0.08 –0.06 –0.04 –0.02 0.00 0.02 0.04 0.06 0.08 0.10
FC1 (22.5%)

Figure 2 Factorial Correspondence Analysis of genotyped Pacific lamprey collections of short-bodied (<660 mm, circles) and long-bodied
(>660 mm, X’s) individuals from three upstream locations in the Columbia River. Individuals that traveled no further than below Bonneville Dam (BB)
were further split into 3 years in which they were collected 2008–2010. Individuals that traveled upstream between Bonneville Dam and John Day
Dam and were last recorded in the main stem Columbia River (BJ or in secondary tributaries BJt) and those that traveled above John Day Dam (AJ)
were pooled together from three migration years. The AJ fate in this case included 10 fish that were detected further upstream above Ice Harbor
Dam. Three Pacific lamprey collections from 2008 that were used in translocations (T, triangles) to the Umatilla River (above John Day Dam) were sep-
arated by statistical week (29, 30, and 31; Table 1). The dashed line is shown to emphasize the genetic differences between short-bodied and long-
bodied collections.

1.0
0.9 throughout the migration season may explain some of the
Proportion of lamprey

0.8
notable differences observed among various samples of fish
0.7
0.6 50%+ that we compared. For example, based on morphological
0.5 (length variation) and genetic (pairwise FST using only the
0.4 three candidate SNPs from the ‘Bonneville’ sample analy-
0.3 Etr_1806
0.2 ses) comparisons, we observed some significant differences
0.1 Above JD between the sample of translocated fish, and the two sam-
0.0 ples of randomly selected fish from different time periods
22 23 24 25 26 27 28 29 30 31 32 33 34 35
Statistical week in the migration season. The sample of translocated fish
was significantly shorter than the sample of randomly
Figure 3 Weekly proportions of Pacific lamprey for presence of minor selected fish from earlier in the season (statistical weeks 25–
allele at Etr_1806 (gray), >50th percentile of length (dashed), and final
27), but showed a nonsignificant difference in length com-
upstream fate above John Day Dam (black), across a 3-year period
pared to the sample of randomly selected fish collected
2008–2010. The above John Day Dam fate in this case included fish
that were detected further upstream above Ice Harbor Dam. from the same time period (statistical weeks 29–31,
Table 7). Average Pacific lamprey length was observed to
be proportional with increasing distance traveled upstream,
in the weekly proportions of Pacific lamprey with the most and the relatively short-bodied fish used in translocations
upstream fate category (above John Day Dam/Ice Harbor were significantly shorter than all three samples of
Dam), appearing to peak when the other declines and vice upstream fated fish. In terms of genetic composition, the
versa. The heterogeneous nature of Pacific lamprey translocated fish were not significantly different from either

1202 © 2014 The Authors. Evolutionary Applications published by John Wiley & Sons Ltd 7 (2014) 1192–1208
Hess et al. Pacific lamprey migration genetics

Table 7. Influence of seasonality and final radio-tag detection location on the length and genetic traits of Pacific lamprey collections relative to Paci-
fic lamprey used in translocations.

ANOVA P-value FST

Final Statistical Average Translocation Random Random Translocation Random Random

Sample reach week length (29–31) (29–31) (25–27) (29–31) (29–31) (25–27)

Translocation – 29–31 648.6 – 0.224 ** – 0.001 0.008

Random – 29–31 656.1 – ** – 0.010*
Random – 25–27 668.6 – –
Fate BJ – 663.7 ** 0.084 0.465 0.009 0.008 0.002
Fate BJt – 669.7 * 0.197 0.559 0.001 0.001 0.000
Fate AJ – 683.4 *** *** *** 0.048* 0.043* 0.012

Lengths and genotypes of fish from the ‘translocation’ sample that were collected at Bonneville Dam during a time period that spanned three statisti-
cal weeks (29–31) in the year 2008 were compared (using ANOVAs and FST for lengths and genotypes, respectively) to the following groups of fish from
the total ‘Bonneville’ sample: (i) Pacific lamprey that were randomly sampled during the same three statistical weeks across 3 years, (ii) Pacific lamprey
that were randomly sampled during an earlier set of statistical weeks (25–27) across 3 years, and (iii) Pacific lamprey that were spatially categorized
by one of the following three final detection fates: between Bonneville Dam to John Day Dam in the main stem (BJ) and secondary tributary (BJt) and
above John Day Dam (AJ). The AJ fate in this case included 10 fish that were detected further upstream above Ice Harbor Dam. FST was based on the
three markers (Etr_1806, Etr_4281, and Etr_5317) associated with body morphology. Significant FST values (*) are indicated at a 0.05 alpha level cor-
rected for multiple comparisons using B–Y method FDR (corrected alpha = 0.02041, Narum 2006). The significant ANOVA P-values are indicated at the
0.05 (*), 0.01 (**), 0.001 (***) alpha levels.

sample of randomly-selected fish, but FST was near 0.0 for (Etr_1806 and Etr_5317) with length and run-timing via
the samples from the same time period, and near 0.01 for analyses of samples with complete metadata on individuals;
the samples from different time periods. Both the sample however, that study did not account for inter-correlation
of translocated and later-season period randomly-selected among predictor variables and interpretation was further
fish were significantly genetically differentiated from the complicated by the known presence of over-wintering
most upstream fate category (above John Day Dam/Ice behavior (Clemens et al. 2010; Hess et al. 2014a).
Harbor Dam), which was not the case for the earlier-season The current study was designed to resolve some of these
period randomly selected fish. issues and results demonstrated that the primary mecha-
nism operating on three of these adaptive markers is related
to adult body length of Pacific lamprey. Further, the pri-
Discussion
mary association of body length with these markers appears
Morphological associations with genetic markers to extend across a broad region that includes both Willam-
Results from the current study clarified the adaptive signifi- ette Falls and Bonneville Dam, because association tests
cance of several loci that had been previously identified as conditioned on location indicated that body length rather
outlier SNPs in a genomewide association study (GWAS) than the alternative predictor variable that was tested (run-
of Pacific lamprey (Hess et al. 2013). Morphology, primar- timing) could explain a significant portion of the residual
ily total adult body length, was found to have the strongest variation. However, while migration fate and timing play
associations with genetic variation at three SNPs (Etr_1806, more secondary roles, they explain minor but significant
Etr_4281, and Etr_5317). The initial study by Hess et al. portions of the remaining genetic variation based on the
(2013) made it possible to categorize 4000 + loci as either Bonneville Dam samples. These candidate loci may be asso-
putatively neutral or adaptive and further characterized ciated with the genetic mechanisms underlying the size dif-
which adaptive loci were significantly associated with sev- ferences observed among migrating Pacific lamprey.
eral predictor variables (geography, dwarf body form, and Because body size is associated with the distance Pacific lam-
run-timing). However, the opportunistic nature of the prey migrate upstream (Keefer et al. 2009, 2013a), this rela-
sampling for that study necessitated the inclusion of collec- tionship appears to explain why Pacific lamprey found in
tions with incomplete metadata or lack of individual trait the interior Columbia River basin are genetically differenti-
data. This constraint and the inter-correlation among pre- ated at these loci from those that spawn in the lower basin.
dictor variables precluded the ability to determine a pri- We note that while our metric of body size (length) was cor-
mary mechanism driving selection of the adaptive markers. related with other size metrics (weight, girth), it seems plau-
Subsequent study by Hess et al. (2014a) provided addi- sible that other unmeasured factors related to both size and
tional support for associations of two of these loci morphology (i.e., shape) also affect migration distance.

© 2014 The Authors. Evolutionary Applications published by John Wiley & Sons Ltd 7 (2014) 1192–1208 1203
Pacific lamprey migration genetics Hess et al.

The SNP locus Etr_1806 was part of another group of 25

Functions of genes associated with genetic markers
linked loci (group B), and this SNP had been observed in
Although the genetic mechanisms associated with each of significant linkage with all 25 loci in the lower Columbia,
these three adaptive loci (Etr_1806, Etr_4281, and and 67% of group-B loci in the interior Columbia but was
Etr_5317) are likely complex, we have some information linked to far fewer loci (0% and 13% in northern and
regarding the reported functions of the genes to which the southern regions, respectively) outside the Columbia River
SNPs were localized. For example, Etr_5317, localizes to the (Hess et al. 2013). Functions associated with genes in
gene DYM (Hess et al. 2013), which encodes a protein nec- which these markers are located include lower limb spastic-
essary for normal skeletal development and brain function ity (KIF1C, Dor et al. 2014; Etr_4193), axonemal motor/
(El Ghouzzi et al. 2003; Denais et al. 2011). The SNP locus, ATPase activity (DNAHC8, Samant et al. 2002; Etr_393),
Etr_4281, aligns with the human homolog, PCDH15 (Hess stabilization of dynamic microtubules (CLASP2, Maia
et al. 2013), which is in the cadherin gene superfamily and et al. 2012; Etr_1257), muscle functioning (TRIM32, Frosk
encodes integral membrane proteins that mediate calcium- et al. 2002; Etr_3295), as well as relatively uncharacterized
dependent cell–cell adhesion. Functions include an essential functions of a signaling protein (WAC, Xu and Arnaout
role in maintenance of normal retinal and cochlear func- 2002; Etr_320) and zinc finger protein (ZNF385D, Lamesch
tion and mutations in this gene result in hearing loss in et al. 2007; Etr_5213).
humans (Ahmed et al. 2008). The other SNP locus, Finally, the Etr_4281 locus was identified as part of a
Etr_1806, which was highly correlated with Pacific lamprey group of 27 linked loci (group C). This locus was observed
morphology, does not appear to localize within any in significant linkage with 100% of the group-C loci in the
described genes but occurs approximately 15 kb from a lower Columbia, and 96% in the interior Columbia, with
genomic region conserved between sea lamprey and lance- 54% outside the Columbia River to the north and south
let (Branchiostoma floridae, Hess et al. 2013). Finally, the (Hess et al. 2013). Functions (and loss of functions) associ-
SNP locus, Etr_2334, appears to be a cryptic adaptive locus ated with genes in which these group-C-SNP loci localize
that had previously been categorized as putatively neutral include signal transduction [RAPGEF2 (Emery et al. 2013),
(Hess et al. 2013), but results from the merged sample Etr_746; PCNA (Webb et al. 1990), Etr_4051; WDR91
‘Bonn + Willamette’ found significant association with (Caldwell et al. 2005), Etr_5841], craniofacial, urogenital,
length. Further, this locus was found to localize to a geno- and respiratory system abnormalities (FRAS1, Hoefele
mic region in sea lamprey that is homologous to the human et al. 2013; Etr_2776), myofibril assembly (NRAP, Mohid-
gene, CC2D2A, which plays a critical role in cilia formation din et al. 2003; Etr_2963), regulation of cardiovascular
and development (Gorden et al. 2008; Doherty et al. 2010). functions and relaxation of smooth muscle tone (PRKG1,
Insight into the genetic mechanisms associated with the Guo et al. 2013; Etr_3055), limb girdle muscular distrophy
three main candidate loci in this study may also be gained (TRAPPC11, B€ ogershausen et al. 2013; Etr_6149), as well
by examining homologous genes and functions associated as the uncharacterized function of a member of a methyl-
with the groups of loci that have previously been found by transferase gene superfamily (THUMPD2, Zhang et al.
Hess et al. (2013) to be highly linked to these 3 SNPs. For 2001; Etr_856).
example, the SNP locus Etr_5317 was part of a group of 53 The homologous genes and functions involved in body
linked loci (group A) that were identified in the GWAS size and muscle development are most obviously relevant
(Hess et al. 2013). This locus was observed in significant to the phenotypic associations that we identified for all four
linkage with 98% of loci in the lower Columbia, 96% in the candidate SNPs (Etr_1806, Etr_2334, Etr_4281, and
interior Columbia, and with 75% and 94% outside the Etr_5317) within the Columbia River. It remains unknown
Columbia River to the north and south, respectively (Hess to what extent the morphological associations of these loci
et al. 2013). Functions associated with genes in which these may apply to Pacific lamprey outside the Columbia River
53 SNPs loci localize include involvement in cartilage basin. We hypothesize that SNPs that localize more proxi-
development (CILP, Nakamura et al. 1999; Etr_5340), and mally to the target of selection in the genome will likely
resistance to oxidative stress (SZT2, Basel-Vanagaite et al. have to be identified to observe this pattern more broadly
2013; Etr_1489). In addition, studies of gene defects (Kalay because of breakdown in these phenotypic associations that
et al. 2011) suggest involvement in seckel syndrome type 5, was noted previously by Hess et al. (2014a).
a rare autosomal recessive disorder characterized by pro-
portionate dwarfism (CEP152, Etr_68), and acromesomelic
Mechanism of selection on morphology
dysplasia (Robinson et al. 2013) which is an extremely rare,
inherited, progressive skeletal disorder that results in a par- At this point, we cannot determine whether the driving
ticular form of short stature (i.e., short-limb dwarfism, force of selection on the candidate SNPs originates from
NPR2, Etr_6363). anthropogenic factors (e.g., main stem hydropower dams,

1204 © 2014 The Authors. Evolutionary Applications published by John Wiley & Sons Ltd 7 (2014) 1192–1208
Hess et al. Pacific lamprey migration genetics

altered hydrograph), or rather from selection due to the sufficient gene flow throughout the range to homogenize
energetic requirements of lengthy upstream migration or the neutral variation of the population. This hypothesis
the differences in habitat present in interior versus streams could be tested by surveying genetic variation within tribu-
closer to the Pacific Ocean. However, it has been suggested taries distributed throughout the Pacific lamprey range. If
there was greater morphological diversity (small-bodied the hypothesis were true, we would predict a consistent
adults were present) in the interior Columbia River basin pattern within each tributary of a gradient of adaptive vari-
historically (Close et al. 2004). The main stem Columbia ation correlated with upstream distance and contrasted by
River dams are known to impose substantial impediments minimal neutral structure at the same scale.
to Pacific lamprey migration, as there is high attrition at It is possible we have ignored other key factors that could
Bonneville Dam and at each of the dams upstream, and influence body size and drive the adaptive divergence
tagged adults successfully passing the dams are consistently observed in this species. No methods are available to reli-
larger than those that do not (e.g., Keefer et al. 2009). Fish- ably determine age in lamprey, and thus, we were unable to
ways at lower Columbia and Snake River dams were not evaluate how maturity, growth rate, and adult body size
designed for adult Pacific lamprey passage (Moser et al. were related to the observed genetic associations, though
2011), and many adults fail to pass fishways (e.g., Keefer we expect that factors affecting maturity in lamprey are
et al. 2013a,b). The genetic associations observed here under strong selection in this semelparous species. Further,
could thus be caused by long-distance migration itself, body size at maturity may be affected by the duration of
selection for traits during difficult passage of artificial fish- time in freshwater or ocean environments, or specialization
ways, or both. The relationship between passage ability at on particular resources. Individuals originating from
fishway barriers and Pacific lamprey traits (especially size) upstream locales (i.e., less productive streams) may repro-
is currently under investigation. If distance is a surrogate duce at older ages and return to freshwater at large adult
for migration investment, then passing dams may reduce sizes. Alternatively, large-bodied Pacific lamprey may
numbers upstream by either imposing high energetic costs return to upstream sites because of some olfactory cue, as
or directly impeding passage of Pacific lamprey with small such cues have been demonstrated to influence migration
body size at barriers requiring high swimming power and/ behavior in Pacific lamprey (Yun et al. 2011) and other
or endurance. Evaluation of the relationships between lamprey species (e.g., Petromyzon marinus; attractants, Li
migration distance, morphological traits, life history, and et al. 2002; and repellents, Wagner et al. 2011). However,
genetic variation in large unimpounded drainages such as any alternative scenario would effectively produce the same
the Fraser River in British Columbia would be especially result that involves nonphilopatric, large-bodied adults
useful for separating the effects of distance and dams on the having heightened propensity for migrating to upstream
gradients observed here. Regardless, the observed patterns locales. It would be ideal if future exploration of drivers of
raise important questions about anthropogenic effects on selection on Pacific lamprey could greatly broaden the
the composition of interior Pacific lamprey populations. types of phenotypes measured at various life stages to ade-
It may initially seem paradoxical to observe adaptive quately treat these possible alternatives.
divergence that is driven by body size and upstream dis-
tance traveled without also observing significant differenti-
Adaptive genetic context for translocation strategies and
ation at neutral loci within the Columbia River basin. The
migratory behavior
lack of neutral genetic differentiation among major rivers
(e.g., Goodman et al. 2008; Hess et al. 2013) may be driven This study has demonstrated a seasonal effect on genetic
by non-specificity in choice of highly mobile hosts during and morphological diversity, and further showed that the
its ectoparasitic feeding mode which results in wide disper- manner in which Pacific lamprey are selected for transloca-
sion in ocean waters (similar to sea lamprey, Waldman tion can result in significantly differentiated genetic and
et al. 2008) and could subsequently be reinforced by selec- phenotypic variation as compared to individuals that voli-
tion against long return migrations to natal streams, a lack tionally migrated to the point of release. If anthropogenic
of sensory capacities to navigate and orient to natal factors are the primary mechanism driving selection on
streams, or other selective forces. Nonetheless, the apparent these volitional migrants, translocations of fish may be one
paradox may be explained by nonphilopatric migration, way to restore historical levels of genetic and morphologi-
continuous distribution, historically high effective popula- cal diversity in the interior Columbia River basin. Alterna-
tion size of this anadromous fish, and on-going selection tively, if it is driven by selection on factors such as
for larger body size during long or difficult migration. migration distance, we would predict that translocations
While Pacific lamprey appear to segregate according to would serve to increase genetic diversity upon which natu-
body length and upstream distance, they have low proba- ral selection would continue to act within the interior river
bility of spawning in their natal stream, which would allow basin.

© 2014 The Authors. Evolutionary Applications published by John Wiley & Sons Ltd 7 (2014) 1192–1208 1205
Pacific lamprey migration genetics Hess et al.

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