DOI: 10.25338/b8p63w
title: Intraspecific variation among Chinook salmon populations indicates physiological adaptation to local environmental conditions

description: Understanding interpopulation variation is important to predicting
 species' responses to climate change. Recent research has revealed
 interpopulation variation among several species of Pacific salmonids.
 Here, we tested for local adaptation and countergradient variation by
 assessing interpopulation variation among six populations of fall-run
 Chinook salmon from California, Oregon, and Washington (USA). Juvenile
 fish were reared at three temperatures and five physiological metrics were
 measured. Statistical associations between the five physiological traits
 and 15 environmental predictors supported our hypotheses of local
 adaptation. Notably, latitude was a poor predictor of population
 physiology. Instead, our results demonstrate that populations from warmer
 habitats exhibit higher thermal tolerance (i.e., critical thermal maxima),
 faster growth when warm-acclimated and greater aerobic capacity at high
 temperatures. Additionally, populations with longer migrations exhibit
 higher metabolic capacity. However, overall metabolic capacity declined
 with warm-acclimation indicating that future climate change may reduce
 metabolic capacity, negatively affecting long-migrating populations. This
 data set contains the growth, critical thermal maxima, metabolic and
 environmental datasets used in our analysis.  Additionally it
 contains mass, length and hematocrit values for 300 fish from which we
 collected tissue samples.
description: Fish (n = 710, 23.9± 4.25 g)
 underwent metabolic trials in one of four, 5 L automated swim tunnel
 respirometers (Loligo, Denmark). The four tunnels were split into two
 paired systems with two tunnels sharing a single sump and heat pump. Water
 for each swim tunnel system was pumped (PM700, Danner USA) from the sump
 into an aerated water bath surrounding each swim tunnel, and then returned
 to the sump. Sumps were supplied with non-chlorinated fresh water from a
 designated well and aerated with air stones. The temperature of the sump
 (and therefore the swim tunnels) was maintained (±0.5°C) by circulating
 water through a heat pump (model DSHP-7; Aqua Logic Delta Star, USA) using
 a high-volume water pump (Sweetwater SHE 1.7 Aquatic Ecosystems, USA). In
 addition, each sump contained a thermostatically controlled titanium
 heater (TH-800; Finnex, USA). Swim tunnels and associated sump systems
 were cleaned and sanitized with bleach weekly to reduce potential for
 bacterial growth. Dissolved
 oxygen saturation within the swim tunnels was measured using fibre-optic
 dipping probes (Loligo OX11250) which continuously recorded data via
 AutoResp™ software (version 2.3.0). Oxygen probes were calibrated weekly
 using a two-point, temperature-paired calibration method. Water velocity
 of the swim tunnels was quantified and calibrated using a flowmeter
 (Hontzcsh, Germany) and regulated using a variable frequency drive
 controller (models 4x and 12K; SEW Eurodrive, USA). The velocity
 (precision &lt;1 cm s<sup>-1</sup>) for each tunnel was
 controlled remotely using the Autoresp™ program and a DAQ-M data
 acquisition device (Loligo, Denmark). Swim tunnels were surrounded by
 shade cloth to reduce disturbance of the fish. Fish were remotely and
 individually monitored using infrared cameras (QSC1352W; Q-see, China)
 connected to a computer monitor and DVR recorder. Oxygen consumption rates for both routine
 and maximum metabolic rates were captured using intermittent
 respirometry(Brett 1964). Flush pumps (Eheim 1048A, Germany) for each
 tunnel pumped aerated fresh water through the swim chamber and were
 automatically controlled via the AutoResp™ software and DAQ-M system. This
 system would seal the tunnel and enable the measurement of oxygen
 consumption attributable to the fish. Oxygen saturation levels were not
 allowed to drop below 80% and restored within three minutes once the flush
 pump was activated. Oxygen saturation data from AutoResp™ was transformed
 to oxygen concentration using the following equation: Where %O<sup>2</sup>Sat is the
 oxygen saturation percentage reported from AutoResp™; αO2 is the
 coefficient temperature-corrected oxygen solubility
 (mgO<sub>2</sub> L<sup>-1</sup>
 mmHg<sup>-1</sup>); and BP is the barometric pressure (mmHg).
 Oxygen concentration (milligrams of oxygen per liter) was measured every
 second and regressed over time, the coefficient of this relationship
 (milligrams of oxygen per liter per second) was then converted to
 metabolic rate (milligrams of oxygen per kilogram per minute, Equation
 3). Where R is the
 calculated coefficient of oxygen over time; V is the volume of the closed
 respirometer; M is the mass of the fish in kilograms and ’60’ transforms
 the rate from per second to per minute. An allometric scaling exponent was
 not incorporated due to similarity in fish sizes and to maximize
 comparability with metabolic data from the Mokelumne Hatchery (CA)
 fall-run population (Poletto et al. 2017). Routine Metabolic Rate Prior to routine metabolic rate (RMR)
 trials, fish were fasted to ensure a post-prandial state. Fish reared at
 16 or 20°C were fasted for 24 hours, while fish acclimated to 11°C were
 fasted for 48 hours. Fish were then transferred into a swim tunnel
 respirometer between 13:00 and 17:00. After a 30-minutes at their
 acclimation temperature the temperature was adjusted at 2°C
 h<sup>-1</sup> to the test temperature (8–26°C). Automated
 intermittent flow respirometry began 30 minutes after the test temperature
 was achieved and continued overnight. Measurement periods ranged from 900
 to 1800 seconds in duration, flush periods were 180–300 seconds. Periods
 varied in length in response to fish size and test temperature to ensure
 oxygen saturation was kept high (&gt;80%) during the trial. A small
 circulation pump (DC30A-1230, Shenzhen Zhongke, China) ensured that water
 was mixed without disturbing the fish. Fish activity was monitored by
 overhead infra-red cameras and measurement periods when the fish were
 active were discarded. RMR was calculated by averaging the three lowest
 RMR values(Poletto et al. 2017). RMR measurements were concluded by 08:00
 ± 40 min. Maximum
 Metabolic Rate A modified
 critical swimming velocity protocol was used to elicit maximal metabolic
 rate (MMR)(Poletto et al. 2017). Tunnel speed was increased gradually from
 0 to 30 cm s<sup>-1</sup> over an ~2 min period and held there
 for 20 min. For each subsequent 20-min measurement period, tunnel velocity
 was increased 10% up to a maximum of 6 cm s<sup>-1</sup> per
 step. Fish were swum until exhausted and unable to swim. Swimming
 metabolism was measured by sealing the tunnel for approximately 16 minutes
 of the 20-minute measurement period. When a fish became impinged upon the
 back screen (&gt;2/3 of body in contact with screen) the tunnel
 velocity was stopped for ~1 minute and then gradually returned to the
 original speed over 2 minutes. A fish was determined to be exhausted if it
 became impinged twice within the same velocity step. At this point, the
 tunnel impellor was stopped to allow for recovery. The highest metabolic
 rate measured over a minimum of 5 minutes during active swimming was taken
 as the MMR. Post-experiment, the tunnel was returned to
 the acclimation temperature and fish were transferred to a recovery tank
 and monitored. In seeking evidence of metabolic collapse at near-critical
 temperatures, some metabolic trials were conducted at temperatures
 exceeding the tolerance of the fish. These mortality events represent
 potential lethal upper limits for sub-acute thermal persistence (Fig. S1).
 Data from fish which did not survive the trial or recovery were not used
 in analysis. After a 24-hour recovery period, fish were euthanized in a
 buffered solution of MS-222 (0.5g/L). Measurements for mass (g), fork
 length (cm) and total length (cm) were taken, and Fulton’s condition
 factor was calculated. Aerobic scope (AS) was calculated as the
 difference between a fish’s RMR and MMR. Thermal optimums (TOPT) were
 defined as the temperature when aerobic scope was maximized, and
 calculated as the root-value of the derivative of the quadratic function
 describing the relationship between AS and test temperature. 
 Growth Data Growth measurements were initiated in mid
 to late spring when all populations would still be rearing prior to
 outmigration. Growth data were gathered every two weeks by measuring a
 sample of 30 fish from each treatment (n=~15 per tank, n = 2149 total
 measurements). Fish were not individually marked and therefore growth rate
 was calculated across individuals. Fish were arbitrarily netted from their
 treatment tank and transferred to an aerated five-gallon bucket until
 measured. Fish were air exposed for ~15–20 seconds to measure mass (± 0.01
 grams, Ohaus B3000D) and fork length (± 0.1 cm) and then placed into a
 second bucket for recovery before returning to their original treatment
 tank. Fish were netted and measured by the same experimenter across all
 sampling days. Condition
 factor was calculated as Fulton's condition factor (K) using the
 equation K = 100*Mass/Fork Length<sup>3</sup>.
 Critical Thermal Maxima
 Critical Thermal Maximum
 (CT<sub>Max</sub>) values were quantified according to
 established methods, briefly described below1. We placed six 4L Pyrex
 beakers in a fiberglass bath tray (1m x 2m x .2m). Beakers were aerated
 with an air stone to ensure both adequate oxygen saturation and
 circulation of water within the beaker. The volume of water in each
 individual beaker (approx. 2.5 L) was calibrated to ensure even heating
 across all CT<sub>Max</sub> beakers (0.33°C/min). Two pumps
 (PM700, Danner USA) were used to circulate water: one pump recirculated
 water across three heaters (Process Technology S4229/P11), while the other
 distributed heated water through the CT<sub>Max</sub> bath via
 a distribution manifold. Experiments began with water temperature set at
 the fish’s acclimation temperatures (11, 16, or 20°C). Fish of appropriate size (n = 377, 12.4 ±
 0.83 cm) were arbitrarily selected from treatment tanks and transferred to
 separate tanks for fasting. To ensure fish were in a similar postprandial
 state, fish reared at 20°C and 16°C were fasted for 24 hours, and 11°C
 fish were fasted for 48 hours to account for their slower metabolic rate.
 Once fasted, fish were individually netted and transferred into individual
 beakers within the CT<sub>Max</sub> heat bath. Fish were given
 30 minutes to acclimate to their CT<sub>Max</sub> beaker after
 which the CT<sub>Max</sub> trial began. During the CT<sub>Max</sub>
 trial, beaker temperature was taken every 5 minutes using a thermocouple
 (Omega HH81A). Thermocouple measurements were calibrated to a Fisherbrand®
 NIST certified mercury thermometer following each trial. Fish were
 observed continually for signs of distress and loss of equilibrium. The
 CT<sub>Max</sub> trial endpoint was loss of equilibrium, at
 which point the temperature of the CT<sub>Max</sub> beaker was
 recorded. Fish were then removed and returned to a recovery bath at their
 acclimation temperature. Fish that did not fully recover within 24-hours
 were not included in analysis (6% of individuals). After the 24-hr
 recovery, fish were weighed (wet mass ± 0.01g) and measured (fork length ±
 0.1 cm). Missing
 values: Only fish that
 successfully completed the Critical Thermal Maximum (CTMax) trials are
 included in this dataset. Fish that did not recover from the trial were
 excluded from analysis, and therefore this dataset. The CTMax trial is
 designed to be survived and therefore failure to recover fully is
 reflective of an abnormality in the fish’s physiology or experimenter
 error, both of which could bias the ultimate results.   Tissue Samples Fish (N
 = ~12) from each treatment group were humanely sacrificed for tissue
 collection. Additionally, one or more capillary tubes of blood were
 collected shortly after fish were sacrificed. Tubes were then spun in a
 centrifuge at ~10,000 RPM for 3 minutes to separate the red blood cells.
 After centrifugation the hematocrit (% red blood cells) was measured and
 the plasma was extracted and flash-frozen in liquid nitrogen. 
 Missing values: Fish varied in size so we
 were unable to get multiple capillary tubes of blood from each fish.
 Therefore some fish only have a single HCT measure, while others have 3.
 Other missing values indicate that the data was not recorded. 
 Becker, C. D. &
 Genoway, R. G. Evaluation of the critical thermal maximum for determining
 thermal tolerance of freshwater fish. Environmental Biology of Fishes 4,
 245–256 (1979). Beitinger, T. L., Bennett, W. A. &
 McCauley, R. W. Temperature tolerances of North American freshwater fishes
 exposed to dynamic changes in temperature. Environmental Biology of Fishes
 58, 237–275 (2000). Fangue, N. A., Hofmeister, M. &
 Schulte, P. M. Intraspecific variation in thermal tolerance and heat shock
 protein gene expression in common killifish, Fundulus heteroclitus.
 Journal of Experimental Biology 209, 2859–2872 (2006).
description: .csv Files can be opened with excel or in R. There are not
 additional software requirements.
description: # Intraspecific variation among Chinook salmon populations indicates
 physiological adaptation to local environmental conditions --- There are 3
 datasets. One that pertains to growth measurements, one that pertains to
 CTMax data and the third contains data regarding metabolic performance.
 The columns and descriptions for these three datasets are below. Only fish
 used in the analysis for the Conservation Physiology manuscript are
 included in this data. This excludes fish which did not recover from the
 metabolic or CTMax trials or fish from other populations which were
 measured during the same experimental seasons, which leads to gaps in the
 individual numbering of trial fish ## Column Specs:
 Con_Phys_2023_Dryad_CTM_Data.csv * SEQ: Numerical number, runs from 1 to
 the end fo the dataset, each row gets its own value * FISH_NUM: Unique
 fish identification number, gaps in sequence are due to fish which did not
 survive recovery, or data from other populations not included in the
 publication associated with this dataset. * HATCHERY: Hatchery
 identification for each fish, Coleman: Coleman National Fish Hatchery,
 Winter-Run: Sacramento River Winter-run from Livingston Stone National
 Fish hatchery, Feather Fall: Feather Fall-run population from the feather
 river hatchery, Feather Spring: Spring-run population from the Feather
 River hatchery * ACCLIMATION_TEMPERATURE: Temperature at which each fish
 was acclimated, 11 16 or 20 * TANK_ID Rearing tank identity for each fish.
 As tanks were reused between years the tank ID code is formatted as
 TANK_POPULATON_YEAR: Population codes are CO: Coleman, WR: Winter-run, FF:
 Feather Fall and FS: Feather Spring * MASS: Fish mass measured in grams *
 STANDARD_LENGTH: Standard length from snout to start of caudal fin,
 measured in mm. Fish from the Coleman population were not measured for
 Standard Length and therefore are NA. * FORK_LENGTH: Fork length from
 snout to fork in caudal fin, measured in mm * TOTAL_LENGTH: Total length
 from snout to furthest extremity of caudal fin, measured in mm *
 CONDITION_FACTOR: Fulton's condition factor calculated using the
 equation K = mass*100/(FL^3) * CTM_CHAMBER: Specific CTM chamber that a
 fish was trialed in, each CTM chamber was assigned a letter identifier,
 A,B,C,D,E,F or G * CTM_DATE: mm/dd/yy of the CTMax Trial * AGE_AT_CTMAX:
 Age of the fish at the CTMax trial (Days) ## Column Specs:
 Con_Phys_2023_Dryad_Growth_Data.csv * SEQ: Numerical number, runs from 1
 to the end fo the dataset, each row gets its own value * HATCHERY:
 Hatchery identification for each fish, Coleman: Coleman National Fish
 Hatchery, Winter-Run: Sacramento River Winter-run from Livingston Stone
 National Fish hatchery, Feather Fall: Feather Fall-run population from the
 feather river hatchery, Feather Spring: SPring-run population from the
 Feather River hatchery * ACCLIMATION_TEMPERATURE: Temperature at which
 each fish was acclimated, 11 16 or 20 * TANK_ID: Tank ID as a
 concatenation of the specific tank code (e.g., I4, E3, H1) and the
 hatchery a fish came from * DATE: Date that a specific fish was measured
 (mm/dd/yyyy) * DAYS: Days into the growth measurement window, starts at 0
 * YEAR: Year of the growth measurements (2017, 2018 or 2019) * MASS: Fish
 mass measured in grams * STANDARD_LENGTH: Standard length from snout to
 start of caudal fin, measured in mm. Fish from the Coleman population were
 not measured for Standard Length and therefore are NA. * FORK_LENGTH: Fork
 length from snout to fork in caudal fin, measured in mm * TOTAL_LENGTH:
 Total length from snout to furthest extremity of caudal fin, measured in
 mm * CONDITION_FACTOR: Fulton's condition factor calculated using the
 equation K = mass*100/(FL^3) ## Column Specs:
 Con_Phys_2023_Dryad_Metabolic_Dataset.csv * SEQ: Numerical number, runs
 from 1 to the end fo the dataset, each row gets its own value * FISH_ID:
 Unique fish identification number, gaps in sequence are due to fish which
 did not survive recovery, or data from other populations not included in
 the publication associated withthis dataset. * AS_EXP: Four aerobic scope
 trials could be conducted at once, one in each swim tunnel. This value
 keeps track of which trials were conducted concurrently. *
 HATCHERY:Hatchery identification for each fish, Coleman: Coleman National
 Fish Hatchery, Winter-Run: Sacramento River Winter-run from Livingston
 Stone National Fish hatchery, Feather Fall: Feather Fall-run population
 from the feather river hatchery, Feather Spring: SPring-run population
 from the Feather River hatchery * ACCLIMATION_TEMPERATURE:Temperature at
 which each fish was acclimated, 11 16 or 20 C * TEST_TEMPERATURE:
 Temperature at which metabolic trials were conducted at. (8, 10, 12, 14,
 16, 18, 20, 22, 23, 24,25 26C) * TANK_ID:Rearing tank identity for each
 fish. As tanks were reused between years the tank ID code is formatted as
 TANK_POPULATON_YEAR. Population codes are CO: Coleman, WR: Winter-run, FF:
 Feather Fall and FS: Feather Spring * TUNNEL_ID: Which of four swim
 tunnels a fish was trialed in. Trials which share a cardinal direction
 (East vs. West) were connected via a shared sump system and therefore test
 temperatures of fish trialed in shared systems were the same. * RMR:
 Routine Metabolic Rate (mgO2/kg/hr) * MMR: Maximum Metabolic Rate
 (mgO2/kg/hr) * AS: Aerobic Scope (MMR - RMR; mgO2/kg/hr) * MASS: Fish mass
 measured in grams * STANDARD_LENGTH: Standard length from snout to start
 of caudal fin, measured in mm. Fish from the Coleman population were not
 measured for Standard Length and therefore are NA. * FORK_LENGTH: Fork
 length from snout to fork in caudal fin, measured in mm * TOTAL_LENGTH:
 Total length from snout to furthest extremity of caudal fin, measured in
 mm * CONDITION_FACTOR: Fulton's condition factor (K) calculated using
 the equation K = mass*100/(FL^3) * AGE_AT_TRIAL: Age of the fish at the
 CTMax trial (Days) ## Column Specs:
 BONUS_Dryad_Tissue_Sampling_Dataset.csv * ACC: Numerical number, runs from
 1 to the end fo the dataset, each row gets its own value * FISH_NUM: A
 unique fish ID value * HATCHERY: Hatchery population the fish comes from *
 ACC_TEMP: Acclimationt temperature the fish was reared at * TANK_ID:
 Rearing tank of the fish * DATE: Date fish was sampled on * SL: Standard
 length from snout to start of caudal fin, measured in cm. Fish from the
 Coleman population were not measured for Standard Length and therefore are
 NA. * FL: Fork length from snout to fork in caudal fin, measured in cm *
 TL: Total length from snout to furthest extremity of caudal fin, measured
 in cm * MASS: Fish mass measured in grams * HCT_1: Hermatocrit reading of
 the first capillary tube (%) * HCT_2: Hermatocrit reading of the second
 capillary tube (%) * HCT_3: Hermatocrit reading of the third capillary
 tube (%) * GONADS: Were gonads present? (Y)es, (N)o, in many cases this
 was not recorded. * NOTES: Observations of the tissue sampling. ##
 Sharing/access Information Contact Ken Zillig 
 for questions about this data.