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Title: Near shore ecology of Grand Canyon fish 

Funding Opportunity Number: 09WRPA0001 

Principal Investigators: Drs. William E. Pine1,4,5, III, J. Korman2

, Karin E. Limburg3, Mike 

S. Allen5

, and Thomas K. Frazer5

Date of Proposed Research: February 1, 2009 – September 20, 2012 

1

 Department of Wildlife Ecology and Conservation, 110 Newins-Ziegler Hall, University 

of Florida, Gainesville, Florida 32611 

2

 Ecometric Research Inc. 3560 W 22nd Avenue, Vancouver, BC V6S1J3 

3

 Environmental and Forest Biology, State University of New York, 249 Illick Hall, 1 

Forestry Drive, Syracuse, NY 13210 

4

School of Forest Resources and Conservation, University of Florida, 7922 NW 71st 

Street, Gainesville, Florida 32653 

5

 PI to whom correspondence should be addressed Bill Pine, email: billpine@ufl.edu 

Telephone: 352 225 1643.

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Section 1 Study motivation - The primary goal of this project is to understand how river 

flow, through its interaction with physical habitat structure, influences the survival rates 

of juvenile native and non-native fishes in the Colorado River in Grand Canyon. Nine 

research questions related to this goal have been identified in the RFP (RFP pages 27-

28). These questions have a hierarchical structure and vary in scope. Some questions 

are fundamental and process oriented in nature (e.g., does river flow conditions alter 

juvenile native fish survival rates?) while others are focused on methodology (e.g., how 

to measure juvenile fish abundance, can small fish be marked?). Some questions, 

though related to the broader goals of the RFP, are quite specific (e.g., what is the 

Colorado River mainstem survival rate for humpback chub emigrating from the Little 

Colorado River (LCR) during freshets?). Some questions will be very difficult to answer 

within the time-frame of this project (e.g., how do biotic and abiotic factors influence 

individual fish growth and survival by habitat type?), or may be quite easy to answer 

with existing data (e.g., what is the feasibility of marking small humpback chub?). 

We propose two key fundamental research questions (RQ) should guide the 

design of this project: 

(RQ1) Do steadier flows during summer and/or fall increase survival rates 

of juvenile native and non-native fish? 

(RQ2) To what extent does physical habitat structure (e.g., sand bars and 

backwaters), in conjunction with flows during these periods, influence 

survival rate? 

We propose to address these research questions and link results to a proposed 

conceptual model for humpback chub Gila cypha (HBC) and other native and nonnative fish (below) by first describing an approach to assess shifts in fish density by 

tracking habitat specific abundance and survival of native and non-native fish in 

response to changing nearshore habitat availability related to and created by the fall 

steady flow experiment. We then detail an approach to determine the source 

populations of juvenile native fish that populate nearshore habitats created by the 

proposed Fall Steady Experimental Flow (FSEF). Our intent is to link this new insight 

into juvenile fish ecology, with a focus on humpback chub, with the flow and habitat 

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management capabilities of the Glen Canyon Dam (GCD) Adaptive Management 

Program (AMP), to create a better understanding of how flow and habitat management 

can be used to cultivate and enhance survival of juvenile native fish and, with time, adult 

native fish populations in Grand Canyon. 

Section 1.1 Conceptual Model – The questions developed in the RFP (RFP pages 27-

28) and RQ1 and RQ2 are part of a broader conceptual model of native and non-native 

fish population dynamics developed over the last 10-15 years in Grand Canyon. Such 

models have a long history of development in the Glen Canyon Dam Adaptive 

Management Program (e.g., Walters et al. 2000), and we summarize essential elements 

for juvenile humpback chub as they relate to this proposal: 

A. Humpback chub juveniles recruit to the mainstem juvenile population from the 

Little Colorado River as very small juveniles during the spring, and larger 

juveniles recruit to the mainstem during monsoon-driven flood events in latesummer and fall, or from mainstem spawning events. 

B. The quantity and quality of juvenile habitat in the mainstem is driven by variation 

in flow and temperature regimes and also channel morphology. Habitat 

characteristics are determined by the monthly average discharge and hourly 

variation in discharge from Glen Canyon Dam, as well as sediment supply in the 

mainstem, and the frequency and timing of flows from Glen Canyon Dam 

designed to create habitat believed to be important for native fish (e.g., 

backwaters). 

C. Survival rates of HBC juveniles in the mainstem depend on the quantity and 

quality of physical habitat, food availability, and the intensity of competition and 

predation from both native and non-native fishes. 

D. Abundance of HBC juveniles will increase with improvements in the quality and 

quantity of habitat because survival rates will be higher. Over the long-term, 

greater juvenile production will increase the abundance of the adult population. 

Section 2 Experimental Design - To resolve uncertainties in the conceptual model 

above, survival rates, growth, and abundance of native and non-native juvenile fish 

must be quantified under contrasting conditions of flow, which, in turn, drives variation in 

habitat availability and quality. The experimental design for this project, which 

determines the extent and timing of contrasts, has already been defined, in large part, 

by the GCD AMP. Over the next five years, there will be hourly variation in flow rates 

(related to diel variations in energy demand) during summer months (Mean Low 

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Fluctuating Flows, MLFF, June-August), and lower, steady experimental flows during 

the fall (Fall Steady Experimental Flows, FSEF, September and October). We will 

quantify abundance, survival, habitat use, growth, and natal source of selected native 

and non-native juvenile fish over three flow periods (summer MLFF, MLFF-FSEF 

transition, FSEF) based on four sampling trips (trip launch month July, August, 

September, October, Table 1) given this experimental flow regime. Due to costs, natal 

source would be determined for only a sub-set of species (likely HBC only) after 

discussions with partner agencies. 

Section 2.1 Sampling methods to estimate fish habitat use, growth, and survival – 

Estimation of juvenile abundance, survival rate, growth rate, and habitat use is 

fundamental to resolving uncertainties in the conceptual model and the two key 

research questions outlined and identified above. We propose sampling trips in late 

July and late August to characterize abundance, habitat use, growth, and survival rate 

of juvenile fish over the summer under MLFF flow fluctuations. These trips would be 

followed by sampling trips in early September and late October to characterize juvenile 

fish responses during the MLFF-FSEF transition, and FSEF period. Differences in 

abundance in each habitat type, between each sampling trips would be used to 

estimate habitat specific, reach-wide survival rates across flow events.

We propose two basic sampling approaches for estimating these characteristics: 

(1) reach-wide abundance estimation (RWAE); and (2) robust-design mark-recapture 

(RDMR) at replicate sites. The RWAE approach follows the two-stage design developed 

by Korman (2009) to track abundance, habitat use, growth and survival of age-0 

rainbow trout in the Lee’s Ferry reach of the Colorado River. Applying this approach to 

native and non-native juvenile fish in the lower Colorado River, we would: 

(1) Select a relatively large study reach that contains multiple habitat types (e.g., 

Kwagunt rapid [River Mile 56] to Lava Chuar rapid [River Mile 65.5]) and stratify 

this reach into sites by habitat type (informed by, and integrated with, ongoing 

GCMRC project 2, Table 2); 

(2) Sample fish at a large number of randomly selected sites within the reach 

using methods established in previous GCMRC efforts and Korman 2009 (e.g., 

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single-pass electrofishing and mini-hoop nets) to quantify catch per effort. Site 

selection would be stratified by habitat type which, in turn, would likely require 

use of multiple gear types. Gear types and number of samples required will be 

informed by, and integrated with GCMRC ongoing projects 1, 3, 5, 6, 7, 11 and 

Trip 1 of this proposal, Table 2 ; 

(3) Quantify capture probabilities by habitat/gear type, species, and fish size 

using short-term, closed, mark-recapture experiments (number of sampling 

events per trip ≥ 2 depending on recapture rate and number of sites from 2 

above, e.g., Korman et al. 2009 [see Appendix]) at a smaller number of sites; 

(4) Convert catch-rates at index sites (from 1 above) to population estimates 

based on capture probabilities estimated from (3 above); and 

(5) Scale up site-specific estimates of population size to a reach-wide estimate 

based on the amount of habitat of each type in the reach. 

Population size by sampling trip would be estimated for each habitat type and will 

be used to examine seasonal and flow-driven changes in habitat use. Fish would most 

likely be marked with a gear, habitat, and sampling pass specific mark using unique 

location and color combinations of Visual-Implant-Elastomer (VIE) (Brennan et al. 2005; 

GCMRC NSE pilot sampling, fall 2008). Ideally, unique marks would be applied to each 

animal (Bailey et al. 2004 for an example with VIE marks). Growth during summerunsteady and fall-steady periods could be quantified based on differences in lengthfrequency distributions over time, as well as by measurements of otolith increments on 

a small sub-sample of fish (as in Korman and Campana 2009 [see Appendix]; see also 

section 2.2.3 and 2.2.4 of this proposal). As in Korman (2009), we would integrate most 

information from the proposed program in a stock synthesis model to jointly estimate 

parameters of interest (including abundance and survival rate by habitat type, growth, 

recruitment to the juvenile population over the growing season) and to identify shifts in 

habitat use. This approach is based on standard closed population mark-recapture 

approaches to estimate abundance and capture probability (analogous to those outlined 

in Otis et al. (1978)) and allows for incorporation of size dependence in capture 

probability (to account for heterogeneity in capture probability by size). The critical 

assumption of the RWAE approach (and all closed population models) is that the 

population can be treated as effectively closed within trips and that differences in 

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abundance across trips are caused by recruitment (LCR and mainstem) and mortality 

only. The approach is unable to separate emigration from mortality. Radio telemetry 

data, and movement rates of marked fish within sites across trips would help evaluate 

the extent of potential emigration from study sites (see section 2.3.2 of methods). 

Immigration would be estimated. Differences in abundance and distribution among 

habitat types across trips would be used to estimate survival and changes in habitat use 

during changing flow conditions from summer MLFF, the MLFF-FSEF transition period, 

and FSEF conditions. If significant changes in abundance are observed, an additional 

sampling trip, if supplemental funds were available, following the return to normal flow 

operations in November could be added to assess fish population responses to the 

transition from experimental steady flows to winter operation flows. Modal shifts in 

length-frequency and direct ageing would be used to evaluate growth. 

 The second approach to estimating growth, survival, and abundance by habitat 

type is based on a robust design mark-recapture approach (RDMR) that would estimate 

habitat specific abundance for each trip using closed models (RWAE), and relax the 

assumption of population closure between sampling trips (Pollock et al. 1982). 

Essentially, the RDMR provides a finer-scale assessment of abundance and survival, 

and is generally similar to RWAE with the following differences: 

(1) Select a series of study sites within a broader reach (e.g., three to four 500-m 

sites between Kwagunt and Lava Chuar rapids). These sites would likely each 

contain a mix of all or most of the habitat types within the broader reach. 

(2) Conduct multi-pass (3-5 pass events) mark-recapture assays in each of the 

study sites during each of the four trips. 

(3) Estimate abundance at each study site on each trip. 

As with the RWAE approach, we would employ a marking strategy to estimate 

abundance within habitat types at each site for each trip. Multiple gear types would 

likely be needed to sample the full range of habitat types and water depths. Under the 

RDMR approach, we would assume that populations within each site are effectively 

closed within trips, but not among trips. We would also assume that changes in 

abundance at the sites across trips represent changes at a reach-wide scale. 

Differences in abundance and distribution in each habitat type across trips will be 

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assessed during summer-MLFF, MLFF-FSEF flow transition, and FSEF periods. 

Survival estimates for each habitat between sampling trips could be estimated using 

open population models (Pollock et al. 1990) following the robust design framework. If 

the recaptures between trips are too low to estimate survival, then the RDMR approach 

basically collapses to the RWAE approach for estimating survival – i.e., these 

approaches are not mutually exclusive in design or analyses employed. Shifts in 

length-frequency and direct ageing using otoliths will be used to evaluate growth (see 

section 2.2.3). 

Section 2.2 Site Selection - We propose to use existing data and models from the 

GCMRC physical science program (Projects 1, 2, and 10, Table 2 to quantify habitat 

availability over the study reach that contains the RDMR sites, habitat availability within 

the sites, and how habitat changes with flow. Such an approach has already been used 

to quantify juvenile chub habitat use in the Colorado River near the LCR (Korman et al. 

2003). The existing GCMRC shoreline GIS database and other surveys can be used to 

stratify habitat into classes such as talus slopes, open sand bars, vegetated sand bars, 

cobble bars, and backwaters. Existing bathymetry and hydrodynamic two-dimensional 

models that cover the entire study reach can be used to predict water temperature, 

depth and velocity in shoreline habitats at daily minimum and maximum flows during the 

summer-unsteady period, and at the average flow during the steady-fall period (Korman 

2003; Projects 1 and 10, Table 2). Velocity and temperature criteria will be used to 

further classify habitat for various species and life stages (e.g., into usable and 

unusable categories, or some finer scale). Additionally, shoreline stability during the 

summer can be quantified based on daily flow variation. For example, in summer, 

backwaters that are barely flooded at the daily maximum flow may be far more stable, 

warmer environments than those subject to complete inundation on each diel flow cycle. 

Similarly, steeper shorelines with cover (e.g., vegetated cut banks) may be more stable 

than low angle, open sand bars or cobble bars, where low velocity littoral areas vary 

more. These characteristics of stability can be related to patterns of fish habitat use 

(density or occupancy) to help evaluate effects of fluctuating flows. A strong 

relationship between fluctuating flows, nearshore habitat use, and otolith growth has 

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been observed for age-0 rainbow trout in the Lee’s Ferry reach of the Colorado River 

(Korman and Campana, 2009). Similar relationships are expected for juvenile native 

and nonnative fish in the Grand Canyon and, in fact, is part of the motivation for the 

current fall steady flow test. We hypothesize that unstable habitat types will be used 

only minimally during the summer-unsteady flow period, but that use of these habitats 

will increase during the fall-steady period when flows are stabilized. If this difference in 

habitat use is ecologically important, we would also predict increase in growth and 

survival during the fall-steady flow period relative to the summer. Information we collect 

as part of Sections 2.1 and 2.3 can be used to test these ideas directly. 

Section 2.3 Assumption evaluation and alternative approaches to assess native fish 

habitat use and response to FSEF - Any mark-recapture approach to estimating 

abundance and density depends on recapturing sufficient numbers of marked 

individuals to draw inferences on the parameters of interest. In many fisheries markrecapture studies, capture probabilities are low (often < 10%, Pine et al. 2003) and 

heterogeneity in capture probability across fish size is often observed (Pine et al. 2003; 

Korman et al. 2009; Coggins 2008). Closed population models generally have fewer 

parameters (and assumptions) than open models and are thus better able to estimate 

parameters of interest (capture probability and abundance) when recaptures are low. 

By design, some closed models can account for heterogeneity in capture probability 

directly (i.e., Mh type models, Otis et al. 1978) or captures and recaptures can be 

stratified by length group and capture probability estimated for each length stratum. 

These are two key reasons why closed population models have been recommended for 

use in fisheries studies to estimate abundance (Pine et al. 2003). One concern is the 

closed model assumption of no emigration from the sites of interest (i.e., habitat specific 

sites) both for the RDMR and RWAE approaches to estimate site specific abundance. 

Korman et al. 2009 successfully used closed model techniques in the Lee’s Ferry reach 

of the Colorado River to estimate capture probabilities of juvenile rainbow trout across 

spatially discrete sites. These authors evaluated closure assumptions by employing a 

site specific marking technique and then sampling up and down-stream of their study 

reach to estimate emigration from each site. Emigration rates were low, ranging from 

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2.2-2.6%, leading these investigators to conclude that rainbow trout populations within 

discrete sites effectively can be considered closed. We would evaluate the closure 

assumption in our mark-recapture experiments using methods similar to Korman et al. 

2009. Additionally, recaptures of fish marked on previous trips will provide useful 

information on growth and movement (e.g., movement into backwaters during periods of 

steady flow) between sampling trips and associated flow conditions. The NSE pilot 

sampling data from 2008 should provide some information on closure and also provide 

information on capture probability which is necessary to fully assess how violation of the 

closure assumption biases abundance estimates. Zehfuss et al. (1999) demonstrate 

declining bias in abundance estimates with increasing capture probability and 

emigration from the study site for Jolly-Seber and robust design models. 

Section 2.3.1 Estimating site occupancy - If catches and recapture rates within trips are 

low, abundance estimates within sites will be highly uncertain. However, as pointed out 

in the RFP, in this case it will be possible to retreat from estimating abundance to 

estimating the probability of site occupancy (by habitat type and over time; MacKenzie 

et al. 2006). Site occupancy, although not as useful as direct estimates of abundance, 

can be used to index habitat use and changes in habitat use over time and with 

associated changes in flow. In all likelihood, the mark-recapture program will be able to 

estimate habitat-specific abundances for some species and size classes, and 

occupancy would be estimated for species and sizes with lower abundance and/or 

capture probability. Total number of site visits required would be based on trip 1 and 

information from ongoing GCMRC projects 3, 6, 7, 11 (Table 2).

Section 2.3.2 Incorporating telemetry information - The potential exists for radical 

changes in capture probability or behavioral shifts by fishes across sampling events 

within a season (~July-October) due to fish growth and possible ontogenetic habitat 

shifts such as fish moving into deeper, less accessible portions of the river. These 

behavioral shifts could also be caused by the designed flow experiments and 

associated changes in available habitat, or biotic interactions (changes in food 

availability or predation risk). To examine these behavioral responses, directly assess 

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habitat use, and test capture-recapture model assumptions such as closure, a small 

sub-set of native and non-native fish (5-10 individuals of 3-4 key species, including 

humpback chubs, > 150-mm TL) will be tagged with telemetry tags and their 

movements, habitat use, and fates assessed directly. 

Implantable, compact telemetry tags coupled with autonomous receivers (which 

monitor a given area continuously for extended time periods for the presence of tagged 

animals) and deployed as a fine scale array within a sample reach could allow the 

telemetered animals to be “virtually” captured by detecting their tags as they move 

through the environment via either the autonomous receiver array, or from boat-based 

receiver units when field crews were onsite. This technology is mature, and thousands 

of telemetered fish and autonomous receivers are currently deployed globally 

(Simpfendorfer et al. 2008; Hedger et al. 2008). However, the use of telemetry 

techniques in Grand Canyon is technologically challenging, given the large amount of 

ambient noise from rapids, moving rocks, and coarse sediments, all of which greatly 

impair the ability to detect acoustic tags. Radio tags have been used successfully in 

Grand Canyon to track movements of adult humpback chubs (Valdez and Hoffnagle 

1999) in response to flood events, but the attenuation distance of radio tags is greatly 

impaired by high conductivity in areas of Grand Canyon near the Little Colorado River. 

Current GCMRC projects 3 and 7 have been involved in several pilot projects exploring 

the utility of these techniques with non-native fishes to assess movement and capture 

probability. Industry cooperator Marlin Gregor, President of Sonotronics Inc. 

(http://sonotronics.com) has been involved in the above projects and has developed 

specific tag and receiver design combinations to maximize the potential of this 

technology in the challenging conditions presented by the Colorado River in Grand 

Canyon. Marlin Gregor has also agreed to accompany on our first sampling trip as we 

test a variety of receiver and tag types. If this pilot sampling during trip 1 reveals poor 

performance of telemetry equipment due to high levels of ambient noise in Grand 

Canyon, we will abandon the telemetry component of this project. We will work with 

cooperators to then identify alternative research efforts such as experiments to assess 

DNR:RNA ratios as a measure of short-term growth as recommended by NSE review 

panel. 

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The incorporation of telemetry information with the mark-recapture sampling is 

experimentally risky, but has huge potential to boost our ability to draw inferences about 

juvenile fish abundance and habitat use. Pollock et al. (2004) showed an example of 

using both tag types to improve estimation of fishing and natural mortality rates using a 

complementary, multinomial approach to the tagging component of the stock synthesis 

model used in Korman et al. in-press and this proposal. We have extensive previous 

experience with these types of telemetry systems and similar applications (Bennett 

2005; Marcinkiewicz 2007; Tetzlaff 2008; Burgess 2008). Coupled with ongoing 

GCMRC projects 3 and 7, and industry cooperator commitment, it is likely that 

technological hurdles could be overcome. Exact tag types, receiver deployment 

strategies, numbers of tags and receivers required would be determined based on insitu range testing of tag and receiver combinations. An array network would not 

guarantee perfect, continuous detection because behaviors of individual animals such 

as occupying habitat types similar to interstitial spaces between rocks, would attenuate 

the signal from the sonic tags. Conceptually, an autonomous receiver array could be 

deployed during a July sampling trip (first trip of sampling season) and a small batch of 

fish tagged and released with telemetry tags. The receivers would then remain 

deployed, monitoring these fish until the next sampling trip, at which time the data would 

be retrieved and an additional batch of fish tagged. Similar procedures would be 

followed for each additional trip, until the last trip of the year when no additional fish 

would be tagged and the receivers would be removed from the study site. Multiple 

batches of fish would be used (a “staggered entry” design, Pollock et al. 1989) because 

of limitations in tag battery life. The staggered entry design would allow specific size 

cohorts of fish to be tracked from the first through the fourth study period coincident with 

the same flow transition periods discussed in Section 2.0. 

Section 2.3.3 Determining origin of recruits – The FSFE will likely almost immediately 

create thermally favorable microhabitats for juvenile HBC and other native fish (RFP, 

page 27) which motivates Question 6 of the RFP (page 27), “Are replicated flows 

September-October steady flows (2008 through 2012) likely to improve the survival and 

recruitment of young humpback chub in the Colorado River ecosystem?” Additionally, 

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while management actions such as FSFE are certainly likely to modify and create 

habitat types, some of which may be favorable to juvenile HBC, where do the juvenile 

HBC come from to colonize these habitats and if they colonize these habitats, how well 

do they survive? As outlined in Sections 2-2.2, we describe approaches to assess 

survival of young HBC (and other species) in response to the FSFE. However, 

recruitment of young HBC and other juvenile fish from the LCR to the mainstem study 

site during July-September (Conceptual Model point A) could confound information on 

patterns in juvenile fish density in different habitats and their survival, unrelated to flow 

events. For example, in the absence of any direct measure of age-0 fish recruitment 

from the LCR to the mainstem population, observed patterns in density, growth, and 

survival of juvenile fish from our mark-recapture study could be attributed to the flow 

experiment, when, in fact, these density changes were actually independent of the 

experiment, and instead related to natural emigration from the LCR to the mainstem. 

Without an understanding of this recruitment from the LCR to the mainstem, the 

estimated growth, survival, and movement among habitat types in the mainstem from 

the tagging data (Sections 2-2.2) could be confounded with new recruits. 

We propose to use otolith microchemistry to determine the origin of juvenile fish 

(mainstem vs. LCR), most likely only HBC because of cost concerns, utilizing available 

habitats across the different flow regimes from July-October. As pointed out in the RFP 

(page 27) there are at least two potential sources of juvenile HBC that use habitats 

available during MLFF and migrate to habitats created by FSFE: (1) LCR spawned 

juveniles that enter the mainstem river in May-June outmigration and (2) LCR spawned 

juveniles that remain in the LCR until they enter the mainstem following July-September 

freshets. A third possible source of HBC juveniles would be mainstem spawned 

individuals who could also be identified through otolith microchemistry. This information 

will be used to compliment and strengthen inference from estimates of habitat specific 

patterns in abundance, movement patterns of telemetered fish, and fish size 

distributions (Sections 2.1-2.3). 

Otoliths are paired structures that form part of the inner ear of teleost fish and are 

commonly used by fisheries researchers to estimate fish age, and increasingly, their 

chemical constituents are used to determine fish movements (Campana 2005). Otoliths 

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are composed of biogenic calcium carbonate that accretes new crystalline and protein 

material daily. Within these accumulating layers, trace elements are incorporated into 

the otolith from the fishes environment, creating a temporal sequence of accumulated 

elements. This represents a chronology of the environment the fish has occupied over 

its life (Elsdon et al. 2008). Within the otolith, the different accumulated elements and 

chemical signatures can be used as a natural tag. These tags can then be linked to 

groups of fish that have similar chemical signatures to show associations of fish with 

their environment in both time and space (reviewed in Elsdon et al. 2008). Chemical 

signatures in an otolith can be examined, for example across a transect, to assess 

patterns in chemical profiles at different ages of the fish. When these chemical 

signatures are paired with information on the chemical profiles of the water in the spatial 

locations where the fish could have lived, movement patterns of individuals and groups 

of fish can be determined. A key assumption is that the chemical signatures from the 

different environments are separable, and that these signatures can be matched on the 

correct temporal scale. To test these assumptions, during 2009 we will test our 

proposed methods on flannelmouth sucker (Catostomus latipinnis) a more common 

native species thought to have similar life history characteristics as humpback chub. 

Samples of flannel mouth sucker are also available from the 2008 NSE pilot sampling 

(M. Yard, USGS-GCMRC, personal communication). We will attempt to analyze at 

least a sample of these otoliths from 2008 to inform 2009 field collection efforts. 

Additionally, we will also examine a sample of previously collected adult humpback 

chubs used in earlier analyses (Hendrickson 1997). 

Recent advances in otolith microchemistry will enable us to assay for a suite of 

major, minor, and trace elements through the use of laser ablation inductively coupled 

plasma mass spectrometry (LA-ICPMS; Figure 1). SUNY-ESF has just acquired a stateof-the-art laser ablation system (New Wave Research UP-193 nm solid state) and has 

connected it to an also new Perkin-Elmer DRC-e ICPMS. This setup, with analytical 

resolution to low ppb and spatial resolution to approx. 10 microns, will be used to collect 

the bulk elemental data, using one of each pair of lapillar otoliths. Other data, 

potentially including strontium and other stable isotopic ratios will be taken on the 

corresponding otolith at a partner institution such as University of Arizona Lunar and 

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Planetary Science Laboratory or the Department of Nuclear Physics at Lund University 

in Sweden (where KL has ongoing collaborations since 1997). In addition, we will 

explore the utility of trace elemental mapping by means of synchrotron-based X-ray 

fluorescence (S-XRF) at the Cornell High Energy Synchrotron Source (CHESS). The SXRF will allow us to explore which elements might be of interest to trace using the LAICPMS. This approach of using the S-XRF to first map multiple trace elements in 

otoliths and the coupling of this information with high-resolution LA-ICPMS work has 

provided new insights into the environmental history of a variety of fish (marine and 

freshwater) and their lifetime movement patterns (see Limburg et al. 2007). Given that 

run times per otolith are long with S-XRF, and time available is limited at the CHESS 

facility, we anticipate that bulk of the work will be accomplished using LA-ICPMS once 

multiple otoliths have been mapped at CHESS. In addition to addressing questions 

related to the nearshore ecology of Grand Canyon fishes proposed here, there are 

obviously additional research questions that these approaches could be applied to using 

otoliths from existing collections. For example, older fish from archival collections (HBC 

otoliths used in Hendrickson 1997) are prime candidates for S-XRF mapping coupled 

with LA-ICPMS analysis to assess movement patterns and provide alternative age 

assessments of larger, older, fish collected at earlier time periods during different river 

conditions. If suitable archived samples were available, such as from HBC spawned 

prior to the Glen Canyon Dam, it would be very interesting to assess the life time 

movement patterns (spawning, rearing, and migratory) or pre- vs. post-dam HBC or 

other native fish. 

As an example of this approach Limburg (1995, 1998, 2001) used ratios of 

strontium (Sr) relative to calcium (Ca) from American shad (Alosa sapidissima) otoliths 

along a transect in the Hudson River, linked with otolith derived age estimates to identify 

the age and size when American shad were transitioning from the Hudson River to the 

Atlantic ocean. Limburg (2001) compared Sr:Ca ratios and size and age structure in 

young-of-year American shad with the Sr:Ca ratios of adult fish from these same 

cohorts migrating back to spawn in the Hudson River in subsequent years. From these 

comparisons Limburg (2001) found that in three of the four year classes analyzed, 

differential mortality had occurred in juveniles, and was related to time of emigration 

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from the Hudson River. A similar approach with humpback chubs over multiple years, 

coupled with Sections 2-2.2 of this proposal, could help us to identify the linkages 

between the timing of out migrating juveniles from the LCR, available rearing habitat in 

the mainstem as determined by flow conditions, and the interaction between available 

habitat and juvenile fish survival. 

Section 2.3.4 Determining growth and survival using otoliths - Measurements of fish 

growth integrates information from a variety of environmental (e.g., temperature) and 

biotic (e.g., food availability) sources into a useful single metric. Growth can be used, 

for example, as a response metric to a variety of management options (DeVries and 

Frie 1996) and also used to provide insight into survival patterns (Lorenzen 1996). For 

larval and juvenile fish, growth information is commonly measured by analyzing size 

frequency distributions or using fish hard parts such as otoliths (DeVries and Frie 1996). 

The use of otoliths to determine juvenile fish growth has been used to address a variety 

of questions for Colorado River native (Bestgen et al. 2006) and nonnative fish (Korman 

and Campana 2009). For example, Korman and Campana (2009) documented a 

strong relationship between fluctuating flows, nearshore habitat use, and rainbow trout 

otolith growth rates in the Lee’s Ferry reach of the Colorado River. As proposed in 

section 2.2, we hypothesize that fish growth may change as a function of shifts in 

habitat utilization driven by flow related changes in habitat availability – thus differences 

in growth may be evident between MLFF, MLFF-FESF transition, and FESF period. 

We propose to assess growth in three ways: (1) by tracking modal progression of 

native and non-native fish size in during each sampling trip, (2) if unique VIE marks are 

possible through recaptures of marked fish, and (3) direct estimation using otoliths 

collected for the microchemistry analyses of HBC described above. The first approach 

is straight forward and simply requires plotting the size frequency of individuals from 

each species and sample trip as a function of length and identifying distinct peaks in the 

distribution. These peaks are then followed in subsequent trips and growth estimated 

by tracking the progression in the modal peaks of each size frequency distribution. This 

approach generally works best if sample sizes for each cohort are large and growth is 

fairly uniform among cohorts such that the size modes for each cohort can be 

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distinguished. If sample sizes are small or growth within a cohort is inconsistent, then 

cohort modes become indistinguishable in the length frequency distribution and 

estimating growth is not possible. This approach is low cost, non-lethal, and would be 

used to estimate growth for all possible fish species. 

The second approach is dependent on being able to mark fish for the capturerecapture experiment using unique marks. The VIE marks we propose to use come are 

available in a variety of colors and there are numerous body locations in which fish can 

potentially be marked. However, it is not until live fish are examined and the color 

patterns and marking locations assessed to determine whether VIE colors can contrast 

sufficiently with natural fish color patterns to offer reliable marking locations. We 

acknowledge the difficulty in creating a unique marking scheme and the potential bias 

induced in the analyses from mistakes made in the marking program thus necessitating 

careful consideration to the design of the fish marking program. 

The third approach is based on the standard technique of visually counting daily 

growth rings on otoliths to estimate age (Pannella 1971; Stevenson and Campana 

1992; Pine and Allen 2001). Following this approach, the second HBC otolith from 

whichever otolith pair is used for the microchemistry analyses (most likely the lapillar 

pair; Hendrickson 1997) would be mounted individually on microscope slide, polished to 

the mid-plane following standard otolith preparation procedures, examined at 400-1250x 

using a compound microscope, and then the daily increment marks on the otoliths from 

the origin to the edge counted 2-4 times. Daily otolith increments have not been 

validated in humpback chubs, but preliminary work by Hendrickson (1997) provides 

strong circumstantial evidence to suggest that the lapillar increments do form with daily 

periodicity. If recapture rates between trips are sufficiently high as evident from trips 1 

and 2 in field season 1, a validation study could be designed by immersing juvenile HBC 

in an alizarin solution to “mark” the otolith, then releasing the fish with the expectation to 

recapture that fish on a future trip. Recaptured fish would then be sacrificed, otoliths 

removed and prepared as described in section 2.3.4, and the otolith rings since the 

immersion in alizarin counted and compared to the actual days elapsed (Devries and 

Frie 1996). Average daily growth rates can then be determined from dividing the fish TL 

when captured by the age. 

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Recent juvenile HBC growth over short time periods, such as just prior to and 

during the transition from MLFF to FSFE and during the FSFE, could be estimated by 

measuring otolith growth increments from the nucleus to a distance along the growth 

axis that would correspond to a point in time (and the fish’s life) during MLFF, and then 

measure from this point to a second point that would represent the time period including 

the MLFF-FSEF transition, and then a third measurement from the MLFF period to a 

time period near the end of the FSEF. The approach outlined would obviously be 

dependent on collecting juvenile HBC near the end of the FSEF such that the growth 

transition could be measured across each of the three flow periods described. 

Obviously these candidate fish would have to survive any changes created by the flow 

events – and it would be impossible to collect growth information on any fish that died 

during the flow events. Alternatively, juvenile HBC could be sampled on each of the 

four sampling events and incremental growth measured from these fish using methods 

described above. However, given the short time period between the flow treatment and 

the timing of the sampling trip, contrasting growth information may be not detectable 

along the post-rostral edge of the otolith because not enough time may have elapsed for 

sufficient otolith deposition along the edge to see or measure. This growth information 

would be linked to the otolith microchemistry results related to natal origin of HBC. For 

example, this linkage could be useful in assessing growth patterns of HBC that were 

early vs. late season emigrants from the LCR to mainstem Colorado River or growth 

patterns of mainstem origin juvenile fish. 

A major drawback of using otoliths to estimate age and growth is that the fish 

must be sacrificed to extract the otoliths. Given the listing status of most native fish in 

Grand Canyon, including HBC, we share concerns that any research associated 

mortality could be deleterious to the population overall, and we acknowledge that 

permits to collect and sacrifice these fish may be difficult to receive. Recruitment trends 

for humpback chubs and other native fish have been increasing over the last 4-5 years 

(ongoing GCMRC projects 5, 6, Table 2; Coggins 2008) reducing the potential for 

population impact from removing a small number of native fish from the population for 

research needs. Exact numbers of fish required would be determined by examining 

otoliths from juvenile humpback chubs incidentally taken as part of routine sampling in 

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prior research efforts (i.e., Near Shore Ecology pilot sampling in August and September 

2008 and backwater sampling efforts in summer 2005). We propose to collect a small 

number of juvenile fish, most likely 5-10 age-0, age-1, age-2, and age-3 humpback 

chubs and also flannel mouth suckers each trip (no more than 50 fish per age class and 

species per year). In addition to providing information from the microchemistry and agegrowth analyses in response to the FSEF, there are at least two other major areas that 

could be assessed by using tissues from these animals. First, age information on these 

small fish would be very useful in informing the age-length key used to assign age-atfirst capture in the standard age-structured mark-recapture program (ASMR, Coggins et 

al. 2006a; GCMRC projects 4 and 6, Table 2) used to assess trends in HBC growth, 

recruitment, and survival (Coggins et al. 2006b; Coggins 2008). This age-length key is 

currently based on a limited set of approximately 60 estimates of HBC length-age 

(Coggins 2008) and this small growth sample, particularly for very small and young fish, 

could be improved with additional growth information possibly leading to reduced bias in 

the recruitment estimates from the ASMR model. Additionally, the previous growth data 

were not collected during the recent time period of warmer water temperatures, which 

may have led to increased HBC growth. Thus, the currently used growth information 

may be negatively biased due to slow growth of humpback chubs in cold-water 

conditions vs. the current warmer-water conditions. 

Fin rays also offer an alternative approach for aging fish to otoliths with the major 

advantage of not having to sacrifice the fish (DeVries and Frie 1996). Using a sub-set 

of fish sacrificed for otolith analyses, we will conduct an exploratory analysis to assess 

the feasibility of the same age and microchemistry assessment on the fin ray sections 

as on the otoliths. This will provide information on the possibly utility of using fin rays as 

a non-lethal technique to determine age of young fish to help inform the HBC stock 

assessment and monitoring program (ongoing GCMRC projects 3 and 4). If successful, 

and necessary, this approach could provide information useful to assessing recruitment 

responses of HBC to planned and unplanned experiments at younger ages (and less 

time lag) than the current 3-4 year response time required before fish recruit to the 

tagging program and recruitment trends assessed as part of ASMR. 

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A final alternative for both the microchemistry and otolith based age and growth 

estimation would be to use a surrogate species for humpback chubs that was thought to 

have a similar life history. Alternative species to humpback chubs could include native 

fish such as bluehead suckers Catostomus discobolus, flannelmouth Catostomus 

latipinnis suckers, speckled dace Rhinichthys osculus or nonnative species such as 

fathead minnows Pimephales promelas. 

Section 3.1 Contingency plans to alternate flow scenarios, expanded spatial replication, 

or inability to secure variance during no-motor season – The proposed sampling 

framework and experimental design (i.e., timing of trips) can easily be modified to 

address alternate flow-related hypotheses. For example, question (9) in the RFP (page 

28), “What happens to juveniles that used the warmed areas when fluctuating flows are 

resumed?”, could be addressed by adding one or two trips (and additional costs) to the 

proposed four-trip schedule. In this case, a trip following the resumption of fluctuating 

flows, (mid-November) and an additional trip one month later, would quantify immediate 

changes in habitat use as well as survival rates under fluctuating flows during early 

winter. A similar strategy, where sampling is conducted before and after a beach 

habitat building flow, could be used to evaluate the effects of flows above power plant 

capacity or normal daily maxima (item 6, RFP page 31). Additional spatial sampling, at 

other known HBC aggregation sites (“Randy’s Rock”, RM 126; “30-mile”, RM 29-32) 

could also be implemented based on results from Section 2 above. The key point is 

that our approach will provide estimates of habitat use, survival, growth, and origin of 

juvenile fish during summer and fall, and that it is relatively easy to expand sampling 

into other times of year to look at other flow-related hypotheses. That said, the 

objectives and research questions that are the focus of the RFP are very challenging. 

Our philosophy is to do a few things well, rather than many things poorly. Given 

available resources, we will focus on population dynamics during summer and fall only 

as outlined above. 

We expect that some elements of the proposed sampling framework would 

become part of the GCD AMP’s long-term monitoring protocol. For example, methods 

developed in this project could be used to estimate juvenile HBC abundance in the 

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mainstem during late summer and late fall. Over many years, these estimates could be 

related to recruitment estimated from the Age-Structure Mark-Recapture (ASMR) model 

(Coggins et al. 2006 a, b). Ultimately, a stock-recruitment relationship between age-0 

chub in the mainstem during both fall and summer, and recruitment determined from the 

ASMR model, would be very helpful in the interpretation of the juvenile data. For 

example, if there is strong density dependence in survival between summer and fall, 

and little density dependence for later life stages, we would expect an asymptotic 

relationship (e.g., Beverton-Holt shaped relationship) between summer and fall juvenile 

abundance, and between summer juvenile abundance and ASMR-based recruitment 

estimates. In this case, there would also be a strong linear relationship between fall 

juvenile abundance and ASMR recruitment. Such analyses are very relevant to current 

policy debates. For example, strong density dependence between summer and fall 

would indicate that it is unlikely that improving habitat conditions during the summer will 

result in substantive changes in juvenile abundance during the fall, and consequently to 

improvements in recruitment to the adult population. It will take many years to establish 

reliable relationships (which will likely be noisy), but we see no alternative for the GCD 

AMP. Ultimately, life-stage specific stock-recruitment functions need to be established 

to fully interpret results from the proposed study in the context of GCD AMP 

management objective for adult HBC. 

Depending on the start of the FSFE, it is likely that one or two of our proposed 

sampling trips would launch after the start of the no-motor season (September 15). In 

this case, we would require a variance from the Park Service for operating motorized 

equipment (boat motors and generators) required for our electrofishing sampling. While 

we intend to use multiple gear types (most likely electrofishing and hoopnets) during 

each of our sampling events, we have high expectations for successful electrofishing 

sampling for use in mark-recapture (Section 2.1) based on results from Korman et al. 

(2009) and fall 2008 Near Shore Ecology pilot sampling. Failure to secure a variance 

allowing us to use electrofishing would increase our dependence on hoopnet sampling 

to track patterns in habitat specific abundances, estimate survival, and track growth via 

changes in length frequency. Gear selectivity patterns between electrofishing and 

hoopnets likely differ, and by deploying both gears on each sampling trip we intend to 

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minimize the confounding that can occur in the data due to changes in gear between 

trips and associated selectivity patterns (i.e., responses could be due to the flow 

experiment or simply to changes in the gear type). Compromises to help meet the Park 

Service’s no-motor requirements are possible. One option would be to create a stash of 

1-2 electrofishing boats in the study reach just prior to the end of no-motor season. Any 

trips that occurred during the no-motor season would then be supported by rowing trips 

out to the sampling sites, where electrofishing boats would be used to complete the 

required sampling effort. At the end of sampling, these boats would be floated or flown 

out of Grand Canyon in cooperation with other sampling efforts with USFWS and AZGF. 

Additional options would be to increase the reliance on telemetered fish and to follow 

the movements and habitat use of telemetered fish using rafts and kayaks during no 

motor season. A final option would be to not grant the variance; thus, electrofishing 

would not be possible. Our proposed closed population models (both mark-recapture 

and occupancy) estimates capture probability with each sampling trip, so if hoopnets 

were the only sampling gear available during the last trip, then our capture probabilities 

would be based on marks and recaptures from hoopnet gear only. Hoopnet catches 

alone would likely result in very low capture probabilities, limiting the use of some markrecapture models. However, habitat occupancy using methods outlined in section 2.3.1 

may be used to estimate occupancy even with low capture probabilities. If no variance 

is granted, extensive comparisons between electrofishing and hoopnet catches based 

on pilot sampling during motor season and previous fish sampling efforts in Grand 

Canyon would be done to develop a better understanding of the selective properties of 

each gear. The style of electrofishing developed and used by Korman et al. (2009) was 

first used in the Lee’s Ferry reach, but was also used as part of the pilot NSE sampling 

during fall 2008 thus limiting the number of comparisons that can be made between 

gear types using historical data. 

Section 4 Conclusions - Certain aspects of the proposed experimental design are 

beyond our control, and will limit the inferences from the resulting data. We propose to 

compare juvenile habitat use, survival, and growth during summer-unsteady and fallsteady flow periods to evaluate the potential benefits of steady flows. However, such an 

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assessment assumes there is no seasonality in the parameters of interest, which we 

know is not likely to be the case. For example, growth rates will likely decline as fish 

increase in size, and as water temperature declines due to reduced solar irradiance. 

Habitat use may also have a flow-independent component that depends on fish size. 

Ultimately, the proposed work, or key elements of the proposed work, will need to be 

repeated under an alternate flow regime. The two logical alternatives are to extend the 

period of steady flows (e.g. July-Oct), or to revert to unsteady flows (MLFFA or greater 

fluctuations) during the fall. Results from the proposed work will be helpful in choosing 

among these alternatives because: (a) we will have a much better sense of our ability to 

detect changes in juvenile survival rates and other life history characteristics; and (b) 

expected and serendipitous insights from the proposed work will provide for more 

refined hypotheses and a more fully articulated conceptual model. 

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Table 1. Proposed sampling schedule and key tasks for Near Shore Ecology of Grand 

Canyon Fish project. MLFF = Mean Low Fluctuating Flows, FSEF = Fall Steady 

Experimental Flows. 

Year Activity Month Trip 

Number

Likely 

river 

flow 

Key tasks or project milestones 

2009-

2011 

Field sampling Late July 1 MLFF Mark-recapture, deploy receiver array, implant 

telemetry tags, manual tracking of telemetered 

fish, collect fish for otolith analyses 

2009-

2011 

Field sampling Late August 2 MLFF Mark-recapture, implant telemetry tags, manual 

tracking of telemetered fish, collect fish for 

otolith analyses 

2009-

2011 

Field sampling Early 

September 

3 MLFFFSEF Mark-recapture, implant telemetry tags, manual 

tracking of telemetered fish, collect fish for 

otolith analyses, possible otolith validation 

experiment 

2009-

2011 

Field sampling Late October 4 FSEF Mark-recapture, retrieve receiver array, implant 

telemetry tags, manual tracking of telemetered 

fish, collect fish for otolith analyses, possible 

otolith validation experiment 

2009-

2011 

Sample 

processing 

October - 

March 

Estimate growth from size distributions, 

estimate abundance and survival from mark 

recapture, assess movement and habitat use 

from telemetry information, and conduct otolith 

analyses 

2009-

2011 

Annual Report April Present annual project updates, submit 

reports, meet with all cooperators to revise 

sampling plans for upcoming field season 

2011-

2012 

Analyze data 

and write report 

May 2011-

Aug 2012 

2012 will include additional isotopic analyses of 

otoliths and funds for mapping chemical 

signature of water 

2012 Final Report September Submit final report and presentation to 

GCMRC staff 

825 

Pine et al. Page 26 Near shore ecology 
826 

827 

828 

829 

Table 2. Existing monitoring and research studies in Grand Canyon as listed in the 

RFP. Numbers in this table correspond to referenced cooperative and complimentary 

research described in this proposal. 

Project Number Project title 

1 Monitoring of biological and physical aspects of backwater habitats 

2 Integrated analyses and modeling: mapping shoreline habitat 

changes 

3 Monitoring mainstem fishes 

4 Stock assessment of native fish in Grand Canyon 

5 Investigate factors affecting the survival rate of juvenile native fishes 

in the mainstem Colorado River 

6 Native fishes habitat data analysis 

7 Nonnative control planning and pilot testing 

8 Status and trends of Lees Ferry trout 

9 Monitoring rainbow trout redds and larvae 

10 Thermal modeling of near shore habitat 

11 Aquatic food base program 

830 

Pine et al. Page 27 Near shore ecology 
Pine et al. Page 28 Near shore ecology 

831 

832 

833 

Optical image Sr map Zn map

Fig. 1. Fish otoliths are located in the semi-circular canals underneath the brain (top left); daily growth 

increments seen in 0.5 mm otolith of young fish (top right); annual rings (bottom left) can be used to 

age adult fish, and elements such as strontium are deposited in proportion to ambient concentrations 

(bottom, middle) whereas zinc (bottom right), in this case, is deposited in a seasonal pattern. The 

analyses shown in the bottom two panels were conducted with a nuclear microprobe. This kind of 

analysis will continue to complement the work we are proposing to do with LA-ICPMS.