Glen Canyon Dam is operated primarily as a load-following hydropower facility, increasing and decreasing dam discharge to match power demand. This periodic “flushing” of the river reduces the residence time of water as well as the availability of certain shoreline habitat types. In contrast, steady flows can increase the retention time of water in littoral areas such as backwaters and low-angle shorelines, and if discharge volume and ambient temperature are appropriate, can locally increase water temperatures. The NSE project evaluated experimental steady flows that occurred from 1 September- 31 October in each year 2009-2011. This study took place between river km 102-106 just downstream of the confluence of the mainstem Colorado and Little Colorado rivers where most prior research on humpback chub in Grand Canyon has occurred. The timing and magnitude of the steady flow experiment was developed by resource managers independent of the NSE team. Experimental flow regimes were about 10% of the unregulated (pre-Glen Canyon Dam) annual fluctuations.
Prior to the NSE project, our understanding of juvenile humpback chub ecology in the mainstem Colorado River was deficient compared with our knowledge of adult humpback chub primarily because of limited sampling of mainstem habitat for juvenile life stages. The NSE project developed a sampling and analytical framework to directly assess juvenile humpback chub survival, abundance, individual growth, and habitat use. These analyses used spatially referenced mark-recapture experiments with multiple gear types and determined provenance (birth river) through otolith microchemistry. This direct assessment of key vital rates complements indirect approaches used to estimate survival through population modeling efforts. For example, age-structured-mark-recapture (ASMR, Coggins et al. 2006) reconstructs juvenile abundance and survival through time from adult population numbers (estimated from mark-recapture) and assumes survival relationships based on life-history characteristics and growth rates. In contrast, the NSE project directly estimates juvenile fish population metrics in terms of abundance, survival, growth, or habitat use, which is useful for rapidly assessing how juvenile humpback chub respond to management actions such as experimental flows. 
Results and Discussion
The NSE project found that annual apparent survival of juvenile humpback chub (size at tagging < 100-mm total length, TL) did not differ significantly between the extant fluctuating flows and the experimental steady flow treatments (Finch et al. in-review A). The NSE project also documented that juvenile humpback chub were able to survive and rear in the mainstem Colorado River even at small sizes of 40-100 mm TL (Finch et al. in-review B). A somewhat surprising finding was that growth in juvenile humpback chub declined during these short-term steady flows versus fluctuating flows even though water temperatures were generally similar (Finch et al. in-review B). Reasons for this counterintuitive growth response are not known, but Finch (et al. in-review B) hypothesizes that food availability in the drift (primarily aquatic insects) is higher in fluctuating flows than in steady flows.
In Grand Canyon the creation, maintenance, and persistence of specific habitat types are considered critical for the persistence and recovery of native fish populations, including humpback chub. Backwaters are thought to be more similar to the Colorado River ecosystem prior to river modification because they are generally warmer and may be less influenced by river stage and dam operations than other mainstem habitat types. The NSE study compared abundance, density and habitat selection patterns between shoreline habitats (cliff, talus, debris fan, sand, backwater) and found that abundance of juvenile humpback chub was consistently highest in talus habitats and lowest in backwater habitats (Dodrill et al., in-review). Juvenile humpback chub showed positive selection for backwater habitats, but the spatial extent of backwater habitats in the NSE study reach was small compared to other habitat types. Additionally, ultrasonic telemetry of larger juvenile humpback chub (about 180-190-mm TL) found that habitat selection and daily movements did not change between fluctuating conditions and the steady flow experiment (Gerig et al., in-review). This suggests that, at least in this reach of the Colorado River, management actions directed at manipulating backwater habitat type will affect only a small proportion the habitats, and the population, of humpback chub. The NSE study reach is in a section of Grand Canyon with steep bank angle, thus the available habitat in this reach is relatively unaffected by changes in river stage associated with the range of flows observed from 2009-2011. Future work could assess whether juvenile humpback chub are similarly robust to changes in a river reach where available habitats are more flow sensitive.
We successfully developed a framework to address humpback chub growth and provenance through the analysis of humpback chub otoliths (ear-stones). Otoliths form part of the hearing and balance system in fishes and grow incrementally as fish grow. Made of calcium carbonate, otoliths absorb trace elements and isotopes from the environment. If these differ amongst environments, analysis of otolith chemistry can shed light on provenance and lifetime habitat use of fishes. Direct querying of this sort permits deeper insight into population ecology and habitat use, because individual fish life histories are tracked retrospectively. Data from tagging programs, on the other hand, are limited to the locations in which fish are captured and recaptured.
Using information on water and humpback chub otolith chemistry we asked:
(1) Do different parts of the mainstem Colorado River system in Grand Canyon, including its tributaries, differ in terms of dissolved water chemistry?
(2) In particular, are there trace elemental and/or isotopic markers that can distinguish the Little Colorado River tributary from the mainstem? If so, then
(3) At what age and size do juvenile humpback chub emigrate from the Little Colorado to the mainstem Colorado River?
(4) Is humpback chub recruitment to the mainstem Colorado River population dependent on reaching a certain size before emigration?
(5) Are any humpback chub spawned in the Colorado mainstem and if so do they survive?
We developed a geochemical atlas of the Grand Canyon reach of the Colorado River, analyzing a suite of trace elements and stable isotopic ratios. Our sampling was limited primarily to summer and fall months in 2009 – 2012. We assayed otoliths of juvenile humpback chub as well as any available adult chub that had been collected as incidental mortalities. We found that the ratio of a few trace elements to calcium – primarily strontium, barium, and selenium, along with the stable isotopic ratios of carbon, oxygen, and hydrogen/deuterium could, as an ensemble, serve to discriminate between the mainstem and the Little Colorado River, which was the focus of the study (Hayden et al., 2012; Limburg et al., in review). Carbon stable isotopic ratios (d13C) were particularly good discriminators because of the substantial fractionation during travertine formation within the Little Colorado River. We also found that the chemical signature of the mainstem was maintained throughout the entire reach of Grand Canyon, with little variation, such that this signature was readily distinguished from many of the tributaries.
We measured lapillar otoliths of humpback chub and derived a relationship between otolith size and fish total length. Using this relationship, we back-estimated the size, as well as age (in days) when humpback chub egressed from the Little Colorado to the mainstem (Limburg et al., in-review). In addition to this evidence from changes in otolith chemistry we discovered changes in the otolith microstructure itself. Daily growth increments of otoliths reduced in width markedly upon fish entry into the mainstem due to the temperature differential which strongly affects otolith growth. We determined a range of sizes and ages at egress amongst juvenile humpback chub captured in the mainstem. However, this range was reduced in adult chub, suggesting that successful recruitment (i.e. survival) is favored by remaining longer in the Little Colorado. We also found that fish captured in the Little Colorado tended to be larger on their first birthdays (determined retrospectively by otolith analysis) than fish in the mainstem. These fish also showed a bimodal distribution of size at age-1 suggesting differential growth within the birth cohort.
We analyzed a small number of juvenile humpback chub that had been captured many kilometers upstream of the Little Colorado confluence with the Colorado mainstem. These individuals turned out to be much older than comparably sized juveniles collected in the Little Colorado River. Chemical analysis of their otoliths suggested a different natal source; one source appears to be in the 30-Mile Spring reach and another may have been an unidentified spring or tributary (Hayden et al. 2012). Such small fish (20-25 mm) for their age (e.g., 70-80 days old) are unlikely to survive and recruit to adulthood in the mainstem Colorado River. 
The NSE project developed a sampling and analytical framework to directly assess juvenile humpback chub population responses to management actions at smaller fish sizes than were previously possible. This framework is important, as the key outcome from many different types of management actions in the Colorado River is to improve survival of juvenile humpback chub, increasing overall abundance and accelerating the population to recovery. The NSE project also documented that small juvenile humpback chub can survive and rear in the mainstem Colorado River. This information is important because adult humpback chub numbers (age 4+) have increased over the past decade, possibly due to improved survival in the mainstem Colorado River (Coggins and Walters 2009).
We identified chemical markers that can distinguish fish use of Little Colorado River from mainstem use. Humpback chub in this reach of Grand Canyon originate overwhelmingly from the Little Colorado. Mainstem adult otoliths showed evidence that longer rearing in the Little Colorado promotes better growth and recruitment. The combination of otolith chemistry and growth increment analysis together produced a good natural marker that could be used as a tag to follow fish movements between the mainstem and Little Colorado River. Further work will be needed to extend this methodology to other humpback chub aggregations within Grand Canyon and possibly to other native fish species assessments.
The results of the NSE project suggest that juvenile humpback chub survival, growth, abundance, and habitat use are robust (did not change) to the fall steady flows observed during 2009-2011. It is likely that more extreme flow treatments (e.g. higher or lower discharges, longer duration) are required before changes in these metrics would be observed. This research demonstrates the apparent flexibility of juvenile humpback chub in habitat selection regardless of fluctuating or steady river flows. Our development and application of methods to assess the growth, survival, and persistence of juvenile humpback chub in the mainstem Colorado River are key new additions to the body of knowledge available for managing the Colorado River and understanding how juvenile fish populations respond to hydropower operations in regulated rivers globally.