Upper Basin Pallid Sturgeon Workgroup Annual Report for Work Completed in 2019

Photo by Ryan Wilson One of many survivors of the 2019 Pallid Sturgeon larval drift experiment, performed in the Missouri River downstream of Fort Peck Dam, that were stocked 5 days post-hatch.

This report documents the work of the Upper Missouri River Basin Pallid Sturgeon Workgroup (Workgroup) during 2019. The report consists of minutes of the annual meeting of the Workgroup Governing Board, minutes of the annual Workgroup meeting held in Billings in March 2019, updates of on-going work and reports completed by members of the Workgroup and other contractors.

In order to limit the size of this document, where reports included large numbers of tables or multiple appendices, much of this data is not included in this report. Users of this Annual Report are encouraged to contact researchers, principal investigators or project leaders for more detailed information.

The Governing Board of the Workgroup during this period consisted of the following individuals. Their work affiliation and the focus areas they represent are indicated.

Zach Shattuck, Chair, Montana Fish, Wildlife and Parks

Luke Holmquist, Montana Fish, Wildlife and Parks, RPMA 1

Mat Rugg, Montana Fish, Wildlife and Parks, RPMA 2

Landon Pierce, U.S. Fish and Wildlife Service, RPMA 3

Tyler Haddix, Montana Fish, Wildlife and Parks, Habitat

Pat Bratten, U.S. Fish and Wildlife Service, Research

Rob Holm, U.S. Fish and Wildlife Service, Propagation

Vacant, U.S. Army Corps of Engineers, Funding*

Ken Staigmiller, Montana Fish, Wildlife and Parks, Fish Health

Kevin Kappenman, U.S. Fish and Wildlife Service, Stocking and Tagging

Ryan Wilson, U.S. Fish and Wildlife Service, Historical perspective, Wisdom and Guidance

TABLE OF CONTENTS

Annual Governing Board meeting minutes – August 14-15, 2019 ...... 1

Annual Workgroup meeting minutes – March 19-20, 2019 ...... 8

Native Endangered Species Recovery (Pallid Sturgeon) RPMA 1 2019 ...... 20

Spawning Readiness, Spawning Location(s), and Habitat Use of Hatchery-origin Pallid Sturgeon in the Missouri River above Fort Peck Reservoir, MT ...... 40

2019 Pallid Sturgeon Population Assessment and Associated Fish Community Monitoring for the Missouri River: Segment 4 ...... 53

Yellowstone River Radio-Telemetry & Intake Passage Summary ...... 55

2019 Pallid Sturgeon Population Assessment Program Annual Report: Segments 5 and 6 ...... 78

Pallid Sturgeon Free Embryo Drift, Dispersal, and Larval Settlement in the Upper Missouri River, 2019 ...... 93

Migrations, Aggregations, and Spawning of Pallid Surgeon in the Yellowstone River, 2019 ..... 128

Genotypic Analyses and Parental Identifications of Juvenile and Sub-adult Pallid Sturgeon in the Missouri River 2019 ...... 143

Determination of Reproductive Indices in Hatchery-origin Pallid Sturgeon in the Missouri and Yellowstone Rivers ...... 155

Pallid Sturgeon Propagation at Gavins Point NFH ...... 162

Pallid Sturgeon Propagation at Garrison Dam NFH ...... 176

Meeting Notes from the

Upper Basin Pallid Sturgeon Recovery Workgroup Governing Board Meeting

August 14-15, 2019

Billings, MT

The following are the notes from the August, 2019, meeting of the Upper Basin Pallid Sturgeon Recovery Workgroup Governing Board. A summary of the action items assigned during the meeting appears at the end of the meeting notes.

Zach Shattuck, Rob Holm, Ryan Wilson, Mat Rugg, Luke Holmquist, Alyssa Fellow, Ken Staigmiller, Landon Pierce, Wayne Nelson-Stastny, Yvette Converse, Kevin Kappenman, Tyler Haddix, Pat Braaten and Bob Snyder were present at the meeting.

Wednesday, August 14

Alyssa and Zach began the meeting with opening remarks. Alyssa encourages WAPA contractors to send in their itemized invoices.

Wayne – Recovery and MRRP updates

No one was hired for the Pallid Sturgeon Technical Coordinator position. It will be re- advertised.

The Middle Basin Workgroup meeting will be in a couple of weeks. The main issue for the Lower Basin Workgroup is hybridization.

Test flows from Fort Peck Dam is currently the biggest MRRP topic Fort Peck AM Framework). The ISRP review of the flow release plan should be available today.

AI – Zach will send David Galat a request for GB and others to participate in the Fort Peck webinar.

The Fish Workgroup meets September 18.

AI – Zach will distribute the webinar invite via email.

Work on habitat interception project is moving forward in the Middle Basin.

The Coalition to Protect the Missouri River doesn’t like the MRRP process. They are meeting with the USACE, USFWS, MRRIC in St. Louis.

The fall Science meeting (November 18) will allow for more discussion than last year’s meeting. The plenary session is scheduled for November 19-20. Dave Marmoreck with ESSA has replaced Rob Jacobsen as head of the Fish Tech Team.

Recovery implementation planning – The Recovery Team is tentatively scheduled to meet in January, 2020. The main topics are hybridization, future of RPMA 3, and, perhaps, inclusion of RPMA 1 in the MRRP process.

Ed Heist has been contracted to look at Pallid Sturgeon population genetic structure and summarize information for USFWS.

AI – Get project scope of work from Yvette.

Use of Gavins Point NFH F2 progeny for conservation stocking – Wild-source progeny and captive broodstock F2s were used for the 2019 larval drift study. Some past crosses are not represented or are under-represented in the Upper Basin stocked populations. Twelve family groups can be stocked in 2020. There will be a recommendation from geneticists (Jeff, Meredith and Ed), using data from Rob, whether to stock these families. The consensus of the UB biologists is to stock missing crosses into RPMAs where they are needed to improve RPMA- specific effective population size.

AI - Biologists will provide comments about the use of F2s to Zach by Monday. Zach will send a memo from the GB soon after.

Rob would like to know the numbers to be stocked by April.

Tyler believes (95% sure) he is finding YOY Pallid Sturgeon that were released during the 2019 larval drift study.

Zach - Update from the Chair

The new ESA changes affect mostly newly-listed species. Wayne stated that the changes to climate change policy and “foreseeable future” language to not affect Pallid Sturgeon. Although there is new language about “transparency of economic analysis”, decisions still will be science-based.

At the June gathering at the confluence improving and coordinating telemetry work was discussed. It was noted that the Upper Basin does a good job of communicating among the different agencies and telemetry teams.

An electronic data sheet is being developed.

Two chubs (sickle fin and sturgeon) have been petitioned for listing. FWP is willing to provide any information that the USFWS needs. USFWS will soon work out the timeline and process for information collection.

Ken – Fish Health

A Pallid Sturgeon health assessment is still in the works. Lacey Hopper leading the effort. Miles City Fish Hatchery has an experimental lot of Pallid Sturgeon from eggs from the captive broodstock to evaluate fin curl management at MCFH.

Rob – Propagation update

Approximately two months post-spawn three broodstock (one male, two female) died a day apart. The rainbow trout forage in their tank also died. Remaining six radio tagged fish were immediately returned to the confluence. Necropsy of the dead fish didn’t reveal any apparent issues. Tissue samples sent off to Bozeman FHC were post-mortem and as a result no cause could be identified histologically. Additional trout were restocked into the two brood tanks and are doing fine. A cause for these mortalities has yet to been identified.

Molly Webb, Hilary Treanor and Wendy Sealey from the Bozeman FTC are doing a food study to investigate larval sturgeon diets that would result in improved larval transition to feed and survival. Three feed types were tested with results pending. The staff at Garrison Dam NFH had a concurrent feed study which identified survival differences between female lots and feed types. None of the three feeds used in the study had remarkably improved survival, although an alternative diet (fed outside the study) did show promise. This work will continue in 2020 using the alternative diet.

The sturgeon building at GDNFH is scheduled to be replaced. GDNFH will not be able to spawn or rear pallid sturgeon in that facility during construction. The isolation room at GDNFH could be used for production fish in limited numbers. MCSFH may be able to help with the holding and spawning of broodstock and potentially production if the fin curl issue is resolved. Currently there is no sign of fin curl in test fish at MCSFH.

Kevin – Stocking and Tagging Update

We are switching back to L3 and R3 scute removal from L7 and R7.

AI – Ryan will send updated scute removal and VIE marking tables to Yvette.

BFTC can put on a small tagging and handling class if needed.

AI - Contact Kevin if there is a need for a tagging and handling class.

Aaron DeLonay is performing telemetry training for the Middle Basin.

Yvette – CPSP update

All Pallid Sturgeon document have been signed. Reports will have to be updated regularly. The CPSP evaluation plan is in an early draft, with some review by a small group.

Landon – RPMA 3 update

High river flows limited sampling to trot lines. Eighty-four HOPS and 6 others (genetics ID pending) were captured. There was a much lower CPUE due to debris, especially below the Niobrara River. Sampling in RPMA 3 will be done every five years.

Over 12 Pallid Sturgeon were captured from Lake Sakakawea. Pallid Sturgeon are also captured in Fort Peck Reservoir.

Thursday, August 15

Luke – RPMA 1 update

2019 was the first time Pallid Sturgeon were documented to move into the Teton River. These fish were non-reproductive.

Marias River flows were supposed to be constant, but USACE requested flows from Tiber Dam be reduced in May and June due to downstream flooding. Flows were reestablished in July.

A discussion of Section 7 consultation for RPMA 1 ensued. The probable process will be to engage the BuRec about the need. There will be a meeting to work with the BuRec in Pallid Sturgeon recovery in RPMA 1.

AI – Try to have Kim Smith or someone else in USFWS attend/listen in to RPMA 1 discussions.

Ryan – RPMA 2 population assessment update

His crew has changed gear to improve adult capture. They caught 24 fish for telemetry: (replaced transmitters in 5, implanted transmitters in 19 “new” fish). Ryan is encouraged about the flexibility in adjusting PopAss 2.0 sampling.

There is a problem reading 20-year old PIT tags with the new HPRlite readers. Ryan suggests crews be prepared to scan fish with new and old readers.

AI – Have BioMark assist with transitioning to new system.

The Middle Basin uses real time data collection and reporting (Sturgeon Information Management System – SIMS). The eventual goal would be to access data via smartphone. The database would be updated daily. The Upper Basin should work to develop a similar system. Any system must be available to all crews, not just PopAss crews.

AI – Mat, Zach and Tyler will approach FWP IT to see how to implement SIMS.

Ryan’s shop will have a trial use of the system this winter.

Mat – RPMA 2 update

Mat didn’t have a lot of Pallid Sturgeon that moved to Intake. Five Pallid Sturgeon (non- reproductive HOPS or wild males) were translocated. Approximately 200 sturgeon larvae were sampled at the mouth of the Powder River (most/all expected to be shovelnose).

The Intake reconstruction project has started. There is a need to figure out a path to implement transition to the new telemetry equipment. A discussion ensued about such a transition. There is a problem with running out of codes using the old system. The new system allows for more codes. The new system will require capturing all telemeterized fish and replacing the existing transmitters. DeLonay is designing the news telemetry system and identifying system needs for the USACE.

AI – Request DeLonay’s report as soon as it is completed.

AI – UB biologists will work through transitioning to the new telemetry system.

A discussion of data collection, recording and sharing ensued.

Tyler – RPMA 2 update

There weren’t high flows in the Missouri below Fort Peck Dam during the critical time period, so adult Pallid Sturgeon did not move into this reach of the Missouri. There were not many larval sturgeon captured until Fort Peck Dam spilled, beginning the second and third week of July.

Benthic trawling yielded lots of SNS juveniles and, hopefully, juveniles from the 2019 larval drift study and the Yellowstone River. Genetics testing of larvae captured during the drift study is the priority for testing by Ed. It appears that 1-day and 5-day old had good survival and capture rates and some have possibly established themselves in the Missouri above Lake Sakakawea.

Pat – Larval drift study update

Water temperature during the 2019 drift study was approximately 14.5° C (relatively cold). One- and five-day old larvae were released. They had good survival and capture of released

5 larvae. DeLonay sampled “dead water” areas. Some free embryos were captured in the slow water zones, but their eventual fates are unknown.

Trawling to detect survivors began the week after the release. Twenty-five millimeter sturgeon have been captured, including some captured in the upper (non-anoxic, flow present) reach of Lake Sakakawea. Fish were captured above and below the Confluence.

Alyssa and Zach – WAPA funding

There is $176,000 available for new projects in FY2020. WAPA pays for genetics identification of PIT tagged recaptures.

AI – Snyder will talk with Lorelyn about MediaWorks budget needs and use of website.

New workplan

The GB expressed a desire to repeat the Rotella survival estimates with possible additional queries. The last estimate of the adult population was performed in 2008 and there are three additional years of data since the last HOPS estimate.

Zach will send out a request for one-page proposals.

Wayne (and the USFWS) could use information to determine if or when SNS are surrogates for Pallid Sturgeon, especially during early life stages.

More work on carrying capacity could be done (temperature and bioenergenics, small fish densities, abundance and distribution of forage species, and expanding on Guy’s carrying capacity report.

Chris Guy is interested in a diet study of Pallid Sturgeon use of chubs.

Workgroup Chair selection

Zach will continue as Workgroup Chair for two more years.

AI – Zach will send out a Doodle poll to select dates for the annual Workgroup meeting.

List of Action Items

Zach will send David Galat a request for GB and others to participate in the Fort Peck webinar.

Zach will distribute the webinar invite via email.

Bob will get project scope of work of Ed’s contract from Yvette.

Biologists will provide comments about the use of F2s to Zach by Monday. Zach will send a memo from the GB soon after.

Ryan will send updated scute removal and VIE marking tables to Yvette.

Contact Kevin if there is a need for a tagging and handling class.

Try to have Kim Smith or someone else in USFWS attend/listen in to RPMA 1 discussions.

Have BioMark assist with transitioning to new system.

Mat, Zach and Tyler will approach FWP IT to see how to implement SIMS.

Request DeLonay’s report as soon as it is completed.

UB biologists will work through transitioning to the new telemetry system.

Snyder will talk with Lorelyn about MediaWorks budget needs and use of website.

Zach will send out a Doodle poll to select dates for the annual Workgroup meeting.

Notes from the 2019 Upper Basin Pallid Sturgeon Workgroup Meeting Northern Hotel Billings, MT March 19-20, 2019

Zach Shattuck opened the meeting. Introductions were made.

Zach Shattuck – 28 years later…

An overview of the progress made since the listing of Pallid Sturgeon was presented and the question of what the status of recovery program will be in 100 years was posited. The annual Adaptive Management cycle was reviewed and the need to “trust the process” was reinforced. Zach stressed the importance of the partnerships that make the Upper Basin Workgroup so effective.

Wayne Nelson-Stastny – MRRP, Workgroup engagement, and recovery implementation strategy (RIS)

The Workgroup should be thinking about new information and issues for presentation at the fall Science meeting. The Fish Science Team will present this information at the spring Adaptive Management meeting.

Pallid Sturgeon Recovery Implementation Plan will be led by: Wayne – Pallid Sturgeon Recovery Leader Landon – Fisheries Tech Lead Ecological Services Tech Lead – TBD

Partnerships are needed to recover Pallid Sturgeon. The “front end” of the recovery implementation strategy is done. However, MRRP doesn’t cover all aspects of recovery. Implementation strategy needs still need to be identified as well as who will do what and how. Species status assessments are more useful for newly-listed species. In the case of Pallid Sturgeon, a Recovery Implementation Strategy is more valuable. The RIS should stimulate funding for recovery actions. It is intended that the start of the process will be within 2019.

Steve Krentz – PIT tag update

Landon sent out a draft transition protocol. Kappenman-Tagging proposals typically move through the Propagation of Tagging committees in the Upper Basin.

Wayne Nelson-Stastny – Stocking and Augmentation Plan update

The S&A Plan is part of a bundle of documents sent to Regional Director for review and acceptance. Approval is anticipated by April 2019.

Tanner Cox – Reproductive Ecology of Hatchery-origin Pallid Sturgeon: A New Hope

The main focus of project is the 97 age class of Pallid Sturgeon. The first reproductive male was found in 2011. The first reproductive female was identified in 2015. In 2015 and 2016, all sampled reproductive females exhibited follicular atresia.

Objectives: • Test hypothesis that fish are going atretic because of a “dummy run”. • Identify spawning locations

Results: While dummy runs may likely be the cause of atresia, other causes are also occurring. While spawning was not confirmed, males’ and females’ location overlapped during the suspected spawning period. Spawning locations were located between Rkm 3100 and 3137.

A new hope: Spawning is occurring and reproductively active fish are exploring the upper reaches of the study area (Marias River).

2019 field season: • Investigate high incidence of atresia o Extent of occurrence o Dummy run • Continue to evaluate spawning location(s) o Upstream distance o Habitat type • Describe reproductive indices o Age at first maturity o Spawning periodicity

Luke Holmquist – Pallid Sturgeon monitoring in RPMA 1

2011 had lowest catch of pallid Sturgeon due to high flows.

Lower reaches of RPMA1 have highest CPUE.

As observed elsewhere, age classes after initial introduction (1997) have slower growth rates.

There are fixed telemetry stations in the Missouri, Teton and Marias rivers that supplement mobile tracking.

Movement and timing of Pallid Sturgeon into Marias offer potential for recruitment.

Future work: • Add more telemetry stations in mainstem and tributaries • Expand tagging program to include Pallid Sturgeon (?) • Perform lab investigation of tagging (retention and related health issues)?

Tyler Haddix – Spawning Migrations of Pallid Sturgeon in the Missouri River Downstream of Fort Peck Dam

Telemetry: With high flow from Milk River and spillway releases from Fort Peck Dam through most of the summer, Pallid Sturgeon ran clear up to the spillway. High flows and conductivity reduced tracking effectiveness.

Larval sampling: High flows increased YOY fish production in the Missouri River. The genetic results of samples collected are pending.

Summary: • Only the second time in 20+ years that reproductively active Pallid Sturgeon were in the upper Missouri River during the spawning season. Whether spawning occurred is undetermined until genetic results are reported.

• Wild female code 36 left the Missouri and spawned in the Yellowstone, but HOPS females 54 and 177 stayed in the Missouri.

• Sampled the most acipenseriforme free embryos since sampling started in 2002.

• 2018 had the second highest captures of young-of-the-year sturgeon using benthic trawl since sampling began.

What should be done next? • Strategic flow test using our best information to see if we can get Pallid Sturgeon to spawn (again-2011) in the upper river close to Fort Peck Dam.

• Keep working on getting warm water withdrawals from Fort Peck Dam .

Wayne Nelson-Stastny – US ACOE scoping comments

Comments are due March 27. Send comments from individuals and Workgroup to Wayne and Zach.

Pat Braaten - Pallid Sturgeon Migrations and Spawning in the Yellowstone River

2018 Scope of Work: • PIT array construction/deployment/testing in the high-flow side channel at Intake

• Pallid sturgeon migrations – timing, spatial extent in the Yellowstone, Missouri, Powder and Milk rivers and at Intake Dam.

• Male Aggregations – locations, persistence

• Spawning – location(s), timing, habitat characteristics

• Hatch verification – free embryos and larvae

Summary: Elevated flows vs telemetry detections/relocations • 2018 was challenging in lower Yellowstone (and Missouri) • Deep water, high conductivity • Some data gaps in timing/locations • Will experiment with higher power transmitters in 2018 (partially preparing for spillway test in future) Migrations • To Intake Dam verified for wild pallid sturgeon and HOPS, but lacking gravid female(s) for the last few years • Within and between rivers (e.g., Missouri to Yellowstone, Yellowstone to Powder) Spawning • Fairview reach verified (functional for spawning, not for recruitment) • Early in some individuals (code 116), later in others (code 36) • Large male aggregation(s) in lower Yellowstone River as spawn-patch indicator(s) Increasing number of telemetered and reproductive HOPS • Good situation, logistically very challenging • One, multiple spawning locations? • Movements between rivers, within river residents, growing “resident” population upstream from Intake

Eric Best -Lower Yellowstone Project Pallid Sturgeon Translocation

Objectives: • Capture, transport, and release motivated pallid sturgeon above Intake Diversion Dam • Monitor movements of translocated individuals and detect any evidence of spawning • Monitor to detect free embryos in the Lower Yellowstone Main Canal and at Intake Diversion Dam

Catch zone is from 0.5 miles below Intake Dam downstream to the mouth of the natural high flow channel. Captured fish are transported through the side channel by boat (if flows allow) or by truck and released upstream of the dam.

Results: Translocation

2 wild males; 1 wild female (odd year spawner) and 4 HOPS (sex unknown; not mature) were translocated. One non-reproductive fish ran up to Cartersville Dam (RM 234 for 42 days and flows up to 70k cfs) and remains above Intake. A wild male ascended the Yellowstone River and then an additional 87 miles up the Powder. This fish was also translocated in 2017 and made a similar ascent.

Larval sampling • Sampling Occurred June 18th – July 10th 2018

• No acipenseriformes detected in Main Canal or Mainstem at Intake in 2017 (2018 samples still being sorted)

Plans for 2019

• Similar to 2018 • May 1st – June 15th (Translocation); Mid-June – Mid-July (Larval sampling) • No sidechannel access during construction or boat ramp during PDFH. Possible temporary boat ramp on Joe’s Island? • Extend “Catch Zone” downstream to increase captures? • Increase tracking post-translocation, especially in the Powder River

Mat Rugg - Native Species Telemetry Project

Mat expressed concern that new dam construction may affect passage over the dam. It is possibly encouraging that Paddlefish and Pallid Sturgeon may use the constructed side channel.

Many more Pallids upstream than was thought just a handful of years ago. Most are smaller comparted to downstream and most are residents of upstream stockings. What are the implications for survival estimates if recapture probability is different up and downstream of Intake? How will these fish fit into PSPAP v2.0?

Future of YSR monitoring project: • Rotella survival estimates continue? o Implications of many upstream Pallids • YSR data compatible with PSPAP v2.0? • Telemetry population maintenance o ~40 implanted fish in 2012 – their battery life ending. How will this be addressed?

Joseph Mrnak - Effects of water velocity and temperature on growth, energy density, activity, settling time, and mortality of endogenous Pallid Sturgeon larvae

Objectives: • Assess the direct effect of water velocity on growth rate, energy depletion rate, activity and survival of endogenous Pallid Sturgeon larvae. • Assess the direct effect of water temperature on growth rate, energy depletion rate, activity and survival of endogenous Pallid Sturgeon larvae.

Results: Velocity • Growth o Not affected by velocity • Energy Depletion, Activity, and Mortality o Greatest in no to low flow Areas with no to low flow may pose a significant source of mortality because of increased activity, higher energy depletion, and mortality.

Temperature • Growth and energy depletion o Greatest at warmer temperatures • Settling time o Shortest at warmer temperatures Warmer water releases may aid survival of larvae by shortening the temporal and spatial drift extent. Increased metabolism led to faster life history transitioning.

Braaten - 2019 Free Embryo Dispersal Experiment

A free embryo drift experiment will be attempted in 2019.

Study design: • Drift and dispersion attributes of free embryos (1, 5, dph) released at RM 1700. • Determine how available models compare predicted with actual drift. • Calibrate and refine flow recommendations and models for management considerations.

A demonstration of a drift simulation model was presented.

Eric Scholl – Density of Pallid Sturgeon and Food Web Dynamics in the Missouri River

Objectives: • Estimate production of the prey base • Assess the potential of food limitation for hatchery-reared pallid sturgeon

Conclusions: • Macroinvertebrate dynamics and food web patterns within reaches are disproportionately influenced by small habitat patches. - Stable (cobble gravel/large rocks/large woody debris) and off-channel habitats often contributed ~50% of total macroinvertebrate production within a study reach and ~50% of macroinvertebrate taxa in sturgeon diets. • Differences in macroinvertebrate production (availability) among reaches related to the dominant habitat type. - Reaches dominated by sand had much lower total macroinvertebrate production than reaches dominated by rocky substrate. • Fort Peck Dam exerts a strong influence on macroinvertebrate diversity and food web complexity. Macroinvertebrate communities increase in diversity and complexity as one moves further downstream from Fort Peck Dam. • Modeling approaches suggest that the dominant fish community requires the majority of macroinvertebrate production.

Opportunities and future directions: • Yellowstone River food web • Energy flows from off-channels to the main channel • Validating macro ecological model by estimating fish densities in the field • Modeling future scenarios of prey fish demand

Chris Guy - Abundance, Biomass, Consumption, and Growth of the Hatchery-Origin Pallid Sturgeon in RPMA 2

Objectives: • Estimate abundance and biomass by year using results from Rotella (2017) • Estimate per capita and total consumption of prey fish • Evaluate weight by stock year • Correlate abundance and biomass with weight

Summary: Abundance peaked in 2010 and then declined; biomass peaked in 2011 and remains level.

Prey fish consumption in 2016 was estimated to be 38,233 kg. ~ 12 million emerald shiners ~ 1.4 million Macrhybopsis spp.

Weight is highly variable within and among year classes.

75th percentile weight weakly related to abundance and biomass

Additional thoughts: • Bioenergetics models for piscivorous pallid sturgeon • Understand the variation in weight within a year class • Prey fish abundance estimates • Need to define targets for growth

Braaten - Long-term Catch Rate Trends for Sturgeon Chubs and Sicklefin Chubs in the Upper Missouri River

Objectives: • Compare catches of sturgeon chubs and sicklefin chubs among years, testing for long-term trends (2004-2016) • Examine biotic (potential predation from HOPS) and abiotic (discharge, temperature) correlates of chub catches

Populations of Sturgeon Chubs appear to be decreasing from 2004-2016. There are fewer and decreasing numbers of Macrhybopsis spp. with increasing numbers of HOPS, although Sicklefin Chubs are not affected as much as Sturgeon Chubs. The Powder River is a strong source of Sturgeon Chubs.

A discussion of sampling chubs to investigate genetic population structure ensued. Zach will set up a chub sampling group within the Workgroup.

Summary: HOPS predation on chubs does occur (Dutton 2019, others), but is it a major factor influencing chub populations? • Lack of correlation with age-1+ sicklefin chubs, negative with age-0 Macrhybopsis spp., and age-1+ sturgeon chubs

Reproduction is occurring annually, but evidence of declining recruitment through 2016: • Age-0 CPUA not associated with Yellowstone River and Powder River conditions

• Age-0 CPUA negatively correlated with Missouri River discharge, HOPS abundance, but declining recruitment likely affected by declining adult stock through time as well (e.g., sturgeon chubs)

Aaron DeLonay - Upper Basin Telemetry Discussion: Improvement, Integration and Implementation

There is a lot of telemetry work in the MRSAMP. There is a need to implement a standardized telemetry data system, especially with the increasing complexity (more tagged fish, more tracking, more fixed stations) and volume of data.

Objectives of project: • Assess existing and available telemetry technologies for the Upper and Lower Rivers • Manual detection and tracking • Data collection platforms • Passive receiver networks • Remote data access and reporting • Technologies for 2-D telemetry monitoring • Biological event detection • Facilitate Implementation

Current technology in the Yellowstone / Upper Missouri River: • Radio Telemetry (150 MHz) • Manufacturer: Lotek Wireless o SRX400*, SRX600*, SRX800 Receivers** • Digitally Encoded Tags o Legacy code set (<212 codes / Freq.) o Potential *512 codes/ Freq., 728 codes/ freq. • Multiple Frequencies (Assigned / Scanned) o 2 freq. pallid sturgeon o (70 wild adults / 130 hatchery juveniles) o 2 freq. native fishes for Intake passage • Passive Radio-telemetry Receivers / Data Loggers o Shore-based enclosures o Solar powered, 12 volts DC o Receivers are not fully communication ready • Manual tracking o Rapid extensive and intensive tracking of high value individuals and locations • Recapture and Reproductive Assessment • Transmitters o MCT2-3L, 16x73 mm, external Antenna, o Expected life: 2929 days (8 years) at 5 sec burst rate o Reproductive cycle time: 2-4 events

Recommendations and Implementation for the Yellowstone / Upper Missouri River: • Radio telemetry is preferred telemetry technology with consistent performance under most conditions. • Update existing telemetry systems in use (Lotek Wireless) migrating to the SRX 800 wide– range (26 MHz) receivers. • Consider migration from 148–152 MHz frequencies to 164–168 MHz. • Update expands the limited code sets now in use (212 unique codes) in receivers and dataloggers to the expanded and robust code sets that provide for 521 or 728 codes. • Provides for advanced sensors data transmission (for example, depth, temperature, activity, accelerometry).

• Test and transition to next-generation higher-powered radio transmitters to increase detection and range (slightly increased diameter, decreased life, increased burst rate). • Optimize passive monitoring stations at important tributary junctions at 10-50 river-mile intervals along the mainstem rivers and at important tributary junctions. • Add QA/QC transmitters or protocols to assess passive network receiver nodes. • Develop and implement near-real time data communication (cellular and satellite) and reporting for passive network receiver nodes. • Identify and test acoustic telemetry tags and receivers for specific scenarios or for critical applications. • Adapt real-time GNSS-linked telemetry tracking data collection platform and uniform updated tag database with electronic data form support.

Jeff Kalie – 2018 Annual pallid sturgeon genetic analysis update

Over 1000 samples were received for analysis. A few triploids were identified (from RPMAs 2,3 and 4).

A 2001 age class HOPS captured in RPMA 3 in 2010 was recaptured in RPMA 4 in 2018, 560 miles from its previous capture site. It had almost doubled in weight.

No Upper Basin fish were spawned in 2018, as none were captured.

There are currently 99 cryo-preserved males in the repository, all pallid sturgeon and unknown origin, including two males that haven’t been spawned from RPMA 2

Deadlines to submit samples for genetics analysis in 2019: • Potential broodfish and other adults o May 3, 2019 (?) • Unknown-origin individuals for spring analysis o June 7, 2019 • Unknown-origin individuals for fall analysis o November 8, 2019

Tyler Haddix - 2018 Montana Pallid Sturgeon Population Assessment Program

More Pallid Sturgeon are being captured upstream of the confluence since 2011. Is this a behavior brought on by increasing density and related impacts (forage depletion in lower reach)?

Fewer Pallid Sturgeon are being captured due to reduced stocking rates and accumulating mortality.

Relative weight (which has remained constant) is not an adequate metric to assess the condition and carrying capacity of Pallid Sturgeon.

The new Population Assessment protocol (V2.0) is designed to perform a population estimate at each sampled bend and within a reach. Trawling will be done as in previous years to maintain the spawning assessment dataset.

Ryan Wilson - Pallid Sturgeon Monitoring in Segment 4 of the Missouri River 2018

Sampling in 2018 was impacted by high flows, debris, and access.

Mean length at age shows early age classes (2002 and older) have grown faster than subsequent age classes. Percent of recaptures is increasing, i.e. not seeing as many new fish. Are we losing fish or are they somewhere else?

More Pallid Sturgeon have been captured in the reach of river immediately above Lake Sakakawea than in all other reaches (range-wide) sampled.

In the 14 years of sampling in Segment 4: ! Over 250,000 Pallids stocked in RPMA 2 ! 3,386 HOPS have been captured in Segment 4 ! 2,611 individual HOPS ! 775 recaptures ! 70% of Segment 4 HOPS from Yellowstone stocking sites

Landon Pierce – Pallid Sturgeon Population Assessment in Segments 5 & 6

Fish condition remains fairly constant at .9. Growth rate of age classes is similar to that seen in other Upper Basin segments.

Population assessment sampling will be performed every 5 years.

The status of stocking Upper Basin fish in RPMA3 was discussed. Landon said there is a need for more discussion with the Upper and Middle Basin workroups and the Recovery Team. The Middle Basin has requested that RPMA 3 not be stocked with Upper Basin-sourced fish because of genetics concerns.

Ed Heist - Improvements in Genetic Analyses for Species Discrimination of Acipenseriformes Unhatched Embryos

Identification of unhatched embryos can refine understanding of time, location, and habitat of Pallid Sturgeon spawning. This project tested eight genetic “kits” to identify which provided the best technique to identify Pallid Sturgeon eggs using mitochondrial DNA. Qiagen micro (QM method) appears to be the most effective kit using 24-hour embryos.

Work is being conducted to use a new SNPs tool to differentiate Pallid Sturgeon from Shovelnose Sturgeon.

Ed is requesting more samples from the captive broodstock and wild fish.

Next steps:

• High through-put sequencing or RAD-Seq using haploids to develop a reference sequence for assembling data from GPMU and CLMU Pallid Sturgeon and Shovelnose Sturgeon. • RAD-Seq to identify SNPs that potentially distinguish between species and MUs. • SNP genotype Pallid Sturgeon, Shovelnose Sturgeon and hybrids range-wide to assess hybridization and stock structure, Ne, etc.

Rob Holm – Garrison Dam National Fish Hatchery 2018 Update

No Pallid Sturgeon were stocked into the Upper Basin because no wild broodstock were captured because of high flows. More than 290,000 Pallid Sturgeon have been stocked into the Upper Basin.

Six males represent 17% of the HOPS in RPMA1. Unequal representation of founding parents has reduced the actual Ne below the possible Ne. A plan for capturing targeted females for 2019 was presented. Some telemeterized fish may be being impacted (by implantation of larger tags or associated handling?), changing their reproductive cycles.

Reducing stress in captured Pallid Sturgeon needs to be stressed with crews. All personnel that handle Pallid Sturgeon need to be trained.

The decision was made to produce genetically important lots for conservation stocking in 2020.

Jeff Powell – Gavins Point National Fish Hatchery 2018 Update

GPNFH staff is working on reduction plan to keep the Upper Basin captive broodstock within the hatchery’s capacity.

GPNFH’s Middle Basin work was presented. They have built a new building for this program. Females have been observed with 1-, 2- and 3-year spawning cycles.

Early life stage mortality: Pallid Sturgeon hatched and held in water from ponds have higher survival that those held in lake, lake/well or reconstructed river water. More work will be done to identify possible chemical causes for the higher survival observed.

GPNFH staff is looking into providing/producing Flathead Chubs for use as forage by adult Pallid Sturgeon held at GPNFH.

Cody Hagemeister – Miles City Fish Hatchery Update

MCFH will be investigating causes for fin curl at that facility. The goal is to eventually use MCFH for production of Pallid Sturgeon for the conservation stocking program.

Lacey Hopper - Pallid Sturgeon Health Assessment Update

Purpose: • Response to April 17th, 2018 memo from USFWS on health/condition of captive-reared & stocked PS. • Standardize PS health assessments between USFWS regions.

Minimum health testing: • MRSIV – PCR (fin clip) • Ranavirus – cell culture (k/s) • VHSV – cell culture (k/s) 18

• Fin Curl Assessment (scored) • Lesions with prevalence/severity (scored)

Reporting: • Standard FWS 3-226 (Aquatic Animal Inspection Report) • Additional detailed/standardized pathology report for all inspections • Database??

Alyssa Fellow – WAPA update

The Upper basin Workgroup MOU has been completed. Alyssa is working on contracts for FY2019. WAPA has spent $6,139,639 on Pallid Sturgeon recovery.

David Trimpe – Bureau of Recreation Update

The USACE has issued a Notice to Proceed on the Lower Yellowstone Fish Passage Project. Construction is expected to begin in the next couple of weeks. However, no work will be completed in the Yellowstone River until after July 1. Reclamation, the USACE, and the Service agreed to an in- water work restriction from April 15 – July 1. Project is to be completed and operational by the fall of 2021.

Reclamation will continue funding projects in RPMA 1 but would like to sit down with FWP, MSU, the Service to evaluate research needs in RPMA 1. This will likely occur in the fall of 2019.

Zach Shattuck – Adopt-a- sturgeon

The Adopt-a-trout program is a possible model that could be modified to increase public outreach and awareness of Pallid Sturgeon in the Upper Basin.

PERMIT REPORT

PERMITTEE: MONTANA DEPARTMENT OF FISH WILDLIFE AND PARKS PERMIT TITLE: NATIVE ENDANGERED SPECIES RECOVERY (Pallid Sturgeon) PERMIT NUMBER: TE06447C-0

LOCATION: Missouri River upstream of Fort Peck Dam, Montana Great Plains Resource Management Unit Formerly known as Recovery Priority Management Area 1 (RPMA1) PERIOD COVERED: January 1, 2019 through December 31, 2019 PROJECT PERSONNEL: Luke Holmquist, Fisheries Biologist, [email protected] Robert Beattie, Fisheries Technician, [email protected]

INTRODUCTION

The Missouri River between Morony Dam and Fort Peck Dam contains the most upstream habitat that supports a population of endangered Pallid Sturgeon (Figure 1) in the Missouri River Basin. This reach was formerly designated as RPMA1 in the original Pallid Sturgeon Recovery Plan (Dryer and Sandovol 1993) and has since been re-designated as part of the larger Great Plains Management Unit under the revised Management Plan (USFWS 2014). However, the former designation continues be used to identify isolated reaches and populations in the context of stocking and sampling efforts, thus we will refer to this reach as RPMA1 in this document. This reach is anthropogenically altered at the downstream portion by the operations of Fort Peck Dam and the subsequent reservoir and additionally at the upstream portion through impacts of multiple dams on the Missouri River and on important tributaries including the Marias River. Most notably Canyon Ferry Dam on the Missouri River and Tiber Dam on the Marias River (both completed in the 1950’s) alter the natural flow, temperature, and sediment regimes in

RPMA1. Holter Dam, Hauser Dam, and the five dams in the Great Falls area (Black Eagle, Rainbow, Cochrane, Ryan, and Morony) are run-of-river structures that impact sediment and temperature regimes however have less impact on the hydrograph. Hydrological alterations and fragmentation of the riverine ecosystem are suspected to be the root cause of population declines across the species range. No natural recruitment has ever been documented for Pallid Sturgeon in RPMA1 (Holmquist 2017). The leading hypothesis for recruitment failure is that if Pallid Sturgeon have successfully spawned (although never documented) their offspring have not had sufficient drift distance to complete the larval drift 20 stage of their life history, therefore have not developed the ability to swim before encountering lethal (anoxic) conditions in the transition zone at the Missouri River interface with Fort Peck Reservoir (Braaten et al. 2008, Guy et al. 2015). The small sample size of reproductive Pallid Sturgeon in RPMA1 has hindered our ability to understand the reproductive ecology of the species as it relates to recruitment and spawning failure. In 2007, the wild population was estimated to be 50 individuals (USFWS 2007) and is assumed to be substantially smaller in 2019. Fortunately, in response to observed recruitment failure in the wild, a conservation Propagation Program was initiated in the 1990’s, that utilized the spawning of wild broodstock to augment the wild population of Pallid Sturgeon. The oldest cohort (1997 year-class) of hatchery-origin Pallid Sturgeon (HOPS) have started to reach sexual maturity in recent years (Holmquist et al. 2018), and as time progresses more HOPS will be reproductively active. The larger sample size of reproductive fish will allow for stronger conclusions to be made regarding spawning behavior and success/failure. Radio-telemetry has proved to be an invaluable tool for monitoring spawning season movement and facilitating recapture of suspected ripe or post- spawn fish for reproductive assessments and will continue to be an important tool for monitoring HOPS in the future.

Including the 1998 stocking event (732 age-1 fish), the Propagation Program has stocked 109,768 fingerlings and 40,987 yearlings representing 14 year-classes (1998 through 2003 year-classes are absent in RPMA1). In recent years stocking numbers have been reduced to 300 per family to increase genetic diversity (Heist et al. 2013). Pallid Sturgeon are captured using set lines, trammel nets, and other gears. Information on catch rates and size are used to inform the evaluation of the stocking program through assessments of growth rates, survival rates, movement, and habitat use. Pallid Sturgeon catch rates prior to 2016 resulted in an estimation of 4,109 HOPS in RPMA1 (95% CI, 3489 to 4731; Rotella 2017), which was much lower than the previous estimate of 7,935 (Rotella 2015).

METHODS

Sampling- Set lines and trammel nets are the primary gear used to capture Pallid Sturgeon, although electrofishing and benthic trawls also catch low numbers of Pallid Sturgeon. Standard fall trammel netting consisted of 20, 7-minute net drifts in the Fort Benton, Coal Banks and Judith Landing sections and 50 drifts in the historical section (1996-present) at Fred Robinson Bridge. For the second consecutive year, high spring discharge (Figure 1) precluded FWP personnel from deploying the 90 standard set lines below Fred Robinson Bridge. These 90 overnight sets typically result in greater than 50% of Pallid Sturgeon caught in RPMA1 on an annual basis. As a result, 30 trammel net drifts were added below

Rock Creek (also done in 2014, 2018) to increase Pallid Sturgeon catch in 2019. Non- standard trammel netting was also conducted in both the Marias and Missouri Rivers as part of targeted netting for sturgeon reproductive assessments. In 2019, benthic trawling and larval sampling efforts were expanded to help improve understanding of Acipenseriform reproduction in RPMA1. All sampled Pallid Sturgeon were measured (fork length [mm] and weight [g] and identified to year-class if possible (using external marks [elastomere or missing scutes] or PIT tag [125 kHz FDX]). Larger (>1000 g) and older fish (older than 2007 year-class) had blood samples and gonadal samples (only when a radio was implanted) collected for assigning sex and stage of maturity in collaboration with the Bozeman Fish Technology Center. Unmarked fish had genetic samples taken, were PIT tagged, and had the L2 scute removed. Relative Condition (Kn) was calculated for all fish using the equation in Shuman et al. (2011) and visually compared among year-classes and sampling years. Year-class representation (proportion of total catch) and growth were also compared among year-classes.

Telemetry- In 2019, 82 Pallid Sturgeon (sixty-six 1997 year-class, eight wild, and eight younger than 1997 year-class) were monitored with radio telemetry. Manual tracking of the entire reach was completed monthly from April through November, and more frequently during the putative spawning season (lateMay through early-July). Fish movements were also continuously monitored by 13 remote land-based receiver stations on the Missouri River from Carter Ferry (RM 2089) downstream to upstream (Roads End; RM 1901.0) of the transition zone, and on the lower Teton and Marias Rivers (Figure 1).

Pilot PIT Tag Study- Shovelnose Sturgeon were implanted with 23 mm 134.2 kHz HDX PIT tags as part of an ongoing pilot study to evaluate the utility of passive PIT tag antennas for monitoring sturgeon movements in prairie streams and tag retention for peritoneal insertion of the 23 mm tags. The pilot study uses Shovelnose Sturgeon as a surrogate for Pallid Sturgeon, because currently the only acceptable PIT tag for Pallid Sturgeon are smaller dorsally implanted 12 mm (125 kHz FDX or 134.2 KHZ FDX) tags, that have very limited utility for passively monitoring fish movement. In 2019, the Pallid Sturgeon Recovery Team initiated the transition to 12 mm 134.2 kHz FDX tags. As in the past, these tags can only be implanted at the fleshy base of the dorsal fin. Due to the smaller size, different tag location and these tags being FDX rather than HDX, the tags will not perform as well as the 23 mm 134.2 kHz HDX PIT tags used in this study.

RESULTS AND DISCUSSION

In 2019, spring discharge was well above median historical flows (Figure 2) and flows remained high throughout the sampling season. In late March, high flows in the Marias River and Teton River (Figure 3 and 4) caused ice jams and flooding in the lower reaches of the Missouri River. This event caused significant fish mortality, with fish stranded in the floodplain not being able to return to the river. Crews confirmed mortalities of two radio tagged HOPS from the 1997-year class, including one gravid female, and one wild male Pallid Sturgeon that became stranded in the floodplain. The anterior portion of the carcasses (head and pectoral fins) were recovered and are currently being stored in a freezer at the MT Fish, Wildlife and Parks field office in Lewistown, MT. Although not confirmed, there were reports of other smaller juvenile sturgeon found stranded and dead once the water receded. For the second consecutive year, spring set line effort was cancelled due to high flow conditions. Standard trammel net sampling in the Fred Robinson section yielded the lowest Pallid Sturgeon catch since 2014. In 2019, only 102 Pallid Sturgeon were captured, the lowest annual catch in more than ten years. Crews collected 61 genetic samples from new Pallid Sturgeon (No existing PIT tag), four of which did not have any visible external markings (missing scutes or elastomer) to allow year-class to be assigned. Genetic results will assign these fish as HOPS or wild and link them back to a year-class and family. To date there have been no wild-born juveniles sampled in RPMA1.

Broodstock and Stocking- As in 2018, there were no broodstock taken to the hatchery from RPMA1 in 2019. All wild adult Pallid Sturgeon spawned in the hatchery in 2018 are already adequately represented in RPMA1 and as a result there was no stocking of any Pallid Sturgeon in RPMA1 in 2019.

Sampling- Of the 102 Pallid Sturgeon caught in 2019 by FWP personnel, 82 were caught with trammel nets. The majority of Pallid Sturgeon captured in trammel nets were in the Robinson (N=34) and Rock Creek (N=32) sections (Table 2). Pallid Sturgeon and Shovelnose Sturgeon CPUE both decreased in the

Robinson section in 2019 and were the lowest recorded for both species since 2014 (Figure 5). Decreased CPUE was observed for both species in the high-water year of 2014 when only 14 Pallid Sturgeon were caught during the Fred Robinson Bridge netting (Table 1). Mean daily discharge varied between 7760 and 7810 cfs during the Robinson trammel netting effort in 2019, only 2011 and 2014 had higher flows at the time of sampling. In addition to trammel netting efforts, a new benthic trawl protocol caught 20 juvenile Pallid Sturgeon in 2019 (Table 2). The new protocol also resulted in the capture of 88 young-of- the-year sturgeon that are awaiting genetic results to assign species as Shovelnose Sturgeon or Pallid Sturgeon. Larval sampling occurred in the Teton, Marias, and Missouri Rivers in

2019. Crews collected 604 free embryos, including 532 from the Missouri River downstream of Fred Robinson Bridge, 67 from the Marias River below the confluence with the Teton River, 1 from the Marias River above the confluence with the Teton River, and 4 from the Teton River (Table 5). Genetic testing will reveal whether the free embryos were Pallid Sturgeon, Shovelnose Sturgeon, or Paddlefish.

Size structure, Relative Condition, and Year-Class Survival- The size distribution of Pallid Sturgeon caught by FWP in 2019 was similar to recent years with the largest percentage having lengths between 450 and 550 mm (Figure 6). The growth trajectories of 2005 year- class and younger fish continues to show much slower growth after age 4 than the length at age for individuals from the 1997 year-class (Figure 7). In 2019, the average length of an age 10 fish from the 2005 through 2009 year-class ranged from 501 mm to 540 mm, while the average length of an age 10 fish from the 1997 year-class was 674 mm (Table 3). Relative condition of hatchery-origin Pallid Sturgeon in 2019 was similar to previous years (Figure 9). In 2019 the most recently stocked year classes (2015 through 2018) had higher relative condition than in years past (Figure 9). We continue to see low representation of Pallid Sturgeon from the 2005 through the 2008 year-classes which represent four of the five largest stocking events, yet each combined make up less than 15% of total catch (Figure 8). Alternatively, the large stocking event of the 2009 year-class has very high representation, making up almost 20% of the total catch in 2019.

Radio Telemetry- In 2019, FWP recorded locations for 82 radio-tagged Pallid Sturgeon (sixty-six 1997 year-class, two 2005 year-class, two 2007 year-class, three 2009 year-class, one unknown year-class, and eight adults). Monthly tracking runs from April through November encompassed 1,808 river miles and 257 hours. Crews recorded 70 wild Pallid Sturgeon locations and 720 HOPS locations. Remote station downloads resulted in 1,047 Pallid Sturgeon detections across the 13 stations. Of the radio-tagged population, 39% were recorded at or above Judith Landing (Table 4). A remote station on the lower reaches of the Teton River recorded a detection from an immature Pallid Sturgeon from the 1997 yearclass. This is the first documented use of the Teton River by any Pallid Sturgeon. As in 2018, FWP collaborated with a Montana State University Graduate student who used radio telemetry to study movements and spawning success of reproductive female 1997 year-class HOPS in RPMA1. This collaborative effort documented that two reproductive females from the 1997 year-class appear to have spawned in 2019 near Fred Robinson Bridge. Discussion of spawning movements and spawning success for 2019 will be reported for Permit Number TE68706C-0 and will not be discussed in any further detail within this document.

Pilot PIT Tag Study- In 2019, crews implanted 23 mm 134.2 kHz HDX tags into the peritoneal cavity of 331 Shovelnose Sturgeon in the Teton, Marias and Missouri Rivers. A 24 concern that has been raised about using peritoneal cavity insertion of PIT tags in sturgeon, is that they may expel tags during spawning. Past years saw 100% retention in recaptures, with no documented tag loss. In 2019, crews captured fourteen Shovelnose Sturgeon that were originally double tagged with a PIT tag and an external cinch tag. All fourteen of those fish retained their PIT tags. Of those fish, one was tagged in 2016, nine were tagged in 2018, and three were tagged earlier in 2019. In March of 2019, high spring discharge and ice flows damaged the PIT readers on the Teton and Marias Rivers. The Teton River remained too high throughout the spring to reinstall the readers, but crews were able to reinstall the readers on the Marias River. The PIT reader at Marias River mile 1.0 recorded 979 detections from 455 individual Shovelnose Sturgeon during the spawning season. This provided valuable information about the timing of Shovelnose Sturgeon spawning movements in the Marias River and could be a valuable tool for tracking Pallid Sturgeon migration into tributaries in the future (Figure 10).

RECOMMENDATIONS

1. Continue to collect data that is necessary for population and survival estimates for Hatchery Origin Pallid Sturgeon, and how this information relates to stocking densities. 2. Continue radio tracking Pallid Sturgeon, conducting reproductive assessments, and improving upon existing knowledge of the reproductive ecology of Pallid Sturgeon in RPMA1. 3. When possible, continue to increase Pallid Sturgeon sampling efforts in upstream sections including the Marias River. 4. Gain approval to utilize 23 mm 134.2 kHz HDX PIT tags to monitor Pallid Sturgeon movements at remote stations in tributaries and side channels. 5. Evaluate the practicality of estimating carrying capacity in RPMA1 as it relates to stocking density and population size necessary for recovery goals to be achieved. 6. When possible, continue expanded trawling and larval sampling efforts to improve understanding of Acipenseriform reproduction in RPMA1. 7. Implement new technology for remote telemetry stations to allow remote data downloading and improve efficiency and response time for tracking and reproductive assessments of high priority radio-tagged Pallid Sturgeon.

ACKNOWLEDGMENTS

The field and office contributions of Grant Grisak, Jason Rhoten, Mike Schilz, Nate Beckman, and Kelson Hickman made this work possible. The many aspects of this collaborative work was funded in part by the United States Fish and Wildlife Service, Montana Fish, Wildlife and Parks, Northwestern Energy, the United States Bureau of Reclamation, and Western Area Power Administration.

REFERENCES

Braaten, P. J., Fuller, D. B., Holte, L. D., Lott, R. D., Viste, W., Brandt, T. F., & Legare, R. G. (2008). Drift dynamics of larval pallid sturgeon and shovelnose sturgeon in a natural side channel of the upper Missouri River, Montana. North American journal of Fisheries Management, 28, 808-826.

Dryer, M. P., & Sandvol, A. J. (1993). Pallid sturgeon recovery plan. U.S. Fish and Wildlife Service, Bismarck, North Dakota.

Guy, C.S., Treanor, H. B., Kappenman, K. M., Scholl, E. A., Ilgen, J. E., Webb, M. A. H. (2015). Broadening the Regulated-River Management Paradigm a case history of the forgotten dead zone hindering pallid sturgeon recovery. Fisheries, 40, 6-14.

Heist, E., M. Bartron, J. Kalie, and R. Leary. (2013) Population Genetics Management Plan for Pallid Sturgeon in the Upper Missouri River Basin. Final Report, Western Area Power Administration. 40 pp.

Holmquist, L. M. (2017). Reproductive ecology of hatchery-origin and wild pallid sturgeon in the Missouri River above Fort Peck Reservoir, Montana. Master’s thesis, Montana State University, Bozeman.

Holmquist, L. M., Guy, C. S., Tews, A., Webb, M. A. H. (2018). First maturity and spawning periodicity of hatchery-origin pallid sturgeon in the upper Missouri River above Fort Peck Reservoir, Montana. Journal of Applied Ichthyology, 00, 1-11.

Shuman, D. A., Klumb, R. A., Wilson, R. H., Jaeger, M. E., Haddix, T., Gardner, W. M., Doyle, W. J., Horner, P. T., Ruggles, M., Steffensen, K. D., Stukel, S., Wanner, G.

A. (2011) Pallid sturgeon size structure, condition, and growth in the Missouri River Basin. Journal of Applied Ichthyology, 00, 1-13.

Rotella, J. Upper Basin Pallid Sturgeon Survival Estimation Project – 2015 Update Report to Upper Basin Pallid Sturgeon Workgroup. Revised 2015.

Rotella, J. Upper Basin Pallid Sturgeon Survival Estimation Project – 2017 Update Report to Upper Basin Pallid Sturgeon Workgroup. 121 pp.

USFWS (United States Fish and Wildlife Service). (2014). Revised recovery plan for the pallid sturgeon (Scaphirhynchus albus). U.S. Fish and Wildlife Service, Denver, Colorado. 115 pp.

FIGURES

Figure 1. Study area map of Recovery Priority Management Area 1 showing remote radio telemetry receiving station locations.

Figure 2. The 2019 Missouri River hydrograph at Fred Robinson Bridge, Montana for mid- March to midOctober compared to the median daily discharge for Pre- Canyon Ferry Dam and Post- Canyon Ferry Dam.

Figure 3. The 2019 Marias River hydrograph at Loma, Montana for mid-March to mid- October compared to the median daily discharge for Pre- Tiber Dam and Post- Tiber Dam.

Figure 4. The Teton River hydrograph at Loma, Montana for mid-March through mid- October from 2006 through 2019.

Figure 5. Long term trends in relative abundance for Pallid Sturgeon and Shovelnose Sturgeon during 50 standard fall trammel net drifts in the Robinson Section of RPMA1 conducted annually since 2001.

Figure 6. Proportion of Pallid Sturgeon within 50 mm length bins for RPMA1 sampling efforts in 20152019.

Figure 7. Mean length (±SD) at age trajectories for the seven oldest year classes of HOPS stocked into

RPMA1.

Figure 8. Proportion of annual HOPS catch from each year-class stocked during the past six sampling years compared with the number of yearlings stocked from each year class for RPMA1.

Figure 9. Mean condition (±SD) by year class of HOPS sampled in RPMA1 for the past six years (20142019) of sampling.

Figure 10. Number of individual fish detected at Marias River PIT tag readers per day compared with the Teton and Marias River discharge (CFS).

TABLES

Table 1. Sampling statistics for the annual sturgeon assessment, near Robinson Bridge, in the Middle Missouri River, MT, 1996-2019, compared with sturgeon captured by FWP 2008 – 2019.

(1996 - 2007) 2008 2009 2010 2011 2012 2013 2014 2015 2016 2017 2018 2019 Pallid Sturgeon

# Sampled 6.8 28 42 39 39 99 106 14 67 61 102 58 34 # Wild 0.6 0 0 0 0 0 0 0 0 0 0 0 0 # HOPS 6.0 28 42 25 39 99 106 14 67 61 102 58 34 Mean #/drift 0.13 0.56 0.82 0.50 0.78 1.98 2.12 0.21 1.34 1.22 2.06 1.16 0.68 All Gears Catch 146 271 347 302 334 451 217 257 229 414 172 102

Shovelnose Sturgeon # sampled 210 222 227 173 125 226 253 80 108 155 181 130 109 mean weight (g) 1477 1693 1612 1838 1924 1884 1871 1862 1842 1797 1904 1950 1858 number/drift 4.2 4.4 4.4 3.5 2.5 4.5 5.1 1.25 2.2 3.1 3.6 2.6 2.2

Effort Summary Drift time (min) 6.8 6.8 7.0 7.0 7.0 7.0 7.0 7.0 7.0 7.0 7.0 6.9 7.0 # of drifts 50 50 50 50 50 50 64 50 50 50 50 50 Drift distance (m) 262 262 303 291 272 215 278 376 320 284 354 272 297 Depth (m) 1.9 1.6 1.7 2.0 2.4 2.0 1.7 2.7 1.8 1.9 1.8 2.1 2.0

Table 2. Pallid Sturgeon total catch by section and sampling gear for RPMA1 in 2019. For trammel netting the subset caught during standard sampling only are in parenthesis.

Trammel Net Set Line* Benthic Trawl TOTAL Marias River 3 (0) - - 3 Morony 0 - - 0 Fort Benton 2 (2) - - 2 Coal Banks 5 (4) - - 5 Judith Landing 2 (1) - - 2 Fred Robinson 37 (34) - 20 57 Rock Creek 33 (32) - - 33 TOTAL 82 - 20 102 *no spring setline effort was conducted in 2019 due to high discharge and ice jams

Table 3. Mean (±SD) fork length at age by year-class for recaptured Pallid Sturgeon in RPMA1 for the past 22 years of sampling.

1997 2005 2006 2007 2008 2009 2010 2012 2013 2014 2015 2016 2017 2018 Age-1 291 (38) 326 (54) 324 (45) 303 (29) 325 (39) 297 (41) 302 (10) 436 324 (28) 338 (68) 295 (12) 341 (49) 400(26) 437

Age-2 387 (59) 342 (40) 364 (31) 335 (33) 354 (32) 350 (46) 380 (25) 404 (8) 381 (19) 446 (29) 330 (29) 423 (41) 414 (28) Age-3 463 (41) 396 (38) 409 (27) 374 (28) 409 (35) 431 (35) 462 (31) 425 (26) 435 (35) 449 (22) 413 (29) 420 (34) - Age-4 435 (32) 425 (28) 422 (24) 429 (42) 459 (42) 467 (35) 454 (28) 452 (30) 455 (33) 462 (38) 433 (34) - - Age-5 482 (30) 440 (36) 464 (53) 466 (34) 480 (28) 466 (26) 467 (28) 477 (23) 473 (14) 510 (63) - - - Age-6 527 (53) 478 (51) 507 (32) 474 (38) 491 (29) 472 (28) 477 (26) 492 (13) 491 - - - - Age-7 529 (34) 521 (32) 515 (30) 465 (31) 485 (28) 486 (30) 493 (24) 529 - - - - - Age-8 558 (24) 523 (41) 534 (32) 480 (36) 500 (42) 498 (26) 540 (33) ------Age-9 613 (69) 533 (53) 527 (32) 469 (57) 509 (30) 532 (36) 524 (37) ------Age-10 674 (83) 540 (70) 524 (36) 501 (48) 536 (18) 537 (47) ------Age-11 686 (83) 550 (92) 532 (37) 555 (80) 524 (31) ------Age-12 727 (129) 582 (74) 570 (44) 552 (57) ------Age-13 762 (62) 557 (71) 616 (74) ------Age-14 854 (134) 527 ------Age-15 866 (162) ------Age-16 878 (127) ------Age-17 974 (105) ------Age-18 1011 (109) ------Age-19 1036 (103) ------Age-20 1053 (109) ------Age-21 1111 (56) ------Age-22 1087 (87)

-underlined bold font denotes the largest mean length at age across all year classes

-underlined italic font denotes largest mean length at age for non-1997 year-classes

Table 4. Number of Pallid Sturgeon (percentage of tagged population) located at or upstream of each remote ground station in 2019. Total tagged population was 82 individuals (8 Wild and 74 HOPS) in 2019.

Station Name River Mile # HOPS (%) # Wild (%) Carter Ferry 2089 1 (1%) - Fort Benton 2074.3 4 (5%) - Teton River Teton 0.4 1 (1%) - Marias RM 3 Marias 3.0 1 (1%) - Marias Confluence Marias 0.5 5 (7%) - Boneyard 2050.4 12 (16%) - Coal Banks Landing 2031.4 21 (28%) 1 (13%) Judith Landing 1984 29 (39%) 3 (38%) Stafford Ferry 1970 34 (46%) 4 (50%) Bird Rapids 1955.8 39 (53%) 4 (50%) Power Plant 1937.9 50 (68%) 7 (88%) King Island 1920.1 62 (84%) 8 (100%) Road's End 1901 74 (100%) 8 (100%)

Table 5. Larval sampling summary of 2019 efforts. Catch per unit effort (CPUE) reflects the number of Acipenseriform free embryos sampled per 100 cubic meters or water filtered.

Sampling Site Dates Paired Samples Minutes Free CPUE Embryos Embryos Marias above June 3-5 10 232 0 0 0 Teton June 12 3 90 0 0 0 July 2-3 9 270 1 0.02 0 Total 22 592 1 - 0 Marias below Teton June 3-5 10 50 0 0 0 June12- 7 50 0 0 0 13 July 1-2 41 410 67 0.35 10 Total 58 510 67 10 Missouri at Robinson June 6 6 30 122 9.60 2 June 10- 18 92 66 1.76 7 11

June 18- 30 151 275 4.65 52 20 June 25- 45 350 69 0.57 125 27 Total 99 623 532 - 186 Teton rivermile June 3 1 10 0 0 0 0.1 June 12 1 5 0 0 0 July 1-2 11 165 4 0.09 0 Total 13 180 4 - 0 2019 Overall 192 1905 604 - 196 Total

APPENDIX A

Editor’s note: Appendix A has not been included in this report for brevity. Please contact Luke for more detailed information.

Spawning Readiness, Spawning Location(s), and Habitat Use of Hatchery-origin Pallid Sturgeon in the Missouri River above Fort Peck Reservoir, MT

2019 Annual Report

Tanner L. Cox and Christopher S. Guy Montana Cooperative Fishery Research Unit, Montana State University

Molly A.H. Webb USFWS, Bozeman Fish Technology Center

Luke M. Holmquist Montana Fish, Wildlife & Parks

Introduction Pallid Sturgeon Scaphirhynchus albus are endemic to the Mississippi River basin, and inhabit the

Missouri River, Mississippi River, and several large tributaries of these rivers (USFWS 2014; Jordan et al. 2016). Pallid Sturgeon were listed as endangered in September 1990 due to declines in abundance, which were hypothesized to be a result of river alterations (U.S. Federal Register 1990). River alterations, such as channelization and construction of large impoundments, have truncated riverine conditions, homogenized available habitat, and reduced population connectivity of Pallid Sturgeon throughout their range (Jordan et al. 2016). Pallid Sturgeon habitat is further altered by the effects upstream reservoirs have on water quality, including changes in temperature regime, flow regime, and sediment loading (Jordan et al. 2016).

River alterations appear to have resulted in recruitment failure for Pallid Sturgeon in the upper Missouri River Basin (USFWS 2014). Successfully spawned Pallid Sturgeon embryos drift for 11 days or more and disperse 245-530 km downstream (Kynard et al. 2007; Braaten et al. 2008). Truncated riverine conditions resulting from the presence of reservoirs reduces available drift distance and allows Pallid Sturgeon embryos to drift into the upstream end of reservoirs (Braaten et al. 2008). The transition zone above Fort Peck Reservoir is anoxic at the water-sediment interface and it is hypothesized that Pallid Sturgeon embryos settle in this habitat and subsequently die (Guy et al. 2015). This phenomenon may result in recruitment failure if Pallid Sturgeon do not spawn far enough upstream to afford adequate drift distance to embryos.

Pallid Sturgeon in the Missouri River above Fort Peck Reservoir appear to experience high rates of follicular atresia, which may also contribute to recruitment failure. Recent research found no evidence of ovulatory success by reproductively-active female (RAF) Pallid Sturgeon in this area, and reproductive assessments of two wild Pallid Sturgeon and three hatchery-origin Pallid Sturgeon determined all RAF Pallid Sturgeon tracked between 2014 and 2016 experienced follicular atresia (Holmquist et al. 2019). Hatchery-origin fish that experienced follicular atresia may have been experiencing their first reproductive cycle. Some fish species experience follicular atresia at the conclusion of their first reproductive cycle. This phenomenon is known as a spawning “dummy run” and consists of elevated sex steroid levels and development of ovarian follicles followed by follicular atresia. It is unknown if the atretic hatchery-origin Pallid Sturgeon observed by Holmquist et al. (2019) experienced a dummy run or experienced ovulatory failure due to other factors. Continued monitoring of maturing hatchery-origin Pallid Sturgeon will allow further explanation of follicular atresia experienced by Pallid Sturgeon in the Missouri River above Fort Peck Reservoir.

In the event of successful spawning, successful recruitment of Pallid Sturgeon in the study area is reliant on adequate free embryo drift distance before reaching an anoxic zone at the upstream end of Fort Peck Reservoir (Guy et al. 2015). Past research observed male aggregations of Pallid Sturgeon at locations that would result in inadequate drift distance (Holmquist et al. 2019). Therefore, further knowledge of Pallid Sturgeon reproductive potential, movement patterns, spawning habitat, and spawning locations is central to informing management strategies for the Pallid Sturgeon population above Fort Peck Reservoir.

Objectives 1. Describe age at first maturity for Pallid Sturgeon in the Missouri River above Fort Peck Reservoir. 2. Describe the spawning periodicity of hatchery-origin Pallid Sturgeon in the Missouri River above Fort Peck Reservoir. 3. Quantify movement rates of reproductively-active Pallid Sturgeon in the Missouri River above Fort Peck Reservoir. 4. Define where Pallid Sturgeon spawning occurs in the Missouri River above Fort Peck Reservoir. 5. Describe habitat characteristics in the Missouri River above Fort Peck Reservoir at aggregation and spawning sites. 6. Determine if the high rate of follicular atresia experienced by maturing Pallid Sturgeon in the Missouri River above Fort Peck Reservoir is due to a spawning “dummy run” or lack of environmental and/or social cues.

Study Area The study area is within the Great Plains Management unit described in USFWS (2014) and consists of the Missouri River from the upstream end of Fort Peck Reservoir to Morony Dam

(river kilometer [rkm] 3,010 to rkm 3,387) and the Marias River from its confluence with the Missouri River to Tiber Dam (rkm 0 to rkm 132). This area represents the northernmost distribution of Pallid Sturgeon in the Missouri River basin (USFWS 2014). Substrate within the

Missouri River above Fort Peck Reservoir is primarily cobble from river kilometer 3,340 to 3,130 (Richards 2011). At river kilometer 3,130, composition shifts to mostly gravel for several kilometers before transitioning into fine and sandy substrate (Richards 2011). Data recorded at the Landusky USGS (06115200) gauging station indicates peak spring discharge occurs between late May and late June. The study location here exhibits more natural habitat than downstream portions of the Missouri River where more hydrologic alterations have accumulated. The study area has not been channelized and continues to be hydrologically influenced by multiple unregulated tributaries (Scott et al. 1997). However, the study area has been influenced by anthropogenic factors (Hesse 1987). The mainstem Missouri River contains nine dams upstream of the study area. Eight of the upstream dams maintain outflow roughly equal to inflow and have little effect on discharge of the Missouri River (DNRC 2014; NWE 2016). Canyon Ferry Dam (rkm 3626) is an exception to this and has regulatory effects on discharge of the Missouri River (Bovee and Scott 2002). In addition to the mainstem impoundments, many tributaries of the Missouri River have been regulated by dams (Hesse 1987). Tiber Dam on the Marias River and Gibson Dam on the Sun River are two regulatory impoundments on tributaries of the Missouri River above Fort Peck reservoir. Both dams have the ability to affect discharge and flow regime within the study area (Bovee and Scott 2002).

Methods Fish Sampling Pallid Sturgeon were sampled using trammel nets between early-May and late-July of 2019. Prespawning season captures facilitated reproductive assessment, and post-spawning season capture of RAF Pallid Sturgeon allowed detection of ovulatory success or failure. Spawning season was defined as the time between the start of the descending limb of the hydrograph and the point at which water temperatures reached 24°C. These times were selected because spawning of Pallid Sturgeon occurs in late spring to early summer on the descending limb of the hydrograph (Bramblett and White 2001; DeLonay et al. 2016).

Additionally, natural spawning temperatures for Pallid Sturgeon are predicted to be 12°C to 24°C (Kappenman et al. 2013). Trammel nets 45.7-m long and 1.8-m deep with 5.08- cm inner bar mesh and 25.4-cm outer bar mesh were used for smaller fish and nets with 10.16-cm inner bar mesh and 25.4-cm outer bar mesh or nets with 10.16-cm inner bar mesh and 20.32-cm outer bar mesh were used for larger fish. Pallid Sturgeon were targeted using radio telemetry. Pallid Sturgeon that were known to be female fish, which had experienced reproductive activity in the past were considered high priority. Pallid Sturgeon that were known to be female fish but without prior confirmation of reproductive activity were targeted after known mature females had been captured. Male Pallid Sturgeon and Pallid Sturgeon of unknown sex were of low priority and were captured opportunistically.

Biological data and samples were collected from captured Pallid Sturgeon. Handling and sampling procedures conformed to protocols developed for Pallid Sturgeon (USFWS 2012). Passive Integrated Transponder (PIT) tags that individually identify fish were scanned for, and age-class markers such as missing scutes and elastomers were noted. Pallid Sturgeon were measured in fork length ± 1 mm and weighed ± 1 g. Blood was collected from the caudal vasculature of fish using a 3-ml syringe. Each blood sample was immediately transferred to a 7ml lithium heparinized vacutainer, stored in a cool environment, and transported to the field station. Blood samples were centrifuged at 3400 rpm for 5 minutes to separate blood plasma from the sample. Blood plasma was transferred to 1.5-ml vials and stored at -20 °C. Ovarian follicle samples were collected from the gonad of female Pallid Sturgeon that were expected to be reproductively active. Ovarian follicles were sampled using the minimally invasive method described in Candrl et al. (2010). If this method was unsuccessful, the gonad was visually examined through an incision, and a histological sample was collected from the Pallid Sturgeon. Histological samples were collected by inserting an otoscope through celiotomy, visually inspecting the gonad, and collecting a biopsy of gonad by inserting a Miltex biopsy cup through the specula of the otoscope (Webb et al. 2018). The Miltex biopsy cup was used to sample from three different parts of the gonad to account for heterogeneity of the gonad. Tissue samples were also collected from the gonad of male Pallid Sturgeon using the method described above. All tools used for collecting ovarian follicle or histological samples were disinfected by soaking in 70% isopropyl alcohol. Tools were rinsed with sterile saline prior to use to prevent irritation to the tissue of the fish. Ovarian follicle and gonadal-tissue samples were preserved in 10% phosphate-buffered formalin.

Reproductive Assessment Blood and gonadal-tissue samples collected prior to spawning season were analyzed at the US Fish and Wildlife Service Bozeman Fish Technology Center. Sex steroids (testosterone [T] and estradiol-17β [E2]) were extracted from blood plasma using methods described in Fitzpatrick et al. (1987). An extraction solvent (2-mL of diethyl ether) was added to tubes

43 with 100-µL of the plasma sample, and the mixture was vortexed. The aqueous phase was removed by snap-freezing with liquid nitrogen, and the extracts from each tube were combined and dried under a stream of liquid nitrogen. The extract was reconstituted in a 1- mL solution of phosphate-buffered saline and gelatin (PBSG) (Fitzpatrick et al. 1986). For T and E2, 10 or 50-µL of reconstituted steroid extract was analyzed by radioimmunoassay (RIA) as described in Fitzpatrick et al. (1986) and modified by Feist et al. (1990). A slightly more concentrated charcoal solution (6.25 g charcoal and 4.0 g dextran/L PBSG) was used for all assays. All samples were analyzed in duplicate to ensure accuracy. Testosterone and E2 concentrations were validated by verifying that serial dilutions were parallel to standard curves. Recovery efficiency was used to correct all RIA results.

Blood plasma sex steroid concentrations were used to assign sex and stage of maturity to Pallid Sturgeon. Concentrations of T greater than 38 ng/mL and E2 less than 0.3 ng/mL were used to indicate a reproductively-active male (RAM) Pallid Sturgeon. Concentrations of T greater than 10 ng/mL and E2 greater than 0.3 ng/mL were used to indicate a RAF Pallid Sturgeon. Pallid Sturgeon that were not classified as reproductively active were classified as non-reproductivelyactive male (NRAM) or non-reproductively-active female (NRAF) if the respective sex of the fish was known. Pallid Sturgeon were classified as non-reproductive and unknown sex (NRAU) if sex had not been previously determined and sex steroid levels were not indicative of a reproductively-active Pallid Sturgeon. A RAM Pallid Sturgeon is defined as a meiotic or ripe male and a RAF Pallid Sturgeon is defined as a vitellogenic or ripe female. A NRAM Pallid Sturgeon is defined as a male Pallid Sturgeon that is not meiotic or ripe. A NRAF Pallid Sturgeon is defined as a female Pallid Sturgeon that is not vitellogenic or ripe. Pallid Sturgeon determined to not be reproductively-active were not expected to spawn during the current season.

Location Data Collection Pallid Sturgeon were tracked from late-May to early-July of 2019. Tracking was conducted using portable Lotek SRX 400 telemetry receivers (Lotek Wireless, Inc., Newmarket ON, Canada). Receivers were connected to a boat-mounted four-element Yagi antenna, a handheld threeelement Yagi antenna, and land-based four-element Yagi antennas that were distributed throughout the study area (figure 1). Boat-mounted and handheld systems were used manually to provide real-time information about transmitters associated with Pallid Sturgeon. Land-based systems autonomously recorded data about each detected transmitter that passed through the field of detection. Transmitters implanted in Pallid Sturgeon transmitted a unique code to the receivers allowing each fish to be identified. Transmitters operated at 149.800 or 149.900 MHz frequencies.

Fish locations were determined using the handheld and boat-mounted telemetry systems during tracking events. When determining the position of a fish, signal directionality and signal strength were used to maximize accuracy. Using the handheld antenna, direction of

44 the fish was determined by rotating the antenna until maximum signal strength was determined. The boat was maneuvered in the direction of the fish until signal strength of the boat-mounted telemetry system decreased as a result of the location of the fish shifting from anterior to posterior of the receiving antenna. Signal strength of the handheld telemetry system was compared when facing port and starboard directions. Similar signal strength indicated that the fish was directly under the boat. After the location of the fish was determined, a GPS location was recorded for the identified fish. Gain of receivers was set to a level that allowed the operator to detect differences in signal strength given directionality and distance to the fish. Blind tests were performed to ensure all operators were accurately locating fish.

Pallid Sturgeon were systematically tracked based on priority given sex and stage of maturity. Female Pallid Sturgeon that were determined to be reproductively-active during the pre-spawn sampling period (described above) were tracked at a minimum of once per two days. Few male Pallid Sturgeon were captured prior to spawning season, and reproductive stage of most male

Pallid Sturgeon was not known at the initiation of tracking so all known mature male Pallid

Sturgeon located within the vicinity of RAF Pallid Sturgeon were tracked once per week. Other Pallid Sturgeon were tracked opportunistically while tracking RAF Pallid Sturgeon. Autonomous land-based telemetry systems were used to aid in searching for undetected Pallid Sturgeon by documenting if a fish had passed within range of the land-based system.

Preliminary Results Twenty-nine 1997 year-class Pallid Sturgeon and one wild male Pallid Sturgeon were captured prior to spawning season. Gonadal-tissue samples were collected from sixteen of the 1997 yearclass Pallid Sturgeon, and blood samples were collected from all captured Pallid Sturgeon. Two of the captured Pallid Sturgeon were determined to be RAFs (Table 1). The RAFs were recaptured after the spawning season, and gonadal-tissue samples were taken. Histological analysis of the gonadal tissue revealed that both fish had post- ovulatory follicles indicating that both RAFs had spawned.

Two RAF Pallid Sturgeon and 14 mature male Pallid Sturgeon were tracked from late- May through early-July. The RAFs were most often located in the lower portion of the study area, with a maximum upstream location of Missouri River kilometer 3208.2 (Figure 2; Table 2). Mature male Pallid Sturgeon were most often located in the lower portion of the study area with only two locations exceeding Missouri River kilometer 3200 (Figure 3).

Preliminary Conclusions One RAF Pallid Sturgeon that spawned in 2019 (Radio ID 8_86) was reproductively active and experienced follicular atresia in 2015 (Holmquist 2017). Spawning at a successive reproductive event after experiencing atresia at the first known reproductive event is consistent with the pattern of a dummy run. Although a dummy run may occur during the first reproductive cycle in Pallid Sturgeon, subsequent follicular atresia experienced during successive reproductive cycles observed in other Pallid Sturgeon during this study in 2018 suggests a lack of environmental cues, social cues, or both needed to spawn successfully. It is unknown if the other RAF Pallid Sturgeon that successfully spawned in 2019 (Radio ID 8_92) had completed a reproductive cycle prior to 2019. These results will be used along with 2018 results to fully evaluate the dummy- run hypothesis. Use of the lower portion of the study area by RAF Pallid Sturgeon is in line with Holmquist (2019) and Richards (2011), which both observed Pallid Sturgeon primarily using lower reaches of the study area during the putative spawning season. However, it is unlike observations made in 2018 when RAF Pallid Sturgeon were documented using upper reaches of the study area including the Marias River. The reasons for this contrast are not well understood. Although discharge of the Missouri and Marias rivers during 2018 was higher than it was during the field season of Richards (2011), Holmquist (2019), and of this study in 2019, other factors could contribute to the observation of RAF pallid sturgeon using upper portions of the study area in 2018.

References Bovee, K. D., and M. L. Scott. 2002. Implications of flood pulse restoration for Populus regeneration on the upper Missouri River. River Research and Applications 18:287-298.

Braaten, P. J., D. B. Fuller, L. D. Holte, R. D. Lott, W. Viste, T. F. Brandt, and R. G. Legare. 2008. Drift dynamics of larval Pallid Sturgeon and Shovelnose Sturgeon in a natural side channel of the upper Missouri River, Montana. North American Journal of Fisheries Management 28:808-826.

Bramblett, R. G., and R. G. White. 2001. Habitat use and movements of Pallid and Shovelnose sturgeon in the Yellowstone and Missouri rivers in Montana and North Dakota. Transactions of the American Fisheries Society 130:1006-1025.

Candrl, J. S., D. M. Papoulias, and D. E. Tillitt. 2010. A minimally invasive method for extraction of sturgeon oocytes. North American Journal of Aquaculture 72:184- 187.

DeLonay, A.J., R. B. Jacobson, K. A. Chojnacki, P. J. Braaten, K. J. Buhl, B. L. Eder, C. M.

Elliott, S. O. Erwin, D. B. Fuller, T. M. Haddix, H. L. A. Ladd, G. E. Mestl, D. M. Papoulias, J. C. Rhoten, C. J. Wesolek, and M. L. Wildhaber. 2016. Ecological requirements for Pallid Sturgeon reproduction and recruitment in the Missouri River— annual report 2013: U.S. Geological Survey, Open-File Report 2015– 1197, 99 p.

DNRC (Montana Department of Natural Resources and Conservation). 2014. Toston Dam (Broadwater-Missouri). Montana Department of Natural Resources and Conservation, Helena, MT.

Feist, G., C. B. Schreck, M. S. Fitzpatrick, and J. M. Redding. 1990. Sex steroid profiles of coho salmon (Oncorhynchus kisutch) during early development and sexual differentiation. General and Comparative Endocrinology 80:299-313.

Fitzpatrick, M. S., G. Van Der Kraak, and C. B. Schreck. 1986. Profiles of plasma sex steroids and gonadotropin in Coho Salmon, Oncorhynchus kisutch, during final ovarian maturation. General and Comparative Endocrinology 62:437-451.

Fitzpatrick, M. S., J. M. Redding, F. D. Ratti, and C. B. Schreck. 1987. Plasma testosterone predicts the ovulatory response of Coho Salmon (Oncorhynchus kisutch) to gonadotropin-releasing hormone analog. Canadian Journal of Fisheries and Aquatic Sciences 44:1351–1357.

Guy, C. S., H. B. Treanor, K. M. Kappenman, E. A. Scholl, J. E. Ilgen, and M. A. H. Webb. 2015. Broadening the regulated-river management paradigm: a case history of the forgotten dead zone hindering Pallid Sturgeon recovery. Fisheries 40:6-14.

Hesse, L. W. 1987. Taming the wild Missouri River: what has it cost? Fisheries 12:2-9.

Holmquist, L. M., 2017. Reproductive ecology of hatchery-origin and wild Pallid Sturgeon in the Missouri River above Fort Peck Reservoir, Montana. Master’s thesis, Montana State University, Bozeman.

Holmquist, L. M., C. S. Guy, A. Tews, D. J. Trimpe, and M. A. H. Webb. 2019. Reproductive ecology and movement of Pallid Sturgeon in the upper Missouri River, Montana. Journal of Applied Ichthyology 35:1069-1083.

Jordan, G. R., E. J. Heist, P. J. Braaten, A. J. DeLonay, P. Hartfield, D. P. Herzog, K. M.

Kappenman, and M. A. H. Webb. 2016. Status of knowledge of the Pallid Sturgeon (Scaphirhynchus albus Forbes and Richardson, 1905). Journal of Applied Ichthyology 32:191-207.

Kappenman, K. M., M. A. H. Webb, and M. Greenwood. 2013. The effect of temperature on embryo survival and development in Pallid Sturgeon Scaphirhynchus albus (Forbes & Richardson 1905) and Shovelnose Sturgeon S. platorynchus (Rafinesque, 1820). Journal of Applied Ichthyology 29:1193-1203.

Kynard, B., E. Parker, D. Pugh, and T. Parker. 2007. Use of laboratory studies to develop a dispersal model for Missouri River Pallid Sturgeon early life intervals. Journal of Applied Ichthyology 23:365–374.

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and SOP agreements with agencies for NWE’s 11 hydropower dams (3 FERC licenses). North Western Energy, Sioux Falls, SD.

Richards, R. R. 2011. Movement of Scaphirhynchus species in the Missouri River above Fort Peck Reservoir, Montana. Master’s thesis, Montana State University, Bozeman.

Scott, M. L., G. T. Auble, and J. F. Friedman. 1997. Flood dependency of cottonwood establishment along the Missouri River, Montana, USA. Ecological Applications 7:677690.

USFWS (United States Fish and Wildlife Service. 2014. Revised Recovery Plan for the Pallid Sturgeon (Scaphirhynchus albus). U.S. Fish and Wildlife Service, Denver, Colorado.

USFWS (United State Fish and Wildlife Service. 2012. Biological procedure and protocols for researchers and managers handling Pallid Sturgeon. U.S. Fish and Wildlife Service, Denver, Colorado.

U.S. Office of the Federal Register. 1990. Endangered and threatened wildlife and plants; determination of endangered status for the Pallid Sturgeon. Federal Register 55:173(6 September 1990):36641-36647.

Webb, M. A. H., J. P. Van Eenennaam, J. A. Crossman, and F. A. Chapman. 2018. A practical guide for assigning sex and stage of maturity in sturgeons and paddlefish. Journal of Applied Ichthyology 00: 1-18.

Table 1. Reproductive status of 1997 year-class female Pallid Sturgeon sampled and tracked during the 2019 field season.

ID Pre-spawn Post-spawn

8_86 Ripe Post-ovulatory

8_92 Ripe Post-ovulatory

Table 2. Sample size, mean (SE), median, maximum, and minimum Missouri River kilometer for RAF Pallid Sturgeon tracked from late-May to early-July 2019.

ID N Mean (SE) Median Max Min 8_86 19 3119.8 (9.4) 3119.2 3208.2 3055.2

8_92 25 3096.6 (5.1) 3090.6 3136.0 3057.0

Figure 1. Map of land-based telemetry stations on the Missouri River upstream of Fort Peck Reservoir, MT (• denotes telemetry station).

Figure 2. Missouri River kilometer ( denotes the mean) by individual RAF Pallid Sturgeon tracked during the 2019 field season.

Figure 3. Missouri River kilometer ( denotes the mean) by individual mature male Pallid Sturgeon tracked during the 2019 field season.

2019 Pallid Sturgeon Population Assessment and Associated Fish Community Monitoring for the

Missouri River: Segment 4

The Bismarck USFWS office has participated in the USACE funded Pallid Sturgeon Population Assessment Program (PSPAP) since 2005. Our office is responsible for sampling segment 4 of the Missouri River from the confluence of the Yellowstone River to the headwaters of Lake Sakakawea. In 2019, the sampling protocols that we had been using for the previous 14 years changed as part of the Missouri River Recovery Adaptive Management program.

Instead of random sampling 12 random bends from April through the end of June as we had done previously, we conducted a weekly segment sweep with telemetry equipment trying to relocate pallid sturgeon implanted with radio tags. After relocating either a wild adult or hatchery origin pallid sturgeon (HOPS) that was needed either for assessment or radio tag replacement, we would then target those fish for capture. During this effort we captured 9 wild adults and 22 HOPS. Five wild adults were hauled to GDNFH for spawning. Ten HOPS were either implanted or reimplanted with radio tags. A blood sample was collected from all 22 HOPS and sent to Bozeman Fish Technology Center to determine the sex and maturity of the fish.

After spring sampling, we assisted with the larval drift experiment that was initiated near Wolf Point, MT. We then began our age-0 sampling by trawling segment 4 with the OT04 when the larval drift experiment was complete. Within the first week of sampling, we suspected the OT04 did not fish as effectively as the standard OT16 that we had been using the previous 14 years. After discussing this with Tim Welker and Mike Colvin, it was decided that we would conduct a gear comparison between the OT04 and the OT16. Both gears were deployed in the same bends on consecutive days. After two weeks of sampling, Mike confirmed what we had suspected that the OT16 captured more fish, the same species and the same size range of fish as the OT04. We completed our sampling using the OT16. One hundred ninety six age-0 sturgeon were captured during this sampling effort. One was genetically confirmed to be a pallid sturgeon from the larval drift release. It was captured on 9/9/2019 near river mile 1553 and was 122mm in length.

When age-0 sampling was complete, we began our targeted sampling for HOPS. Each week we would randomly sample two bends on the first day. On the second day we would conduct a telemetry sweep of each bend and proceed to sample the habitats in the bends that had the highest probability of having pallid sturgeon present. We continued this sampling for an additional two days. On the last day we again performed a telemetry sweep of the bends to determine if any radio tagged pallids were still present in the bend. Forty five HOPS and one wild adult were captured during this targeted effort. In addition we also conducted a mark

53 recapture study with shovelnose sturgeon. Ninety six shovelnose sturgeon were marked with floy tags and one was recaptured.

Yellowstone River Radio-Telemetry

Intake Passage Summary

2019

Jordan Pesik1, Mathew Rugg1, Eric Best2, Trevor Gust2, & David Trimpe2 1 Montana Fish, Wildlife & Parks 2 U.S. Bureau of Reclamation

Submitted to U.S. Bureau of Reclamation Montana Area Office March 2020 Agreement No. R18AP00281

ABSTRACT Construction on the Lower Yellowstone Intake Diversion Dam Fish Passage Project began in 2019 and is expected to take three years to complete. The first of its kind, this highly anticipated project is expected to improve fish passage upstream at Intake Diversion Dam, particularly for the federally endangered Pallid Sturgeon. With the closure of the historic Joe’s Island Side Channel circumventing Intake dam, fish passage was expected to be limited to over the dam or via translocation for the duration of construction. However, rising waters in May reopened the side channel for a period and permitted passage by Paddlefish and Shovelnose Sturgeon. Blue Sucker, Paddlefish and Shovelnose Sturgeon all had upstream passages over the dam in 2019. While no Pallid Sturgeon passed naturally, 12 individuals were translocated upstream in 2019, including two male wild-origin Pallid Sturgeon. Pallid Sturgeon, Paddlefish and Shovelnose Sturgeon all demonstrated use of habitats in the Powder River during the early summer spawning period. Use of the Powder River in 2019 provided new insights on the behaviors of Paddlefish and Shovelnose Sturgeon. This is also the first year a wild-origin Pallid Sturgeon has overwintered upstream Intake. Telemetry efforts for the remainder of the construction period will be focused on reimplanting Pallid Sturgeon carrying transmitters with an expiring battery and preparing for the second phase of the overarching study: post-construction evaluation of the success of the bypass channel.

ABREVIATIONS/ACRONYMS cfs – cubic feet per second

FWP – Montana Fish, Wildlife and Parks

HOPS – Hatchery-Origin Pallid Sturgeon

JISC – Joe’s Island Side Channel PDR – Powder River

RM – river mile(s)

RPMA2 – Pallid Sturgeon Recovery Priority Management Area II

TNG – Tongue River

USBR – United States Bureau of Reclamation

USACE – United States Army Corps of Engineers

WGFD – Wyoming Game and Fish Department

WOPS – Wild-Origin Pallid Sturgeon

YSR – Yellowstone River

INTRODUCTION Following years of litigation, the Intake Diversion Dam (Intake) fish passage bypass channel and new dam project on the Lower Yellowstone River broke ground in 2019. Located 16 miles northeast of Glendive, MT, this highly debated project was proposed by the U.S. Bureau of Reclamation (USBR) and U.S. Army Corps of Engineers (USACE) in response to the listing of Pallid Sturgeon (Scaphirhynchus albus) as a federally endangered species in 1990. Pallid Sturgeon once inhabited and readily accessed the warm water reaches of the Yellowstone River from its confluence with the Missouri River to the confluence of the Bighorn River almost 300 river miles upstream (Nelson and Jaeger 2006). Following the completion of Intake in 1909, spawning migrations of Pallid Sturgeon (and other native species) up the Yellowstone River have been greatly limited by Intake. Spawning is consistently documented in the lower ten miles of the Yellowstone River, but no recruitment has resulted from those spawning events. The current theory of why there has been no documentable recruitment of Pallid Sturgeon in the Yellowstone River since their listing is the lack of drift distance between spawning locations downstream of Intake and the anoxic

57 headwaters of Lake Sakakawea on the Missouri River. In recent years, those Pallid Sturgeon residing, translocated (since 2017), or able to naturally pass above Intake have demonstrated wide use of the available habitats in the Yellowstone River, and an affinity for the Powder River during the spawning window. Spawning in the Powder River or Yellowstone River above the Powder River confluence would greatly increase the drifting distance for larvae, which may provide enough developmental time for recruitment.

As of 2014, wild-origin adult Pallid Sturgeon (WOPS) and hatchery-origin Pallid Sturgeon (HOPS) juveniles have been occasionally documented using Joe’s Island Side Channel (JISC) to swim up and around Intake, and only once have they traversed up over the dam (Rugg et al., 2019). Since adult Pallid Sturgeon have been relocated frequently in the river below and up to Intake, increasing upstream passage past Intake presents itself as a viable solution to the problem of Pallid Sturgeon recruitment.

Since other species in the Yellowstone River may also be affected by Intake, four native species besides Pallid Sturgeon, including Blue Sucker (Cycleptus elongatus), Paddlefish (Polyodon spathula), Shovelnose Sturgeon (Scaphirhynchus platorynchus), and Sauger (Sander canadensis), were included in the four-year pre-bypass channel construction analysis of fish passage at Intake. These species were all found to pass Intake, but with varying degrees of success and route preference. Paddlefish mostly passed via JISC, Blue Sucker preferred to pass over the dam, and both Shovelnose Sturgeon and Sauger showed no preference of passage route. Movements from all species but Blue Sucker indicated Intake was a barrier to upstream movements (Rugg et al., 2019). Therefore, increased upstream passage past Intake will likely have beneficial ramifications not limited to Pallid Sturgeon.

In what is projected to be a three-year construction period, excavating the new bypass channel around Intake dam began in May 2019. The highly anticipated bypass channel is expected to improve upstream passage past Intake by Pallid Sturgeon and other native species. While construction is underway, telemetry efforts will focus on monitoring Pallid Sturgeon activities upstream of Intake in the Yellowstone and Powder Rivers, replacing the expiring radio transmitters in WOPS, and preparing for the second phase of the study: the post- construction analysis of the success of the bypass channel. The purpose of this report is to summarize telemetry data from 2019: construction year one.

METHODS Intake is located at RM 71 on the Yellowstone River, which is only a portion of the geographic area concerning this population of Pallid Sturgeon. Pallid Sturgeon Recovery Management Priority Area II (RPMA2) includes the Missouri River from Fort Peck dam to the headwaters of Lake Sakakawea, the Yellowstone River, and the tributaries in these river reaches (Figure 1). Management and telemetry efforts are in action on the Missouri River, but they will not be included in this summary.

Montana Fish, Wildlife & Parks (FWP) Fisheries Region 7 and USBR crews monitored Lotek Wireless Inc. radio-implanted Pallid Sturgeon (Scaphirhynchus albus), Blue Sucker (Cycleptus elongatus), Paddlefish (Polyodon spathula), Shovelnose Sturgeon (Scaphirhynchus platorynchus), and Sauger (Sander canadensis) with ground-based receiver stations and manual relocation operations via boat or aircraft. In 2019, FWP crews managed an array of 12 receiver stations between Seven Sisters Fisheries Access Site (Yellowstone River [YSR] RM 39) and the Bighorn River confluence (YSR RM 295), which included adding a second station at Intake to monitor only Pallid Sturgeon radio-tag frequencies, and a station at Stipek Fisheries Access Site (YSR RM 83) upstream of Intake. There was additionally a station on the Powder River at Locate, MT (Powder River [PDR] RM 31), that was monitored by FWP crews. Blue Sucker, Paddlefish, Shovelnose Sturgeon and Sauger data from a station managed by the United States Geological Survey at the Yellowstone River Confluence (YSR RM 1) was also included in this report. Manual relocations of fish in the Yellowstone River and its tributaries occurred from early-April through late- October in 2019.

At the beginning of April, a rock crossing with three small culverts was constructed across the historic Joe’s Island Side Channel (JISC) to be a bridge for heavy machinery crossing the channel. Consequently, flows and fish passage through the channel were impeded. Fish passage at Intake through JISC was thought to have concluded in 2018, since construction plans included filling the existing side channel with excavations from the soon-to-be bypass channel. However, the rise in river discharge in mid-May 2019 topped over and eroded a channel around the constructed crossing in JISC, enabling fish passage around Intake via JISC until July 1, 2019, when the road was rebuilt and the channel was permanently resealed with an earthen dam at the head of the channel. In addition to JISC, fish were able to pass over Intake dam, and USBR crews translocated

Pallid Sturgeon over Intake dam during the May pre-high-water period. This was the third year in a row that USBR translocated Pallid Sturgeon from the reach below Intake. The capture reach downstream of Intake was extended an additional mile downstream (to YSR RM 69) in 2019 to atone for expected reduced natural passage during the bypass channel construction years.

RESULTS Hydrograph

Yellowstone River discharge in 2019 near Glendive, MT, was above the historic median from iceout in late-March to the spring rise in early-May. Thereon, the discharge curve mirrored the historic trend, peaking at 60,900 cfs on June 10, 2019. The descending limb of the hydrograph occurred about two weeks later than normal, but by September the hydrograph reached historical averages. A pulse in September bumped the hydrograph about 3,000 cfs above normal for the remainder of the year (Figure 2).

Like the Yellowstone River, the Powder River discharge in 2019 near Locate, MT, was above the historic median throughout the year. There were primarily two peaks in discharge during 2019. The first peak occurred during the ice-out event on 24 March 2019, peaking at 18,200 cfs. The second peak occurred on 2 June 2019, peaking at 7,040 cfs. Discharge was higher than 1,000 cfs from 22 May 2019 through 21 July 2019, which was almost double the timeframe as the average year (Figure 3).

Hatchery-Origin Pallid Sturgeon

There were 96 radio-tagged HOPS relocated in the waters of the Yellowstone River during 2019 (Table 1). Of those 96, 18 were newly radio- tagged HOPS in 2019, including one individual upstream of Intake. Five individuals (codes 149.620-40, 149.620-98, 149.620-140, 149.620-143, and 149.620-158) began the year in the Yellowstone River between Intake (YSR RM 71) and Terry, MT (YSR RM 135), and code 149.760-190 was tagged in this reach in late September (Figure 4). While 20 Pallid Sturgeon encountered Intake during 2019, no natural upstream passages were documented. Ten HOPS (codes 149.620-66, 149.620-69, 149.620-112, 149.620-133, 149.620-147, 149.620-155, 149.620-156, 149.620160, 149.620-167, and 149.620-177) were translocated from the 2.3-mile reach below Intake to Stipek

Fishing Access Site (YSR RM 83) by the USBR. Four of the sixteen HOPS residing/translocated upstream Intake in 2019 ventured upriver to the Powder River confluence (YSR RM 147; codes 149.620-98, 149.620-112, 149.620-140, and 149.620-147). While 149.620- 140 was last relocated at the Powder River confluence in June, the remaining fish continued further upstream. Code 149.620-147 swam to YSR RM 157; codes 149.620-112 and 149.620-98 swam to YSR RMs 229 and 233, respectively (almost to Cartersville diversion dam, YSR RM 235). By the end of October, only five HOPS had moved downstream over Intake (codes 149.620-66, 149.620-69, 149.620-156, 149.620-160, and 149.620-167).

Wild-Origin Pallid Sturgeon

There were 32 radio-tagged WOPS relocated in the Yellowstone River during 2019 (Table 2), and only two fish swam up to Intake. Both these male fish (codes 149.760-11 and 149.760-131) were translocated upstream Intake (YSR RM 71) to Stipek Fishing Access Site (YSR RM 83) by the USBR in lateMay (Figure 5). Only code 149.760-11 was translocated in previous years (2017, 2018) of this study. Once released, these individuals continued their upstream migrations to and into the Powder River (YSR RM 147). Both fish were in and around the Powder River almost the entire month of June. Then their paths diverged and code 149.760-11 left the Powder River headed back down to the Missouri River and code 149.760- 131 made a large run up the Powder River. Code 149.760-11 swam to PDR RM 48 during 2019, while first-time translocated Pallid, code 149.760-131, swam over 81 river miles up the Powder River (the true extent was unable to be documented by FWP tracking crews on that day). Code 149.760131 finally left the Powder River in mid-July, but instead of following the path of his WOPS predecessors, he remained in the Yellowstone only a couple miles downstream of the mouth of the Powder River throughout the summer, fall, and into winter.

Blue Sucker

There were 35 radio-tagged Blue Sucker swimming in the waters of the Yellowstone River during 2019, and 33 of those individuals began the year in either in the Missouri River or the Yellowstone River below Intake (Table 3). No new Blue Sucker were tagged this year. All 31 Blue Suckers that encountered Intake swam upstream over the dam (100% passage). Intake upstream passages occurred from April through early-June in Yellowstone River discharges ranging 61 from 11,000 cfs to 60,000 cfs (Figure 6). Twenty-one fish swam as far upstream as the Powder River confluence (YSR RM 147), fourteen up to the Tongue River confluence (YSR RM 183), and seven beyond the Tongue River confluence up to RM 229 of the Yellowstone River. Only one Blue Sucker was tracked into the Tongue River (TNG RM 0.2). Blue Sucker began migrating down over Intake dam in August, though most activity occurred in October.

Paddlefish

Fifty-seven Paddlefish were relocated in the Yellowstone River in 2019 (Table 4); radio transmitters were implanted into eight new paddlefish this year. Thirty-three fish swam from Lake Sakakawea up to Intake, and 11 fish passed upstream (33.3% passage); six fish swam up over the dam and five fish passed via JISC during the subsequent 42-day timeframe when the construction access road crossing was breached in mid-May. Fish began passing Intake in late-May when Yellowstone River discharge reached about 30,000 cfs, though most passages occurred in early-June when discharge exceeded 50,000 cfs (Figure 7). Ten of the eleven fish that passed Intake swam up to the Powder River (YSR RM 147). While no Paddlefish were recorded in the Yellowstone River upstream of the Powder River confluence, six Paddlefish were found upstream in the Powder River as far as RM 15. All paddlefish swam back downstream over Intake dam by late-July, once discharge had dropped to about 20,000 cfs.

Shovelnose Sturgeon

There were 47 Shovelnose Sturgeon relocated in the Yellowstone River in 2019 (Table 5). Eight new Shovelnose Sturgeon were radio tagged this year. Eighteen fish encountered Intake during the year, but only three fish passed upstream (16.7% passage): two fish over the dam and one fish via JISC. The two fish swam over the dam in January and mid-August, when discharge was less than 11,000 cfs, whereas the JISC passage took place in late-May at a discharge around 30,000 cfs (Figure 8). Ten Shovelnose Sturgeon swam upstream to the Powder River confluence and three fish were relocated up in the Powder River as far as RM 97.5. Three fish swam in the Yellowstone River as far upstream as the Tongue River confluence, but no fish were found within the Tongue River during the year. However, two of those three fish continued swimming upstream past the Tongue River confluence and were found as far as YSR RM 224.

Sauger

None of the Sauger tags implanted from past years were still active by the beginning of this sampling year and no new Sauger were implanted with radio tags this year.

DISCUSSION Fish Passage at Intake

During 2019, no Pallid Sturgeon passed upstream Intake of their own free will. This was to be expected from the findings of the previous four-year study (Rugg et al., 2019), that Pallid Sturgeon almost exclusively passed via Joe’s Island Side Channel (90% via JISC). Consequently, we expect little to no natural passage of Pallid Sturgeon until the completion of the project and the bypass channel flows freely. As many Pallid Sturgeon (HOPS and WOPS combined) were translocated in 2019 as both 2017 and 2018 combined. Only two of those Pallid Sturgeon (HOPS code 149.620-66 and WOPS code 149.76011) had been translocated in prior years.

Blue Sucker passed upstream Intake with 100% passage, Paddlefish with 33.3% passage, and Shovelnose Sturgeon with 16.7% passage during 2019. Blue Sucker and Shovelnose Sturgeon had very similar passage rates to the results from the pre-construction study (98.3% and 19.5%, respectively). While Blue Sucker passed entirely over the dam, two of the three passing Shovelnose Sturgeon went through JISC during the brief period it was flowing. Paddlefish passed at a higher rate during 2019 than the average from the pre-construction study (33.3% vs 20.8%; Rugg et al., 2019). Roughly 50% of Paddlefish (5 of 11) passed through JISC when the opportunity presented itself. Now that JISC is permanently filled, the only natural passage will be over Intake diversion dam until the man-made bypass channel is completed, and passage rates for both Paddlefish and Shovelnose Sturgeon may decrease during the construction period. Blue Sucker may also see decreased passage with construction beginning on the dam itself Summer 2020.

Telemetry Upstream Intake

Blue Sucker, Paddlefish and Shovelnose Sturgeon behaved similarly in 2019 as years during the pre-construction study, though with two notable

63 distinctions involving the Powder River. First, while we have known for years that Paddlefish spawn in the Powder River (from fish larvae captured during benthic larval sampling in early-June and sightings of adults by fisherman on the river) this was the first year telemetered adults had been relocated in this important tributary of the Yellowstone River. Historically, eyewitness records indicate Paddlefish may migrate up the Powder River into Wyoming waters, almost as far as the Clear Creek confluence (PDR RM 242; Personal Comm., Mike Backes [FWP] and Gordon Edwards Jr. [WGFD], 28 November 2018). Since only 20% of Paddlefish are able to currently pass Intake on any given year, few individuals are assumed to make it this far. However, success of the Intake bypass project may make the Powder River a destination for many anglers during the Paddlefishing season as larger numbers of Paddlefish are able to migrate to the Powder River and congregate to spawn. Additionally, annual sightings in Wyoming waters (dependent on sufficient flows) may become a reality in the years to come, prompting the potential for Paddlefish regulations to be created in Wyoming.

Second, telemetered Shovelnose Sturgeon have been recorded in the Powder River in past years, though none have gone more than ten miles upriver. This year, however, two individuals traveled over 80 river miles up the Powder, with the furthest going almost 100 river miles. Though we had only telemetered this extensive migratory behavior up the Powder River in Pallid Sturgeon to this point, Shovelnose Sturgeon have been documented far up the Powder River for decades. Wyoming’s Game and Fish Department (WGFD) historically captured Montana-tagged Shovelnose Sturgeon over 295 river miles up the Powder River to the confluence of Crazy Woman Creek, Wyoming (Personal Comm., Gordon Edwards Jr., WGFD, 19 February 2020). The reverse has even been true; Shovelnose Sturgeon tagged in Wyoming have been recaptured by the FWP in the Yellowstone River near Rosebud, MT (Annear 1992). The furthest Shovelnose Sturgeon have been documented by WGFD up the Powder River thus far is 396 river miles, though anecdotal evidence has placed them up to the Schiffer diversion four miles downstream from Kaycee, WY, on the Middle Fork Powder River and another 50 miles upstream from current documentation (WGFD 2015). Shovelnose Sturgeon were only captured this far up the Powder River in June around peaks in discharge and most were ripe with milt or eggs. Summarily, the consensus is these fish migrate up the Powder River to spawn on years with adequate flow and reside the remainder of the year in the Yellowstone River.

Since Pallid Sturgeon exhibit similar seasonal migratory patterns to Shovelnose Sturgeon, Pallids may have historically traveled farther up the Powder River than what we have seen. Our lack of documentation may simply be a factor of the quantity of Pallid Sturgeon able to access the Powder River during the short

64 window of optimal flows, of which the completed Intake bypass channel and an increased population of upstream residents will likely improve. Six of the twelve translocated Pallid Sturgeon in 2019 remained upstream Intake going into winter, including one WOPS. These individuals doubled the current population of tagged residents upstream. Increased abundances of Pallid Sturgeon upstream Intake holds the promise of locating preferred habitats and finding aggregations of Pallid Sturgeon, particularly during the spawn window (May - July). With more Pallids residing upstream and HOPS hopefully beginning to mature in the next few years, the likelihood of witnessing a spawning event in the upper Yellowstone or Powder Rivers (a milestone in Pallid Sturgeon restoration) will also increase. Subsequently, efforts to understand the significance of the environmental conditions at these preferred locations and to protect these critical habitats will need to be made.

Moving Forward

Telemetry efforts upstream Intake will continue to be a high priority for the duration of this construction period and will continue into the post- construction phase of the study. A concerted effort is also underway during the interim to reimplant Pallid Sturgeon, particularly WOPS, with new radio tags in preparation for the second phase: post-construction evaluation. The reasons behind replacing radio tags in Pallid Sturgeon are two-fold. First, many radios are nearing or at the end of their battery life expectancy. Due to the rarity of these fish, failure to replace the radio tags before the current radios expire reduces the likelihood of recapture to almost nonexistent and valuable data is lost. Therefore, reimplantation of as many radio tags in Pallid Sturgeon (especially WOPS, who number around 100 individuals remaining in the whole system) as possible during these interim years is requisite to be able to evaluate the success of the completed bypass channel. Even with a functioning radio tag, recapture is not guaranteed and may take several days of effort from multiple crews to capture a single targeted fish. Second, we required four radio frequencies of tags during the pre- construction study phase to encompass all five species, including two specifically designated for Pallid Sturgeon. The time required to cycle through all the frequencies when tracking fish proved to be inefficient and costly as it resulted

in missed fish. This was especially true for station receivers which had the added step of cycling through each antenna before switching to the next frequency. To combat this issue, we plan on using a different code-set of radio tags, one that has 728 unique identification numbers (IDs) per frequency as opposed to the 212 unique IDs in the current code-set. This change will allow us to reduce the

number of frequencies down to two (one frequency for Pallid Sturgeon and the other for all other species), cutting cycling time in half. However, work during the code-set transition period will prove time consuming, not only in tag replacement, but also for tracking fish since our current receivers can only identify tags from frequencies in a single code-set at a given time. Telemetry efforts for Pallid Sturgeon will require a minimum of two receivers, one per code-set, with the potential need for a second crew to relocate the other native species with non- Pallid radio tag frequencies. Work the next few years will be tedious but should increase telemetry efficiency in the years that follow.

LITERATURE CITED Annear, T. 1992. Shovelnose Sturgeon Investigations; Summary Report 1983 to 1991. Project IF-3091-179001. Wyoming Game and Fish Department.

Nelson, M., and M. Jaeger. 2006. Historical distribution of pallid sturgeon in the Yellowstone River: an oral retrospective. Montana Fish, Wildlife and Parks. Glendive, Montana.

Rugg, M., J. Pesik and D. Trimpe. 2019. An Evaluation of Fish Passage at Intake Diversion Dam: Bypass Channel Pre-Construction. U.S. Bureau of Reclamation. Billings, Montana.

(WGFD) Wyoming Game and Fish Department. 2015. Annual fisheries progress report on the 2014 work schedule. Wyoming Game and Fish Department, Fish Division. Cheyenne, Wyoming.

TABLES Table 1. Summary of hatchery-origin Pallid Sturgeon (HOPS) upstream movements in the Yellowstone River in 2019. Totals represent the number of individuals who demonstrated a particular upstream movement.

Farthest Upstream Upstream Movements Total Extent (RM) Date Range Recorded in Yellowstone River 96 To Intake 20 Move Past Intake 10 Via Joe's Island Side Channel 0 N/A Over Dam 0 N/A Translocated 10 5/4/2019 - 5/30/2019 To Powder River 4 Into Powder River 0 N/A To Tongue River 2 Into Tongue River 0 N/A Move Past Tongue River 2 232.8

Table 2. Summary of wild-origin Pallid Sturgeon (WOPS) upstream movements in the Yellowstone River in 2019. Totals represent the number of individuals who demonstrated a particular upstream movement. (*) FWP crews traveled up to RM 81 on the Powder River and did not relocate this individual. Therefore, the true location of this fish was somewhere above RM 81 on that day.

Farthest Upstream Upstream Movements Total Extent (RM) Date Range Recorded in Yellowstone River 32 To Intake 2 Move Past Intake 2 Via Joe's Island Side Channel 0 N/A Over Dam 0 N/A Translocated 2 5/27/2019 - 5/31/2019 To Powder River 2 Into Powder River 2 >81* 6/3/2019 - 7/21/2019 Move Past Powder River 0 146.8

Table 3. Summary of Blue Sucker upstream movements in the Yellowstone River in 2019. Totals represent the number of individuals who demonstrated a particular upstream movement.

Farthest Upstream Upstream Movements Total Extent (RM) Date Range Recorded in Yellowstone River 35 To Intake 31 Move Past Intake 31 Via Joe's Island Side Channel 0 N/A Over Dam 31 4/5/2019 - 8/21/2019 To Powder River 21 Into Powder River 0 N/A To Tongue River 14 Into Tongue River 1 0.2 6/6/2019 - 6/20/2019 Move Past Tongue River 7 229.4

Table 4. Summary of Paddlefish upstream movements in the Yellowstone River in 2019. Totals represent the number of individuals who demonstrated a particular upstream movement.

Farthest Upstream Upstream Movements Total Extent (RM) Date Range Recorded in Yellowstone River 57 To Intake 33 Move Past Intake 11 Via Joe's Island Side Channel 5 6/4/2019 - 6/23/2019 Over Dam 6 5/28/2019 - 7/2/2019 To Powder River 10 Into Powder River 6 14.7 6/4/2019 - 6/17/2019 Move Past Powder River 0 146.8

Table 5. Summary of Shovelnose Sturgeon upstream movements in the Yellowstone River in 2019. Totals represent the number of individuals who demonstrated a particular upstream movement.

Farthest Upstream Upstream Movements Total Extent (RM) Date Range Recorded in Yellowstone River 47

To Intake 18

Move Past Intake 3

Via Joe's Island Side Channel 1 5/31/2019

Over Dam 2 1/1/2019 - 8/19/2019 To Powder River 10

Into Powder River 3 97.5 5/15/2019 - 7/19/2019 To Tongue River 3

Into Tongue River 0 N/A

Move Past Tongue River 2 223.8

FIGURES

Figure 1. Map of Yellowstone River and major tributaries with river mile markers. Dams and 2019 radiostation receivers are plotted.

Figure 2. Yellowstone River hydrograph of mean daily discharge in 2019 (red and blue lines) and historic median daily discharge (orange line) at Glendive, MT USGS stream-gauging station (Gauge No. 06327500; YSR RM 92).

Figure 3. Powder River hydrograph of mean daily discharge in 2019 (red and blue lines) and historic median daily discharge (orange line) at Locate, MT USGS stream-gauging station (Gauge No. 06326500; PDR RM 31).

Figure 4. Hatchery-origin Pallid Sturgeon (HOPS) movements in the Yellowstone River (YSR; teal and maroon lines) superimposed on the mean daily discharge of the YSR at Glendive, MT during 2019 (black line). Individuals originally tagged in the YSR downstream of Intake Diversion Dam (Intake) and in the Missouri River between Fort Peck Dam and Lake Sakakawea are categorized as “Intake” (I) fish (teal line). Individuals originally tagged upstream of Intake are categorized as “Glendive” (G) fish (maroon line). Various YSR landmarks are posted at their respective river miles.

Figure 5. Wild-origin Pallid Sturgeon (WOPS) movements in the Yellowstone (YSR; teal line) and Powder (PDR; blue line) rivers superimposed on the mean daily discharge of the YSR at Glendive, MT (black line) and the PDR at Locate, MT (gold line) during 2019. Individuals were originally tagged in the YSR downstream of Intake Diversion Dam and in the Missouri River below Fort Peck. Various YSR landmarks are posted at their respective river miles.

Figure 6. Blue Sucker movements in the Yellowstone (YSR; teal and maroon lines) and Tongue (TNG; red line) rivers superimposed on the mean daily discharge of the YSR at Glendive, MT (black line) and the TNG at Miles City, MT (purple line) during 2019. Individuals originally tagged in the YSR downstream of Intake Diversion Dam (Intake) and in the Missouri River between Fort Peck Dam and Lake Sakakawea are categorized as “Intake” (I) fish (teal line). Individuals originally tagged upstream of Intake are categorized as “Glendive” (G) fish (maroon and red lines). Various YSR landmarks are posted at their respective river miles.

Figure 7. Paddlefish movements in the Yellowstone (YSR; teal line) and Powder (PDR; blue line) rivers superimposed on the mean daily discharge of the YSR at Glendive, MT (black line) and the PDR at Locate, MT (gold line) during 2019. Individuals were originally tagged in the YSR downstream of Intake Diversion Dam and in the Missouri River between Fort Peck Dam and Lake Sakakawea. Various YSR landmarks are posted at their respective river miles.

Figure 8. Shovelnose Sturgeon movements in the Yellowstone (YSR; teal and maroon lines) and Powder (PDR; orange line) rivers superimposed on the mean daily discharge of the YSR at Glendive, MT (black line) and the PDR at Locate, MT (gold line) during 2019. Individuals originally tagged in the YSR downstream of Intake Diversion Dam (Intake) and in the Missouri River between Fort Peck Dam and Lake Sakakawea are categorized as “Intake” (I) fish (teal line). Individuals originally tagged upstream of Intake are categorized as “Glendive” (G) fish (maroon and orange lines). Various YSR landmarks are posted at their respective river miles.

2019 Pallid Sturgeon Population

Assessment Program Annual Report:

Segments 5 and 6

Prepared for the U.S. Army Corps of Engineers – Missouri River Recovery Program

By: Landon L. Pierce, Daniel A. James, and Dylan R. Turner

U.S. Fish and Wildlife Service Great Plains Fish and Wildlife Conservation Office

Pierre, SD December 2019

Executive Summary We sampled ten bends in Segments 5 and 6 of the Missouri River (i.e., from Fort Randall Dam, SD to the headwaters of Lewis and Clark Lake, NE-SD) for three consecutive days with trotlines. This sampling strategy deviated from the Scope of Work because we hypothesized that large amounts of debris from flooding on the Niobrara River would greatly reduce trammel net sampling efficiency in Segment 6 (i.e., downstream of the Niobrara River). We caught 90 pallid sturgeon and 120 shovelnose sturgeon. We recaptured three pallid sturgeon during the week of initial capture, but we did not recapture any shovelnose sturgeon. Relative abundance of pallid sturgeon during random sampling (i.e., day one) was low relative to previous years, whereas shovelnose sturgeon relative abundance in 2019 was high compared to previous years. High amount of detritus and shifting sand likely reduced sampling efficiency in Segment 6 by covering hooks (detritus) and burying trotlines (shifting sand). Body condition of stock-length (330-629 mm) pallid sturgeon was intermediate compared to previous years, while condition of quality- and preferred-length pallid sturgeon (630-839 mm and 840-1,039 mm, respectively) was high compared to previous years. Overall, the new sampling strategy was a reasonable workload and the non-random sampling on days two and three had higher pallid sturgeon catches compared to day one, but additional guidance on the preferred non-random sampling strategy may increase consistency among crews.

Background

As part of the Pallid Sturgeon Population Assessment Program (PSPAP), we monitor the status of pallid sturgeon in Segments 5 and 6 of the Missouri River (i.e., from Fort Randall Dam, SD to the headwaters of Lewis and Clark Lake, NE-SD). The original PSPAP (hereafter “PSPAP V.1) was designed to detect changes in pallid sturgeon and native target species populations in the Missouri River using a suite of sampling gears throughout the year (Welker et. al 2016). As part of the development of the U.S. Army Corps of Engineers (USACE) Science and Adaptive Management Plan (SAMP; Fischenich et al. 2018), the PSPAP was redesigned to provide the information needed to evaluate progress towards the fundamental objective and sub-objectives for pallid sturgeon (Colvin et al. 2017; Welker 2018). Monitoring in Segments 5 and 6 during 2019 under the redesigned PSPAP (hereafter PSPAP V.2) focused on providing the information necessary for evaluating sub-objective two (i.e., maintain or increase numbers of pallid sturgeon as an interim measure until sufficient and sustained natural recruitment occurs), while also maintaining continuity with PSPAP V.1 data and providing relevant demographic data for the pallid sturgeon population model (Colvin et al. 2018).

To meet the objectives of PSPAP V.2, a targeted sampling approach using gears expected to maximize pallid sturgeon catch was implemented in Segments 5 and 6 during spring of 2019. The sampling strategy outlined in the Scope of Work for 2019 required one day of random trammel net sampling to maintain continuity with legacy PSPAP V.1 data,

79 followed by one day of non-random trammel net sampling and two days of non-random trotline sampling. However, our sampling deviated from the Scope of Work because we expected that flooding on the Niobrara River would result in high amounts of debris in Segment 6 that would prevent effective sampling with trammel nets. In consultation with Dr. Tim Welker (USACE) and Dr. Mike Colvin (Mississippi State University), we collectively agreed to conduct one day of random trotline sampling followed by two days of non-random trotline sampling. This revised sampling strategy was expected to maximize sampling efficiency given post-flood river conditions, while also being consistent with the sampling strategy deployed in lower Missouri River Segments 8-14. In this report, we summarize the conditions and sampling results from 2019 sampling and discuss our experiences that may improve PSPAP V.2 sampling in the future.

Methods We sampled ten river bends with trotlines following methods described in Welker (2018) from April 22, 2019 to May 10, 2019. Two bends were sampled the first week (one crew) and four bends were sampled per week during weeks two and three using two crews (i.e., two bends per crew per week). Eight trotlines were deployed per bend each day for three consecutive days. Trotline locations on day one followed a stratified-random sampling design (i.e., randomly deployed within designated macrohabitats) to maintain continuity with PSPAP V.1, whereas locations on days two and three targeted locations expected to increase pallid sturgeon captures based on previous sampling efforts (i.e., within week and during previous years). Water depth and surface water temperature were recorded at all deployment locations. Velocity and turbidity measurements were recorded at all pallid sturgeon capture locations.

We processed pallid sturgeon according to Welker (2018), including measuring of forklength and weight, checking for and implanting passive integrated transponder (PIT) tags, documenting external hatchery marks, and collecting genetics samples from individuals of unknown origin (i.e., hatchery or wild). Blood was collected from six individuals for analysis of sex steroid levels at the U.S. Fish and Wildlife Service Bozeman Fish Technology Center. All shovelnose sturgeon were measured (fork-length), and individuals caught during days one and two were tagged with individually numbered floy tags. We enumerated the numbers of individuals caught per trotline for all non-sturgeon species.

Results

Sampling Conditions

Water conditions (i.e., gage height, discharge, and temperature) were generally similar among weeks over the course of the three-week 2019 sampling season, but gage height was high during sampling and varied considerably prior to sampling in response to flows from the

Niobrara River and other tributaries. Gage height (available at https://nwis.waterdata.usgs.gov/sd/nwis/uv?) measured at Greenwood, SD (Segment 5, the mostupstream gage available) ranged from 8.71 to 8.88 m (28.6 to 29.1 ft; flood stage = 9.14 m or 30.0 ft) during sampling (Figure 1). Gage height at Springfield, SD (Segment 6, mostdownstream gage available) was at or above flood stage [3.05-3.30 m (10.0-10.8 ft); flood stage = 3.05 m (10.0 ft)] throughout sampling (Figure 1). Fort Randall Dam discharge ranged from 1,308 to 1,359 m3/s (46,200-48,000 ft3/s) during week one, from 1,371 to 1,424 m3/s (48,40050,300 ft3/s) during week two, and from 1,359 to 1,422 m3/s (48,000-50,200 ft3/s) during week three. Mean surface water temperatures at gear deployments increased from 3.8 °C during week one, to 5.3 °C during week two, and to 6.6 °C during week three. Gage height varied considerably prior to sampling (March and April) due to the precipitation event that occurred during mid-March. Gage height at Greenwood, SD increased from 6.55 m (21.49 ft) on 3/12/2019 to 7.80 m (25.59 ft) on 3/14/2019 due to local precipitation (Figure 1), then decreased to 6.20 (20.35 ft) on 3/22/2019 due to reduced Fort Randall Dam releases to minimize downstream flooding due to high Niobrara River flows. Meanwhile, gage height at Springfield, SD increased from 2.52 m (8.27 ft) on 3/12/2019 to 5.40 m (17.72 ft) on 3/14/2019 due to local precipitation and Niobrara River flooding, then decreased to below flood stage [3.05 m (10.0 ft)] from 3/19/2019 to 4/5/2019 (Figure 1).

Pallid Sturgeon We caught 90 unique pallid sturgeon and recaptured three individuals (n=2 captures each). Forty pallid sturgeon were caught in Segment 5 and 50 were caught in Segment 6. Pallid sturgeon catch per trotline was 0.28 fish/20-hook night on day one, 0.48 fish/20-hook night on day 2, and 0.41 fish/20-hook night on day three (Table 1). Relative abundance of pallid sturgeon on day 1 (random sampling) was low relative to previous years (Figure 2). All pallid sturgeon were confirmed to be of hatchery origin (Table 2), and the 2008-2010 year class were the most represented year classes (n=15-19; Table 3). Pallid sturgeon ranged in length from 490 to 971 mm and ranged in weight from 340 to 3,685 g. Body condition of stock-length (330-629 mm; see Shuman et al. 2006) pallid sturgeon caught in 2019 [0.91

Shuman et al. (2011) Kn; 1.00 Randall et al. (2017) Kn] was similar to 2018 [0.89 Shuman et al. (2011) Kn; 0.96 Randall et al. (2017) Kn] and intermediate to previous sampling years (Figure 3). Condition of quality- and preferred length pallid sturgeon (630-839 mm and 840-

1,039 mm, respectively) caught in 2019 [0.95-0.96 Shuman et al. (2011) Kn; 1.06-1.12 Randall et al. (2017) Kn; Table 4] was high relative to previous years (Figure 3).

Shovelnose Sturgeon We caught 120 shovelnose sturgeon; 89 from Segment 5 and 31 from Segment 6. We tagged 74 individuals, including two individual that had lost floy tags from a previous year. Eighteen individuals were recaptured with floy tags from previous sampling efforts, but we did not recapture any individuals tagged in 2019 (Table 5). Shovelnose sturgeon relative abundance was 0.48 fish/20-hook night on day one, 0.58 fish/20-hook night on day two, and 0.44 fish/20hook night on day three. Relative abundance of shovelnose sturgeon on day 1 (random sampling) was high relative to previous years (Figure 4). Shovelnose sturgeon ranged in length from 544 to 770 mm.

Discussion High water levels and detritus (due to Niobrara River flooding) in Segment 6 likely affected sampling efficiency and relative abundance estimates in 2019. Numerous trotlines in Segment 6 likely had reduced effectiveness because hooks were covered in detritus and lines appeared to be buried in shifting sand. These issues may explain the low relative abundance of pallid sturgeon in 2019 compared to previous years, as catch of pallid sturgeon in Segment 6 during 2019 (0.25 fish per trotline) was lower than previous years (0.28-1.32 fish per trotline; unpublished data). Meanwhile, shovelnose sturgeon relative abundance in 2019 was high compared to previous years, but most (n=89; 74%) shovelnose sturgeon were caught in Segment 5 during 2019 where Niobrara River flooding likely did not affect sampling efficiency. This finding may suggest that sampling efficiency for shovelnose sturgeon or shovelnose sturgeon abundance was higher in Segment 5 than Segment 6. In addition to issues related to sampling efficiency, low catches of pallid sturgeon in Segment 6 may have been a result of changes in fish use of this reach during high water. For example, we hypothesize that the relatively uniform habitat conditions (i.e., depth and velocity) observed in Segment 6 Bend 9 may have resulted in lower sturgeon use and catch (i.e., two pallid sturgeon and zero shovelnose sturgeon) in this bend during 2019. Although the increased levels of detritus likely reduced trotline sampling efficiency, we suspect that trotlines were still more efficient than trammel nets in these conditions because the high detritus levels observed would likely prevent trammel nets from fishing effectively and would have substantially increased the amount of time required to clean and repair nets prior to redeployment.

In addition to the effects of the Niobrara River flood on trotline sampling efficiency, the flood also affected the time required to accomplish standardized sampling and perform gear maintenance. The flood prohibited reasonable access to all of the boat ramps in Nebraska that we would typically use to sample the randomly-selected bends for 2019 (i.e., Verdel Landing, Niobrara, and Santee). As a result, travel time (both automobile and boat) was higher in 2019 than under normal conditions. Finally, the high levels of detritus in Segment 6 resulted in increased gear repair because many trotline clips and hooks were damaged during sampling.

The workload of PSPAP V.2 sampling in Segments 5 and 6 (i.e., eight trotlines per bend, two bends per day) was reasonable, but additional subsamples may be feasible in future years. We completed most of our sampling in 8-10 hours per day and likely could have deployed more trotlines in this time under normal conditions (i.e., without extra travel time due to flooding). However, there is a balancing act that must be considered when determining the appropriate number of trotlines to deploy per day (for both the minimum number required and deployments done at crew leader discretion) because additional deployments on one day may limit the number of lines that can be deployed the following day. For example, if the crew leader increases the number of subsamples on day two due to low catches on day one and catch is substantially higher on day two, then the crew may not have sufficient time to redeploy trotlines for day three after processing large numbers of fish. Sampling with multiple crews provided several benefits and should be considered when personnel needs allow. Sampling with two crews allowed us to complete our standardized sampling in three weeks rather than the five weeks that would be required if one crew was used, and likely improved our ability to make comparisons among weeks because there was likely less temporal and temperature variability among weeks. Furthermore, this compressed sampling schedule is more consistent with our PSPAP V.1 trotline sampling and likely makes 2019 data more comparable to legacy data. Sampling with two crews per week also provided an opportunity for cross training among crews that will likely improve crew efficiency and pallid sturgeon catch (e.g., by learning what habitat features other crew leaders consider potential areas of high abundance) in the future. The primary negative aspect of sampling with two crews is that this approach requires additional personnel and, therefore, may not be consistently repeatable in the future or in other segments. For example, required telemetry sweeps prior to sampling in Segments 7-14 may limit opportunities to sample with multiple crews because personnel maybe occupied with telemetry obligations. Given the potential benefits identified of sampling with multiple crews, we recommend that this approach be considered in the future when possible.

The non-random sampling design of PSPAP V.2 increased catch of pallid sturgeon, but additional discussion and guidance on the appropriate strategy for selecting non-random sampling locations may be warranted. Our increased catch and relative abundance estimates for pallid sturgeon on days two and three relative to day one suggest that capture efficiency (or catchability) was higher during non-random sampling than during random sampling, assuming true abundance was constant throughout sampling. These increases in catch were likely, in part, a result of targeting habitats where trotlines fished effectively (i.e., not buried) and where pallid sturgeon were captured on previous sampling days. However, our sampling with two crews led to questions and discussions about “what is the most appropriate non-random sampling strategy?” We discussed two potential strategies (generalized below, though many strategies may exist) for determining non-random sampling locations based on results of previous sampling within the week: 1) re-deploying trotlines that caught pallid sturgeon near

83 the location sampled the previous day, and moving lines that did not catch pallid sturgeon or did not fish effectively to new areas in the general vicinity of the original deployment (e.g., upstream or downstream portion of the bend), and 2) re-deploying trotlines that caught pallid sturgeon near the location sampled the previous day and moving trotlines that did not catch fish to locations that caught pallid sturgeon during previous sampling (i.e., saturating local areas). We think that trade-offs exist between these two philosophies. Option one may provide less-biased population estimates of the bend because mark and recapture locations are distributed throughout the bend, whereas recapture locations in option two are limited to localized areas of high abundance. As a result, option one may represent bend-level population estimates and option two may represent habitatspecific population estimates. Meanwhile, option two will likely provide more pallid sturgeon captures for characterizing the population (e.g., condition, size and age structure, etc.). Ultimately, the best approach for non-random sampling will likely depend on prioritizing program objectives, and guidance for sampling crews should increase consistency among crews and improve the data collection and analyses of PSPAP V.2.

Although “Big Questions” were not developed in the SAMP for Segments 5 and 6 (Fischenich et al. 2018), data from this reach may help address hypotheses related to Big

Questions in the Upper and Lower Missouri River. First, most, if not all, pallid sturgeon in

Segments 5 and 6 are of hatchery origin, and may help address hypotheses related to Big Question 6 - Population Augmentation. Second, the data collected in Segments 5 and 6 may help inform and parameterize the pallid sturgeon population model. Finally, the relatively high body condition of pallid sturgeon during 2019 may provide information to improve our understanding of factors that affect pallid sturgeon condition (Lower River Big Question 7 – Fish Condition). For example, we observed that condition increased from 2018 to 2019 and hypothesize that high water levels in the summer and fall of 2018 may have increased foraging opportunities (e.g., increased prey availability). This hypothesis could be formally evaluated to improve our understanding of factors that affect condition. Acknowledgments We thank Jason Kral, Jennifer Johnson, Cole Moderegger, Kenton Asao, Paul Pierce, and Hannah Kast for assistance in the field. We thank the U.S. Army Corps of Engineers for funding.

References Colvin, M. E, S. Reynolds, R. B. Jacobson, L. L. Pierce, K. D. Steffensen, and T. L. Welker. 2018. Overview of Pallid Sturgeon Assessment Framework Redesign Process: U.S. Geological Survey Open-File Report. https://doi.org/10.3133/ofr20181166.

Fischenich, J. C., K. E. Buenau, J. L. Bonneau, C. A. Fleming, D. R. Marmorek, M. A. Nelitz, C. L. Murray, B. O. Ma, G. Long, and C. J. Schwarz. 2018. Science and Adaptive

Management Plan: Missouri River Recovery Program, U.S. Army Corps of Engineers ERDC.

Randall, M. T., M. E. Colvin K. D. Steffensen, T. L. Welker, L. L. Pierce, and R. B. Jacobson. 2017. Assessment of adult pallid sturgeon fish condition, Lower Missouri River- Application of new information to the Missouri River Recovery Program: U.S. Geological Survey Open-File Report 2017–1121, 103 p., https://doi.org/10.3133/ofr20171121.

Shuman, D. A., R. A. Klumb, R. H. Wilson, M. E. Jaeger, T. Haddix, W. M. Gardner, W. J. Doyle, P. T. Horner, M. Ruggles, K. D. Steffensen, S. Stukel, G. A. Wanner. 2011. Pallid sturgeon size structure, condition, and growth in the Missouri River Basin. Journal of Applied Ichthyology 27: 269-281.

Shuman, D. A., D. W. Willis, and S. C. Krentz. 2006. Application of a length-categorization system for pallid sturgeon (Scaphirhynchus albus). Journal of Freshwater Ecology 21:71-76.

Welker, T. L. 2018. Pallid Sturgeon Population Assessment Project Appendix A: Standard operating procedures defining the sampling strategy U.S. Army Corps of Engineers, Omaha District, Yankton, SD.

Welker, T. L., M. R. Drobish, and G. A. Williams (editors). 2016. Pallid Sturgeon Population Assessment Project, Guiding Document, Volume 1.8. U.S. Army Corps of Engineers, Omaha District, Yankton, SD.

Tables Table 1. Pallid sturgeon caught with trotlines in Segments 5 and 6 of the Missouri River during 2019, by sampling day. Daily total includes all fish captured that day (new and recaptures from previous sampling days).

Sample bend Sample Day 1 Day 2 Day 3 Segment Bend week Total Total Recaptures Total Recaptures 5 4 4/29/2019 2 3 0 1 0 5 5 4/29/2019 1 2 0 1 0 5 12 4/22/2019 3 1 0 5 0 5 13 4/22/2019 3 6 0 3 0 5 16 5/6/2019 5 4 1 1 0 6 3 5/6/2019 7 7 0 6 1 6 5 5/6/2019 0 3 0 3 0 6 9 5/6/2019 0 1 0 1 0 6 10 4/29/2019 1 4 0 4 0 6 11 4/29/2019 0 7 0 8 1

Total 22 38 1 33 2 Fish per trotline 0.28 0.48 0.41

Table 2. Pallid sturgeon caught with trotlines in Segments 5 and 6 of the Missouri River during 2019 by origin. Numbers represent unique individuals caught in 2019 (i.e., only one record per individual fish).

Origin Total Hatchery 90 Wild 0 Unknown 0 Total 90

Table 3. Pallid sturgeon caught with trotlines in Segments 5 and 6 of the Missouri River during 2019, by year class. Numbers represent unique individuals caught in 2019 (i.e., only one record per individual fish).

Year Class Total 1997 1 2001 5 2002 3 2003 1 2004 5 2005 2 2006 2 2007 5 2008 19 2009 15 2010 17 2012 4 2013 1 2014 3 2015 2 2016 4 2017 1

Grand total 90

Table 4. Mean body condition (Kn) of pallid sturgeon caught with trotlines in Segments 5 and 6 of the Missouri River during 2019, by length category. Values were calculated using the standard weight equations from Shuman et al. (2011) and Randall et al. (2017). Stock length = 330-629 mm; Quality length = 630-839 mm; Preferred length = 840-1,039 mm.

Mean Kn Length Shuman et al. Randall et Category 2011 al. 2017 Stock 0.91 1.00 Quality 0.95 1.06 Preferred 0.96 1.12

Table 5. Shovelnose sturgeon caught with trotlines in Segments 5 and 6 of the Missouri River during 2019, by sampling day. Daily total includes all fish captured that day (new and recaptures from previous sampling days).

Sample bend Sample Day 1 Day 2 Day 3 Segment Bend week Total Total Recaptures Total Recaptures 5 4 4/29/2019 9 2 0 5 0 5 5 4/29/2019 10 14 0 8 0 5 12 4/22/2019 5 9 0 4 0 5 13 4/22/2019 0 8 0 9 0 5 16 5/6/2019 4 2 0 0 0 6 3 5/6/2019 6 9 0 4 0 6 5 5/6/2019 3 2 0 3 0 6 9 5/6/2019 0 0 0 0 0 6 10 4/29/2019 1 0 0 1 0 6 11 4/29/2019 0 1 0 1 0

Total 38 47 0 35 0 Fish per trotline 0.48 0.59 0.44

Figures

Figure 1. Missouri River gage height at Greenwood, SD (Segment 5; top) and Springfield, SD (Segment 6; bottom) from March 12, 2019 to May 11, 2019. Sampling occurred April 22, 2019 – May 11, 2019. Data available from https://waterdata.usgs.gov/sd/nwis/uv?.

1.6

1.4

1.2

1.0

) 0.8 hook nighthook

- *

0.6 * fish/20 Pallid sturgeonPallid CPUE 0.4

0.2 (

0.0 2009 2010 2011 2012 2013 2014 2015 2016 2017 2018 2019 2020

Year

Figure 2. Relative abundance (CPUE) of pallid sturgeon caught with trotlines in Segments 5 and

6 of the Missouri River during Pallid Sturgeon Population Assessment Program sampling from 2010 to 2019. Asterisks indicate non-random sampling strategy. Error bars represent two standard error.

Figure 3. Body condition (Kn) of pallid sturgeon caught in Segments 5 and 6 of the Missouri River during Pallid Sturgeon Population Assessment Program sampling from 2003 to 2019.

Condition was calculated using the standard weight equations from Shuman et al. (2011) and Randall et al. (2017). Stock length = 330-629 mm; Quality length = 630-839 mm; Preferred length = 840-1,039 mm. Preferred-length fish (with weight recorded) were not caught prior to 2007.

1.0

* 0.8

* 0.6

) hook night - 0.4 fish/20

Shovelnose sturgeon CPUE sturgeon Shovelnose 0.2

(

0.0 2009 2010 2011 2012 2013 2014 2015 2016 2017 2018 2019 2020 Year

Figure 4. Relative abundance (CPUE) of shovelnose sturgeon caught with trotlines in Segments 5 and 6 of the Missouri River during Pallid Sturgeon Population Assessment Program sampling from 2010 to 2019. Asterisks indicate non-random sampling strategy. Error bars represent two standard error.

Pallid Sturgeon Free Embryo Drift, Dispersal, and Larval Settlement in the Upper Missouri River, 2019

Pat Braaten, U. S. Geological Survey, Columbia Environmental Research Center, Fort Peck Project Office, Fort Peck, Montana 59223. Email: [email protected]

Colt Holley, U. S. Geological Survey, Columbia Environmental Research Center, Fort Peck Project Office, Fort Peck, Montana 59223

Background

One of the leading hypotheses for lack of pallid sturgeon Scaphirhynchus albus recruitment in the Upper Missouri River basin integrates reproduction, early life stage behavior, and anthropogenic alterations. Specifically, the larval drift hypothesis (LDH) posits that “Under existing conditions, there is an insufficient length of free-flowing riverine habitat available between spawning (hatch) and settling locations in fragmented river reaches for pallid sturgeon free embryos to complete ontogenetic development, transition to benthic-oriented larvae, and survive.”

Early life stage processes are focal attributes of the LDH. Pallid sturgeon free embryos at hatch emerge from the spawning and incubation sites, and immediately initiate downstream dispersal in the river currents (Kynard and others, 2002; Kynard and others, 2007). All aspects of the dispersive life stage are not fully understood, but work to date suggests downstream dispersal will persist for several days until exogeneous energy reserves are exhausted or nearly so, and at that point, dispersing free embryos transition from drifting to settling on benthic habitats as larvae (Kynard and others, 2002; Kynard and others, 2007; Braaten and others, 2012b). Duration and distance of free embryo dispersal are dependent on temperature, rate of ontogenetic development, and river channel hydraulic conditions (for example, water velocity), and although variation occurs, free embryos may disperse hundreds of miles downstream from spawn and hatch locations prior to settling (Braaten and others, 2012a). Fragmentation in the Upper Missouri River system resulting from dam construction and associated reservoirs is identified as a major factor negatively affecting pallid sturgeon early life stages. For example, Lake Sakakawea on the Missouri River truncates the length of lotic habitat available to dispersing free embryos. It is hypothesized that premature termination of dispersal during ontogenetic development in combination with unsuitable environmental conditions in reservoirs (Guy and others, 2015) are factors contributing to mortality of naturally produced pallid sturgeon free embryos.

The LDH and long-distance dispersal attributes of free embryos are primary drivers guiding restoration planning in the Yellowstone River and adjacent Upper Missouri River. The precursor of the LDH “Under existing conditions” is highly applicable to both river systems. The Yellowstone River provides environmental conditions (for example discharge, temperature) supportive of pallid sturgeon reproduction. Spawning is known to occur almost annually as evidenced by spawning aggregations of males and females, and verifications of dispersing free embryos below spawning sites (DeLonay and others, 2014; DeLonay and others, 2016a; DeLonay and others, 2016b; DeLonay and others, 2016c; DeLonay and others, In Review-a; DeLonay and others, In Review-c, b). However, verified spawning occurs almost exclusively in the lower eight miles of the river. Free embryo dispersal distance between the lower Yellowstone River spawning locations and suitable riverine conditions towards the headwaters of Lake Sakakawea (about 60 miles) is likely insufficient to support ontogenetic development and survival of dispersing free embryos and settled larvae. Currently, Yellowstone River restoration programs are progressing to substantially increase free embryo dispersal distance. These programs aim to facilitate pallid sturgeon passage around an irrigation dam, and provide spawning access to upstream areas of the Yellowstone River (Braaten and others, 2015; Fischenich and others, 2018). Spawning and hatch in the upper Yellowstone River system could provide increased free-flowing habitat for ontogenetic development and dispersal of free embryos.

For the Missouri River, existing conditions are hypothesized as mostly unsuitable to support spawning by pallid sturgeon due to regulated flow releases through Fort Peck Dam and altered temperatures resulting from hypolimnetic releases. Spawning has been verified only in 2011 when elevated flows stimulated spawning in the river below Fort Peck Dam (DeLonay and others, 2014). Although thermally and hydrologically impaired under normal flow operations, the Missouri River from Fort Peck Dam downstream to suitable habitat in the headwaters of Lake Sakakawea provides about 225 miles of free-flowing river that could potentially support free embryo dispersal, ontogenetic development, and settlement of larvae if spawning occurred in the upper river reaches. Similar to the Yellowstone River, enhancement of the Missouri River downstream from Fort Peck Dam is planned to increase the likelihood of spawning, and provide recruitment in the Upper Missouri. However, different from Yellowstone River enhancement measures, enhancements for the Fort Peck reach focus on managing flow scenarios and temperatures to initially stimulate migrations into the upper reaches, promote spawning, then provide conditions supportive of free dispersal and larval settlement (Fischenich and others, 2018). Although the Missouri River provides an extended length of riverine habitat, resource managers are challenged by a complex question: Is there sufficient riverine distance at some combination of flow and temperature regimes to support complete ontogenetic development and survival of pallid sturgeon early life stages in the in the Missouri River between Fort Peck Dam and Lake Sakakawea?

The 2019 free embryo drift and dispersal study was designed and implemented to support multiple science and management needs for the Missouri River downstream from Fort Peck Dam, but applications also extend to other rivers and reaches in the Missouri River basin. The study expanded on earlier investigations, and included refinements based on previous work. In brief, the 2019 study assumed that implementation of managed flows and temperatures in upcoming years would promote spawning, hatch, and drift-entry near the mouth of the Milk River and outflow from the Fort Peck spillway near river mile (hereafter RM) 1760.0. A similar study conducted in 2016 (DeLonay and others, In Review-c) also followed this rationale, whereby 0- to 2-day post-hatch (dph) pallid sturgeon free embryos were released just downstream from the Milk River confluence. The 2016 study depicted initially moderate survival of free embryos during the first few hours after release, then low survival through the duration of the experiment. Reasons for low survival were not specifically identified, but potentially included cool water (for example, ~14.6 oC) at time-of-release as contributions of warm Milk River water were minimal, the spillway was not flowing, and Missouri River flows were sourced almost exclusively from hypolimnetic releases.

Similar conditions were anticipated for 2019 early in the season. Rather than risk losses of free embryos due to unknown factors in the absence of enhanced flow and temperature conditions, 1-dph and 5-dph pallid sturgeon free embryos were used in 2019 and released near Wolf Point near RM 1700.0. Water temperature near Wolf Point is typically about 16.0-18.0 oC during late June and early July. The 1-dph free embryos were used to approximate age and developmental state of 0-dph free embryos originally hatched at or near RM 1760.0, based on river velocities and about a 1-day travel time between the locations. The 5-dph free embryos were not used to simulate fish hatched between the Milk River and Wolf Point; rather, this group was used for comparative purposes (for example, compare advection and dispersion to 1-dph free embryos). In addition, both age groups were used to evaluate high-resolution particle tracking models that had been developed for a reach of the Missouri River downstream from Wolf Point (described below).

The goal of this project was to discern processes of pallid sturgeon drift and dispersal dynamics applicable to management and restoration potential in the Missouri River below Fort Peck Dam. Objectives were: 1) quantify drift and dispersal attributes of pallid sturgeon free embryos (for example, vertical position in the water column, lateral location in the river channel, drift rate, drift rate relative to velocity, dispersion) through a 150-mile reach of the Missouri River, 2) quantify pre-settlement dispersal distance of pallid sturgeon free embryos, 3) identify settling locations where pallid sturgeon transition from drifting free embryos to benthic larvae, 4) identify and quantify hydraulic elements driving free embryo dispersal and

larval settlement, and 5) test existing particle tracking and advection/dispersion models and develop more refined models.

Methods

Free Embryo Sources

Wild-caught pallid sturgeon and captive broodstock were used to produce free embryos of approximately 1- and 5-dph. The 1-dph free embryos were produced from wild pallid sturgeon spawned at Garrison Dam National Fish Hatchery (Garrison) and captive broodstock spawned at Gavins Point Dam National Fish Hatchery (Gavins; Braaten_Table 1). Spawning for 1-dph production at both locations occurred on June 25 to synchronize hatch timing as best as possible. The 5-dph free embryos were produced from captive broodstock spawned at Gavins on June 20. Embryos from captive broodstock spawning events were transferred to Garrison where they were incubated through hatching and initially reared. Embryos as sources for the 1-dph age class started to hatch at about 10:00 a.m. on June 30, and continued hatching through early morning of July 1 when packaged for shipping to the study site. Embryos as sources for the 5-dph age class started to hatch at about 2:30 a.m. on June 25, and hatch was completed by about 11:00 a.m. on June 27. Free embryos for the 5-dph age class were reared at temperatures of 17.7-19.0 oC prior to the July 1 packaging and shipment date. Collectively, an estimated 771,707 1-dph and 200,786 5-dph free embryos were packaged on the morning of July 1 for shipment to the Missouri River release site (Braaten_Table 1). Prior to release, age-1 dph free embryos were exposed to about 18.6 cumulative thermal units (CTU) and 5-dph free embryos were exposed to about 90.0-108.0 CTU (some variation to account for temporal variation in hatch).

Free embryos from Garrison that were packaged in plastic bags and coolers arrived at the Missouri River release site near Wolf Point, Montana on July 1 at 1:00 p.m. After transferring coolers, boats with coolers and fish anchored at different points laterally across the river channel where the release location was defined as RM 1701.3 (48.06747, - 105.53221). Water in the packaged free embryos (19.8 oC) was much warmer than river water temperature (14.2-14.3 oC) owing to a cold front that had affected the area. As such, free embryos were acclimated to river temperatures for about one hour in a process that involved release boats repeatedly dumping river water over the bagged free embryos in coolers, emptying the cooler water, then adding new river water. After acclimation, free embryos were released at the river surface from about 2:002:02 p.m.

Main Channel Sampling for Dispersing Free Embryos

Main channel sampling for dispersing free embryos was conducted during July 1-8 at six sites, beginning at RM 1691.6 and extending downstream to RM 1551.0 (Braaten_Table 2). Two sampling protocols were implemented, where one sampling protocol involved sites 1 and 2 on the July 1 release date and the second protocol involved sites 2-6 on subsequent dates.

For the first protocol, sampling at site 1 on July 1 (9.7 RM downstream from the release location) was conducted by three boats positioned in different portions of the river channel. Sampling at site 2 (21.6 miles downstream) during late evening of July 1 through 7:16 a.m. on July 2 was similarly conducted by three boats positioned across the river channel. Boats at each site were equipped with two rigged (and spares) rectangular frame nets (frame: 0.5-m high, 0.75m wide; net: 3.0-m long, 1,000-µm net mesh, cod-end collecting cup). One net was deployed to the river bed to sample for fish in the lower 0.5-m of the water column (hereafter bottom net). The other net was deployed to sample in the middle of the water column (hereafter midwater net). Nets were fitted with a General Oceanics Model 2030R flow meter from which water velocity and volume of water filtered could be estimated. Downrigger weights or a sounding weight affixed to the net frame maintained net position at the specified sampling location. Nets were simultaneously deployed, and fished for five minutes. Upon retrieval, contents of the collecting cups were emptied into black rubber trays. The trays provided a black background contrast to aid seeing the grayish-white free embryos. Tray contents were sorted, and Acipenseriformes free embryos were extracted and placed in numerically coded individual vials containing 95% non-denatured ethanol. If a sample contained numerous free embryos, the entire sample (organic matter, free embryos) was transferred to Whirl-Pak™ bags and preserved in a 10% formalin solution for later processing in the laboratory. All bags were labeled according to boat number, sampling time, and net position consistent with data sheets. After free embryos were picked from the sample or the sample was preserved, nets were quickly deployed to initiate another sample. Sampling at site 1 was initiated in advance of predicted time-of-first arrival (TOFA). The TOFA from site 1 was used to estimate dispersal rate, and predict travel time and TOFA to site 2. In addition to TOFA, catches from site 1 provided initial estimates for time-ofmass-arrival (TOMA) and potentially time-of-last-arrival (TOLA) for the dispersing population that could be used to estimate travel times to other sites. Travel time of the dispersing free embryos was based on hours post-release (hereafter HPR) starting from release at 2:00 p.m. on July 1.

The second protocol was implemented for sites 2-6 beginning late morning July 2 and extending to July 8 (Braaten_Table 2). Under this protocol, 1-2 boats rigged with paired rectangle nets (as described above) were used to conduct 10-minute samples for dispersing 97

free embryos. One bottom net was deployed during every sample, while the second net alternated between bottom sampling, midwater sampling, or sampling at the surface. The midwater or surface deployments were implemented when sample times occurred near the top of the hour as described in the following scenario. For example, a sample implemented around 11:30 a.m. would include two bottom net samples fished simultaneously. The next sample deployed about 12:00 p.m. would include a bottom net and a midwater net fished simultaneously. At 12:30 p.m., both nets would be simultaneously deployed to the bottom. The sampling regime around 1:00 p.m. would include a bottom net and a surface net fished simultaneously. Upon completion of sampling, sample contents were removed from the cod- end collecting cups and transferred to the black rubber trays. Acipenseriformes free embryos were extracted from the detritus, then individually preserved in numbered vials containing 95% non-denatured ethanol.

Free Embryo Entrainment in Low-velocity Habitats

A 13.5-mile reach of the Missouri River from RM 1692.5 to RM 1679.0 was a focal area for assessing free embryo entrainment and potentially retention in low-velocity habitats (Braaten_Figure 1). This reach was the focus of intensive hydrology work in 2018 that resulted in development high-resolution particle simulations of free embryo transport and dispersal. Prior to the free embryo release, particle transport through the reach (hereafter referred to as the Simulation Reach) was simulated at a discharge of 10,230 ft3/s – a discharge anticipated during the 2019 dispersal study. The simulation identified 11 habitat complexes of low-velocity or backwater conditions where particles (and potentially free embryos) could be entrained and retained.

Reconnaissance of the 11 low-velocity habitat complexes throughout the Simulation Reach was conducted a few days prior to the free embryo release, and two methodologies were identified to sample the habitats. First, to assess entry from the main channel flow field into the habitat complex, 0.5-m diameter plankton nets (750-µm mesh, 1.5-m long) were secured to the river bed using fence posts at inlet points to the habitat. These nets (hereafter inlet nets) were set on July 1, and retrieved on July 2. Water velocity at the net-mouth was recorded when the nets were deployed and retrieved using a Marsh McBirney flow meter. An estimate of water volume sampled with inlet nets was obtained based on measured water velocity, net-mouth area, and duration of deployment. Seining was the second method used, and this method assessed potential free embryo retention in the habitats. Owing to the small size of free embryos, fabrication of a small-mesh seine was necessary. The seine was comprised of two nets sewn together in length. The anterior net (0.32-cm mesh) was 1.2-m wide by 1.2-m high at the opening, and tapered down to 0.5-m diameter through a length of 1.8-m. The posterior net (same specifications as plankton net above) was attached to the 98

posterior end of the anterior net. The seine was pulled through the low-velocity or no-velocity areas of the habitat when depths were suitable for seining. Seine area was estimated based on seine width and length of area seined. Acipenseriformes free embryos collected in inlet nets and from seining were placed in numerically coded individual vials containing 95% non- denatured ethanol.

Transition from Dispersing Free Embryos to Settled Benthic Larvae

Sampling for pallid sturgeon that had potentially transitioned from dispersing free embryos to settled benthic larvae was conducted on nine weekly sampling events from July 16 (about 1-week after dispersal netting in the mainstem was completed) to September 11. On the initial sampling event during July 16-19, 18 Missouri River locations were sampled in a 60- mile reach extending from RM 1599.3 (flowing river upstream from the Yellowstone River confluence) to RM 1538.0 (low to no flow area in Lake Sakakawea). This initial sampling event was targeted to assess the spatial distribution of initial larval settlement. The eight subsequent weekly sampling events targeted four Missouri River locations between RM 1583.6-1591.8 upstream from the Yellowstone River confluence (hereafter UTC), and four locations between RM 1552.1-1581.2 downstream from the Yellowstone River confluence (hereafter DTC).

Sampling for settled larvae was conducted with a benthic beam trawl (2.0-m wide, 0.5- m height, 5.5-m length, 3.81-cm outer chafe net, 0.32-cm inner fish retaining net). The trawl was deployed in three river-bend habitats at each location including the inside bend, outside bend, and channel crossover (Braaten and Fuller, 2007). Trawls were deployed for a target of 4.0 minutes. Trawl distance (m) was also measured to facilitate estimating area of the river bed sampled (m2). All larval Scaphirhynchus spp. captured were measured to the nearest 1.0 mm total length (TL, excluding caudal filament). A fin clip or the whole fish (if too small to obtain a fin clip) was placed in numerically coded individual vials containing 95% non- denatured ethanol.

Analysis

Several aspects of the 2019 drift and dispersal project (for example, comparing densities of 1- and 5-dph free embryos among bottom, midwater and surface sampling locations, comparing dispersal rate between age groups, comparing dispersal rates to hydraulic conditions, quantifying free embryo dispersion, quantifying pre-settlement dispersal distance, identifying settling locations for larvae) are dependent on identification of recaptured fish as the original 1- or 5-dph released free embryos. Free embryos captured as part of the mainstem dispersal sampling are in the process of genetic testing using single nucleotide polymorphism (SNP) markers to differentiate specimens as pallid sturgeon, shovelnose sturgeon 99

Scaphirhynchus platorynchus or paddlefish Polyodon spathula, and to further identify pallid sturgeon as the original 1-dph and 5-dph released free embryos (Eichelberger and others, 2014). Lacking genetic verifications at time of report preparation, only general information on drift and dispersal are reported. These trends are based on initial laboratory identifications. For example, the older 5dph age group at time of release were larger and more developed than the 1-dph fish, and exhibited concentrated melanophore pigmentation towards the tail characteristic of their advanced developmental state. In most cases, the 5-dph free embryos could be discerned from other free embryos. Comparatively, the 1-dph free embryos were smaller and lacked the heavily pigmented tail. Although distinct from the 5-dph free embryos, the 1-dph free embryos could not always be unequivocally identified as pallid sturgeon due to the likelihood that similar-sized, naturally produced paddlefish and shovelnose sturgeon free embryos were also present in the mainstem fish collections. The mass-capture of free embryos at downstream sites did verify the pulse of dispersing free embryos consistent with the mass- release (see Progress below), but also included other unknown Acipenseriformes. Thus, results are presented in terms of presumed 1dph free embryos, presumed 5-dph free embryos, and unknown free embryos.

Definitive information on free embryo advection and dispersion (incorporating estimates of TOFA, TOMA, TOLA) is not yet available as genetic results are needed to identify species and age class of pallid sturgeon through the entire sampling times of the catch curves. The TOFA and TOLA (especially necessary for quantifying dispersion) could not be unequivocally determined as uncertainty in species identification and time-of-arrival exists in the tails of the catch curve distributions where a single unidentified pallid sturgeon free embryo may occur with one (or more) naturally produced shovelnose sturgeon free embryos. Unlike TOFA and TOLA, the TOMA arrival is less affected by small numbers of non-pallid sturgeon free embryos as the detected mass of free embryos is mostly driven by the original mass of released free embryos. A such, the TOMA was used to discern initial trends in advection through the study reach. For this analysis, the HPR that corresponded to 1- and 5- dph TOMA was quantified from the catch curves at all sites. Two or more peaks of mass arrival occurred in some cases, and HPR was averaged across TOMA. The HPR was converted to dispersal velocity in miles per hour based on downstream distance of the sites (Braaten_Table 2). Linear regression was used to test for relationships between free embryo dispersal velocity (response variable) and downstream distance (predictor variable).

Total length (nearest 0.1 mm total length) was measured on all free embryos preserved in ethanol prior to shipping for genetic analysis. Length measurements are in-progress for specimens preserved in formalin, mainly corresponding to hundreds of specimens collected at site 1 shortly after release. Lacking genetic verification of species and pallid sturgeon age group, lengths are preliminary and used to describe only general trends for the dispersing 100

population of free embryos. Length-frequency plots were constructed, and visually inspected for apparent length gaps between the 1- and 5-dph free embryos. Lengths for both age groups were summarized by median length, the third quartile of the length distribution, and maximum length. Although the median, and to a lesser extent the third quartile, would be influenced by additions of small naturally produced free embryos (for example, shovelnose sturgeon), maximum length of the dispersing population of released free embryos would not be influenced by natural inputs except potentially at sites downstream from the Yellowstone River.

In contrast to dispersal collections for the main channel, genetic analyses are completed for settled Scaphirhynchus spp. sampled with the benthic beam trawl during mid- July through mid-September. Fin clips were initially screened using SNP markers, and fish identified as potential pallid sturgeon were further genetically analyzed to identify species and test if the larvae were sourced from the original 1- or 5-dph free embryos (Eichelberger and others, 2014).

Progress

Main Channel Sampling for Dispersing Free Embryos

Discharge in the Missouri River changed markedly prior to and during implementation of the study. During June 20-26, discharge (measured at USGS gage 06177000 near Wolf Point, Montana) varied slightly between 9,120-9,750 ft3/s. Discharge increased after June 26 due to rain events and increased flows from the Milk River, and on the July 1 release day, discharge at the Wolf Point release site was 12,300 ft3/s. Discharge continued to increase and remained elevated (12,500-13,900 ft3/s) during the study.

Sampling at site 1 (RM 1691.6) on the July 1 release date was conducted during 2.757.06 HPR (Braaten_Table 2). The sampling regime yielded 921 presumed 1-dph free embryos, 262 presumed 5-dph free embryos, and 59 unknown free embryos (Braaten_Figure 2). The TOFA for 1- and 5-dph free embryos occurred simultaneously at 4.65 HPR. For 1-dph free embryos, the TOMA spanned 4.90 HPR to 5.15 HPR. The TOMA for 5-dph free embryos was 5.47 HPR to 5.75 HPR. The full range of dispersion including the slowest drifting tail of the distribution and TOLA was not quantified at this site as crews departed before nightfall. Through termination of sampling, free embryos were collected at 6.77 HPR (1-dph) and 7.03 HPR (5dph). The dispersing population of free embryos that included presumed 1-dph fish had 101

a median length of 9.0 mm, third quartile of 9.4 mm, and maximum length of 10.6 mm. The presumed 5dph fish had a median length of 14.3 mm, third quartile of 14.8 mm, and maximum length of 15.5 mm (Braaten_Figure 3).

Site 2 (RM 1679.7) was located at the downstream terminus of the Simulation Reach, thus it was critical to quantify advection and the full range of dispersion for comparisons to model predictions. Sampling initially occurred from 7.50 HPR (just after sunset on July 1) through 17.27 HPR (7:16 a.m. on the morning of July 2). Sampling then persisted through 39.75 HPR (5:45 a.m. on July 3) to quantify the potential for extended dispersion (Braaten_Table 2; Braaten_Figure 4). The catch at this site totaled 926 free embryos that were tentatively identified as presumed 1-dph (629 fish), presumed 5-dph (240 fish), and unknown (57 fish). The TOFA was 9.60 HPR for 1-dph free embryos, and 10.60 HPR for 5-dph free embryos. The TOMA arrival for 1-dph free embryos spanned 11.30-11.92 HPR during the early morning of July 2. The 5-dph free embryos exhibited mass arrival at 11.92-12.15 HPR. Final captures occurred at 31.30 HPR for presumed 1-dph free embryos (2 fish), and 16.10 HPR for a presumed 5-dph fish. The dispersing population of free embryos that included presumed 1-dph fish had a median length of 9.5 mm, third quartile of 9.9 mm, and maximum length of 10.8 mm. The presumed 5-dph fish had a median length of 14.5 mm, third quartile of 15.0 mm, and maximum length of 16.0 mm (Braaten_Figure 3).

Mainstem sampling for dispersing free embryos at site 3 (RM 1620.0) near Culbertson, Montana, extended from 24.0 HPR on July 2 through 85.77 HPR on July 5 (Braaten_Table 2; Braaten_Figure 5). The catch included 262 presumed 1-dph free embryos, 254 presumed 5- dph free embryos, and 79 unknown free embryos. The initial free embryo collected at 29.28 HPR was tentatively identified as a 1-dph pallid sturgeon, but this fish occurred well in advance of other suspected 1-dph fish. The TOFA for the 5-dph pallid sturgeon occurred at 43.80 HPR. The TOMA mass for presumed 1-dph pallid sturgeon occurred during 54.13- 54.87 HPR. Mass arrival for presumed 5-dph free embryos also occurred at 54.13-54.87 HPR. Extended sampling at site 3 suggested the possibility of 1-dph free embryos occurring at 67.19 HPR, and 5-dph free embryos occurring through 66.13 HPR. The dispersing population of free embryos that included presumed 1-dph fish had a median length of 10.9 mm, third quartile of 11.5 mm, and maximum length of 12.8 mm. The presumed 5-dph fish had a median length of 16.4 mm, third quartile of 16.8 mm, and maximum length of 17.7 mm (Braaten_Figure 3).

Site 4 (RM 1587.0) was the most downstream sampling station prior to receiving potential free embryo inputs from the Yellowstone River. Sampling at site 4 was conducted from 42.72 HPR on July 3 to 125.82 HPR on July 6 (Braaten_Table 2; Braaten_Figure 6). A total of 240 free embryos was collected, partitioned among 109 presumed 1-dph free embryos, 102

92 presumed 5-dph free embryos, and 39 unknown free embryos. Initial sampling on July 3 during 42.72-53.65 HPR yielded a few free embryos, but none were tentatively identified the 1- or 5dph released pallid sturgeon. Initial samples on July 4 were suggestive of 1-dph (66.70 HPR) and 5-dph free embryos (66.75 HPR), thus indicating that the released free embryos arrived at site 4 sometime between 53.65 HPR and 66.70 HPR. The TOMA occurred at 72.42 HPR for presumed 1-dph free embryos and 80.73 HPR for presumed 5-dph free embryos. The last capture of a suspected 1-dph free embryo occurred at 96.40 HPR; whereas, a presumed 5- dph free embryo was captured 125.13 HPR just prior to termination of sampling (125.82 HPR). The dispersing population of free embryos that included presumed 1-dph fish had a median length of 11.0 mm, third quartile of 12.0 mm, and maximum length of 13.0 mm. The presumed 5-dph fish had a median length of 16.7 mm, third quartile of 17.3 mm, and maximum length of 19.4 mm (Braaten_Figure 3).

Site 5 (RM 1577.0), located about five miles downstream from the Yellowstone River confluence, was not included as a sampling site in the original sampling plan. However, logistical constraints necessitated establishment of this site. Specifically, sampling was planned to occur at site 4 (discussed above) and at a site near Williston, North Dakota (~RM 1551.0). Elevated water levels in Lake Sakakawea flooded all boat ramps in the Williston area, thus no access was available at the planned lower sampling site. Although access was available at the boat ramp adjacent to the Yellowstone River confluence, use of the confluence ramp to access the Williston sampling site required about a 1-hr boat trip each way to navigate the 30-mile reach of river. Rather than lessening sampling time by boating two hours each day, site 5 was established as a sampling location that also complemented limited sampling that occurred near Williston (see site 6 below).

Sampling at site 5 was conducted on multiple occasions between 68.15 HPR and 168.20 HPR (Braaten_Table 2; Braaten_Figure 7). Located downstream from the Yellowstone River, the catch at this site (239 total free embryos) potentially included progeny from natural spawning events in the Yellowstone River as well as naturally spawned and released free embryos from the Missouri River. The majority of free embryos collected at this site were identified as unknown (58%, 138 individuals); whereas, 70 free embryos were classified as presumed 1-dph pallid sturgeon and 31 classified as presumed 5-dph pallid sturgeon. Catches of presumed 1-dph and 5dph free embryos occurred early in the sampling regime at this site (68.15 and 69.99 HPR, respectively). The TOMA for presumed 1-dph free embryos occurred during 74.76-77.20 HPR, slightly offset from site 4 but expected based on proximity of the sites and travel time. Thus, sampling at site 5 likely detected the mass of 1-dph fish that had been detected at site 4. The greatest number of presumed 5-dph free embryos at site 5 occurred at 94.57 HPR (4 fish). Concluding samples at site 5 were characterized by catches of mostly unknown free embryos. The dispersing population of free embryos that included presumed 1-

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dph fish had a median length of 10.9 mm (less than site 4 due to additions of small free embryos from Yellowstone River inputs), third quartile of 11.7 mm (less than site 4), and maximum length of 12.9 mm (similar to site 4). The presumed 5-dph fish had a median length of 17.2 mm, third quartile of 17.8 mm, and maximum length of 18.3 mm (Braaten_Figure 3).

During July 6-8, one boat navigated to site 6 (RM 1551.0) near Williston while the other boats maintained sampling at site 5. Three sets of samples were conducted at site 6 spanning 116.20-167.82 HPR (Braaten_Table 2; Braaten_Figure 8). A total of 115 free embryos was collected, partitioned between three presumed 5-dph pallid sturgeon and 112 unknown free embryos. The presumed 5-dph free embryos were sampled consecutively at 120.33, 121.38, and 122.50 HPR, and these fish measured 16.4, 17.8, and 18.3 mm (Braaten_Figure 3). The unknown free embryos exhibited a median length of 8.8 mm, third quartile length of 10.0 mm, and maximum length of 13.6 mm. Although catches suggest a lack of age-1dph free embryos at site 6, dispersal to and beyond the site may have occurred, but were entirely or mostly undetected. Specifically, sampling at this site could not be initiated until about 116.0 HPR due to access issues. Presumed 1-dph free embryos were detected at site 5 at about 68.0-76.0 HPR, and it is possible that the leading edge, mass, and majority of the trailing edge of the dispersing population of 1-dph free embryos moved past site 6 before sampling was initiated – about 48 hours after this group was detected at site 5. One unknown free embryo (13.6 mm, essentially isolated from all other lengths in the distribution; Braaten_Figure 3) may be a 1-dph pallid sturgeon representative of the trailing edge of the population.

The TOMA varied between presumed 1- and 5-dph free embryos from site 1 through site 5. The mean TOMA estimates for presumed 1-dph free embryos were 5.0, 11.7, 54.43, 72.42 and 75.99 HPR for sites 1-5, respectively. For presumed 5-dph free embryos, mean TOMA estimates were 5.62, 12.04, 54.43, 80.73, and 94.57 HPR for sites 1-5, respectively. There were significant inverse relationships between mass dispersal velocity and dispersal distance for 1-dph free embryos [r2=0.82, P=0.035, N=5; mass dispersal velocity in miles per hour=1.93-0.0037(distance in miles)] and for presumed 5- dph free embryos [r2=0.96, P=0.004, N=5; mass dispersal velocity in miles per hour=1.83-0.0044(distance in miles)]. Thus, for both age groups, dispersal velocity for the main mass of fish decreased with increasing dispersal distance.

Free Embryo Entrainment in Low-velocity Habitats of the Simulation Reach

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The 11 habitat patches of low-velocity habitat or backwater conditions identified by particle simulation models in the Simulation Reach (Braaten_Figure 1) changed markedly between reconnaissance surveys and implementation of the experiment on July 1. Specifically, the progressively increasing discharge in the Missouri River changed some habitats from lowvelocity backwaters to complete flow-through habitats connected to the main channel. As a consequence, eight of the original 11 low-velocity habitat complexes were sampled (Braaten_Table 3). Three habitat complexes (1, 7, 9) had complementary inlet sampling and seining, and five complexes (2, 3, 6, 10, 11) were suitable only for inlet net sampling.

A combined total of 22 free embryos was sampled from all habitat complexes, and all specimens resulted from inlet net collections (Braaten_Table 3). Inlet nets collected 12 free embryos at complex 1, nine free embryos at complex 3, and one free embryo at complex 9. Four free embryos were likely 5-dph fish based on heavy pigmentation and large sizes (11.9, 12.6, 13.2, 14.9 mm); whereas, the remaining 18 free embryos were smaller (7.2-10.8 mm) likely representative of the 1-dph released fish.

Transition from Dispersing Free Embryos to Settled Benthic Larvae

The initial week of trawling during July 16-19 extended from the completely flowing portion of the Missouri River at RM 1599.3 downstream to the slow-flowing portion of Lake Sakakawea at RM 1538.0 (Braaten_Figure 9). The spatially expansive trawling regime yielded captures of 38 settled Scaphirhynchus sp., where capture locations extended from RM 1588.9 downstream to the lowermost sampling location at RM 1538.0. Genetic analyses of the 38 larvae identified 15 pallid sturgeon, and all pallid sturgeon larvae were captured in the Missouri River UTC spanning RM 1584.1-1587.8 (Braaten_Table 4). Additional genetic analysis identified the 15 larvae as survivors of free embryos originally released as 5-dph. For this initial week of trawling, the settled larvae averaged 29 mm (25-33 mm; Braaten_Table 4).

During the eight subsequent weekly trawling events from July 22 to September 11, 68 additional settled larvae captured from trawling sites were genetically confirmed as pallid sturgeon (Braaten_Figure 10, Braaten_Table 4). Genetic analyses identified all 68 pallid sturgeon larvae as representative of free embryos originally released as 5-dph. The majority of settled larvae during this sampling time frame (55 of 68 larvae, 81%) were captured in the Missouri River UTC. Growth of the larval pallid sturgeon progressed through summer as individuals were 105-110 mm during late August and September.

The 83 genetically confirmed pallid sturgeon larvae captured by the USGS and MFWP crews were complemented by an additional capture by the USFWS (R. Wilson, USFWS, Bismarck, North Dakota). While performing otter trawl sampling on September 9, 2019, the 105

USFWS crew captured a 112-mm larvae near Williston, North Dakota (RM 1551.7) that was genetically confirmed as sourced from the 5-dph cohort of released free embryos. Collectively, sampling by all crews in 2019 yielded 84 settled larval pallid sturgeon from the 5-dph cohort of released free embryos. No settled larvae representative of the 1-dph cohort of released free embryos have been sampled to date.

Discussion

Implementation of the 2019 free embryo drift and dispersal experiment was successful on multiple fronts including production of nearly one million free embryos from wild and captive broodstock, accident-free practices for fish and hydrology crews over long periods of deployment, and substantial data acquisition on free embryo dispersal and river channel hydraulics. Whereas multiple successes were evident, one success critical to science gains centered on free embryo survival, especially for the 1-dph fish. Work in 2016 utilized free embryos varying from 0- to 2-dph released in cool water (14.6 oC), and despite acclimation of the 2016 free embryos, minimal survival was documented through the initial 24 hours of the study. In planning for the 2019, it was anticipated that water temperature would be warmer at the Wolf Point release site to provide thermal conditions more suitable for survival. However, the cold front in the days prior to the 2019 release date suppressed temperature to levels cooler than in 2016. Despite this unforeseen situation, the 1- and 5-dph free embryos acclimated to ambient river temperatures exhibited not only short-term survival, but also long-term survival under cooler than normal water temperatures.

Lacking genetic results for the dispersing free embryos, multiple aspects of the 2019 experiment cannot be definitively quantified at this time. However, preliminary results provide initial information on dispersal from several perspectives. Catch curves for sites 1-5 extending 120 miles downstream from the release point depict a catch periodicity (for example, period of minimal catch, period of increasing catch, period of mass catch, period of declining catch to minimal numbers) that would be expected from a mass-release of free embryos at a single point in time. Free embryos exhibiting developmental characteristics of the 1- and 5-dph released fish were identified from the catches to indicate that both age groups survived during downstream dispersal. Maximum catches decreased with increasing dispersal distance as would be expected owing to natural mortality and increasing dispersion. In addition, length progressions for both age groups indicated continued development and growth with increasing dispersal distance.

The experimental population of 1- and 5-dph free embryos exhibited increasing dispersion with increasing dispersal distance as evidenced from the broadening catch 106

distributions through time and distance. For example, observed dispersion of 5-dph free embryos increased from about 2.4 hours to about 58.4 hours between sites 9.7 and 110.0 miles downstream, respectively. Dispersion as observed from the field data should be regarded as nominal estimates based on three considerations. First, full extent of the dispersing population was not quantified at all sites (for example, site 1). Second, genetic results are needed to assign all free embryos to point-in-time captures. Individuals representing TOFA and TOLA are critical towards quantifying the full extent of dispersion. Third, although sampling persisted over extended periods of time at several sites, temporary retention of free embryos in upstream areas or other factors of slowing dispersal may have contributed to delayed passage times beyond sampling times implemented during the study.

Whereas dispersion for the 1- and 5-dph free embryos increased with increasing dispersal distance, initial results from 2019 presented evidence that dispersal velocity (advection) of the free embryo mass decreased with increasing dispersal distance. For example, based on the regression model, dispersal velocity for the mass of 1-dph free embryos decreased 20% from about 1.89 miles per hour when sampled 9.7 miles downstream from the release site to about 1.52 miles per hour when sampled 110.3 miles downstream from the release site. Dispersal velocity for the 5-dph free embryos decreased about 25% from 1.79 miles per hour (9.7 miles downstream) to 1.34 miles per hour (110.3 miles downstream). Declining velocity for the two age groups of free embryos occurred within a hydrologic setting where river channel velocities averaged about 2.0 miles per hour between the free embryo release site and about 100 miles downstream to nearly the Yellowstone River confluence. Working in the same reach of the Missouri River, Braaten and others (2012b) similarly detected declining dispersal velocity with increasing distance for 5-13 dph pallid sturgeon free embryos dispersing through 66 miles. The similarity of results between studies despite different discharges (2012 study=6,400 ft3/s, 2019 study=12,300 ft3/s) indicates that slowing dispersal velocity with increasing dispersal distance is an attribute exhibited by pallid sturgeon free embryos across a range of discharges. In addition to quantifying changes in dispersal velocity with distance, the mass dispersal velocity model also identified differences in dispersal velocity between the age classes. For example, catch curves at most sites illustrated that mass arrival tended to be earlier for presumed 1-dph free embryos than presumed 5-dph free embryos. The earlier mass arrivals corresponded to faster dispersal velocities. Dispersal velocity for the free embryo mass was 1.93 and 1.73 miles per hour initially at site 1 for the 1- and 5-dph free embryos, respectively, and remained faster for the 1-dph fish as they dispersed downstream.

Elevated catches and catch-curve patterns among sites indicated substantial free embryo dispersal in the main channel flow field. Whereas downstream dispersal is an innate characteristic of pallid sturgeon free embryos (Kynard and others, 2002; Kynard and others, 107

2007), sampling in the low-velocity habitat complexes indicated that some free embryos can be swept from the main channel flow field into lower velocity and potentially slack-water habitats. Within the Simulation Reach, sampling in eight of 11 pre-selected low-velocity habitat complexes verified entry for 22 pallid sturgeon free embryos into these habitats. However, mass retention of free embryos within the habitats was not verified as evidenced from the limited seine sampling that did not capture any free embryos in the low-velocity and backwater areas. These results tend to contrast Marotz and Lorang (2017) who asserted that most drifting free embryos are swept from the thalweg into low velocity habitats where they stall. If mass entrainment and stalling occurred in low-velocity and backwater areas, few or no free embryos would be expected from mainstem sampling at downstream sites. However, this expectation was not observed as free embryos persisted in mainstem samples collected 80-110 miles downstream. Whereas mass stalling was not observed, entrainment for a portion of the dispersing free embryos into lowvelocity areas can occur. But, behavioral or advective processes likely return free embryos to the main-channel flow field (Kynard and others, 2007). The cumulative effect of slow-zone entry followed by main channel re-entry processes over long distances likely probably contributes to increasing dispersion through time and space, where the slowest-drifting fish have experienced the greatest level of slowing dispersal.

Transition from Dispersing Free Embryos to Settled Benthic Larvae

Mainstem river sampling through site 3 (81.3 miles) was indicative of continued dispersal by both age groups of pallid sturgeon. At site 4 (110.3 miles downstream) and continuing downstream, river hydrology changed from upstream sites and dispersal patterns diverged between the 1 and 5-dph free embryos. Sampling at site 4 was conducted during July 3-6 when elevated discharges of 13,700-14,500 ft3/s occurred in the Missouri River and discharges of 38,300-38,900 ft3/s occurred in the Yellowstone River. The initial 3.0-4.0 miles of the Missouri River upstream from the Yellowstone River confluence exhibited backup water conditions, and although flow was discernable, water velocities were very low.

Sampling at site 4 (5.0 miles upstream from the confluence) through about 7:40 p.m. on July 3 failed to collect free embryos characteristic of the 1- and 5-dph pallid sturgeon. Initial samples during the morning of July 4 indicated the presence of the 1- and 5-dph pallid sturgeon, thus initial arrival to site 4 occurred between the night of July 3 and early morning of July 4. Sampling throughout the day and early night of July 4 verified continued dispersal of the 1-dph free embryos. Sampling at site 5 (10 miles downstream from site 4) on July 4 yielded increasing numbers of presumed 1-dph pallid sturgeon through termination of sampling. These results suggest that the original 1-dph released pallid sturgeon continued dispersing through sites 4 and 5 at an age of about 4-dph and a maximum length of about 13.0 mm.

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Whereas downstream dispersal continued for the original 1-dph free embryos, mainstem dispersal sampling suggested that pallid sturgeon originally released as 5-dph free embryos transitioned from dispersing to settling as larvae in the backwater reach of the Missouri River upstream from the Yellowstone River confluence and potentially in areas farther upstream. For example, dispersal sampling yielded fewer presumed 5-dph free embryos at site 5 relative to site 4, and although sampling effort and sampling times were not fully consistent between sites, fewer 5-dph free embryos at site 5 suggested the likelihood of some settlement between sites. Three presumed 5-dph free embryos were also collected during limited sampling at site 6 to suggest the possibility that initial dispersal may have continued down to RM 1551.0.

Verification of settlement for the original 5-dph larvae resulted following genetic confirmation, and supported results from dispersal sampling to indicate that substantial numbers of original 5-dph free embryos settled in the backwater area upstream from the Yellowstone River confluence and areas farther upstream. Quantitatively, 83% of the 84 genetically confirmed pallid sturgeon was sampled from the Missouri River upstream. Trawl results from the first week of trawling (for example, about one week after dispersal netting was completed) provided initial inferences on the spatial extent of settling; larvae were collected from RM 1584.1 to 1587.8 despite spatially extensive sampling from RM 1599.3 to RM 1538.0 Braaten_Figure 9). Sampling in subsequent weeks yielded larval pallid sturgeon as far upstream as RM 1591.8 on three separate sampling occasions (Braaten_Figure 10). This same river mile location was sampled during the initial trawling event, but no pallid sturgeon were collected from the site. Thus, it is unknown whether larvae were present but not captured at this location during the first week of trawling or if captures on subsequent weeks represented movements of small pallid sturgeon among sites and weekly sampling intervals. In addition, it is possible that larvae initially settled but were undetected in areas farther upstream, then gradually dispersed downstream as larvae. For example, previous work suggests that the transition from drifting to settling is initiated at about 18.0 mm (Snyder, 2002; Braaten and others, 2008; Braaten and others, 2012b), similar to the approximate settling length inferred from the length-frequency distributions. The presumed 5-dph free embryos sampled at site 3 exhibited lengths extending to 17.7 mm. Thus, a portion of the original 5-dph free embryos at site 3 was likely close to settling, and some individuals may have settled between site 3 and site 4.

In summary, the transition from dispersing free embryos to settled benthic larvae was verified for free embryos originally released at 5-dph (~median release length=14.3 mm; Braaten_Figure 3). The transition from dispersing to settling for the original 5-dph fish

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occurred about 100-110 miles downstream from the release location based on known captures, but settlement may have been initiated at slightly less distances. Settlement for original 1-dph free embryos (~median release length=9.0 mm; Braaten_Figure 3) was not verified from trawl collections during 2019.

Completion of genetic analysis for the dispersing free embryos will facilitate analyses of multiple objectives contained in this study. For example, the analyses will address comparisons in vertical drift location (bottom, midwater, surface) within and between age groups, comparisons in dispersal velocity between age groups to include TOFA, TOMA and TOLA related to river velocity conditions, and growth and development for both age groups in the context of water temperature regimes encountered during downstream dispersal. Age group determinations will also facilitate quantifying age-specific advection and dispersion estimates, critical for evaluating performance of particle tracking models developed for the Simulation Reach. More broadly, dispersal attributes of the free embryos will be applied used to calibrate existing hydrologic models that are used to predict dispersal, settlement, and survival of pallid sturgeon under a range of management scenarios.

References Braaten, P.J., Elliott, C.M., Rhoten, J.C., Fuller, D.B., and McElroy, B.J., 2015, Migrations and swimming capabilities of endangered pallid sturgeon (Scaphirhynchus albus) to guide passage designs in the fragmented Yellowstone River: Restoration Ecology, v. 23, no. 2, p. 186-195.

Braaten, P.J., and Fuller, D.B., 2007, Growth rates of young-of-year shovelnose sturgeon in the Upper Missouri River: Journal of Applied Ichthyology, v. 23, no. 4, p. 506–515.

Braaten, P.J., Fuller, D.B., Holte, L.D., Lott, R.D., Viste, W., Brandt, T.F., and Legare, R.G., 2008, Drift dynamics of larval pallid sturgeon and shovelnose sturgeon in a natural side channel of the Upper Missouri River, Montana: North American Journal of Fisheries Management, v. 28, no. 3, p. 808–826.

Braaten, P.J., Fuller, D.B., Lott, R.D., Haddix, T.M., Holte, L.D., Wilson, R.H., Bartron, M.L., Kalie, J.A., DeHaan, P.W., Ardren, W.R., Holm, R.J., and Jaeger, M.E., 2012a, Natural growth and diet of known-age pallid sturgeon (Scaphirhynchus albus) early life stages in the Upper Missouri River basin, Montana and North Dakota: Journal of Applied Ichthyology, v. 28, no. 4, p. 496–504.

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Braaten, P.J., Fuller, D.B., Lott, R.D., Ruggles, M.P., Brandt, T.F., Legare, R.G., and Holm, R.J., 2012b, An experimental test and models of drift and dispersal processes of pallid sturgeon (Scaphirhynchus albus) free embryos in the Missouri River: Environmental Biology of Fishes, v. 93, no. 3, p. 377–392.

DeLonay, A.J., Chojnacki, K.A., Jacobson, R.B., Albers, J.L., Braaten, P.J., Bulliner, E.A., Elliott, C.M., Erwin, S.O., Fuller, D.B., Haas, J.D., Ladd, H.L.A., Mestl, G.E., Papoulias, D.M., and Wildhaber, M.L., 2016a, Ecological requirements for pallid sturgeon reproduction and recruitment in the Missouri River: A synthesis of science, 2005-2012: U.S. Geological Survey Scientific Investigations Report 2015-5145, 224 p.

DeLonay, A.J., Chojnacki, K.A., Jacobson, R.B., Braaten, P.J., Buhl, K.J., Elliott, C.M., Erwin, O., Bulliner, E.A., Candrl, J.S., Schaeffer, D.B., Fuller, D.B., Rugg, M.L., Haddix, S.M., Hunziker, J., Best, E., Haas, J.D., Mestl, G.E., and Heist, E.J., In Review-a, Ecological requirements for pallid sturgeon reproduction and recruitment in the Missouri River: Annual report 2017: U.S. Geological Survey Open-File Report 201x-xxxx.

DeLonay, A.J., Chojnacki, K.A., Jacobson, R.B., Braaten, P.J., Buhl, K.J., Elliott, C.M., Erwin, S.O., Bulliner, E.A., Candrl, J.S., Schaeffer, T.W., Fuller, D.B., Backes, K.M., Rugg, M.L., Haddix, T.M., Haas, J.D., Mestl, G.E., and Heist, E.J., In Review-b, Ecological requirements for pallid sturgeon reproduction and recruitment in the Missouri River: Annual report 2015: U.S. Geological Survey OFR 201x-xxxx, 171 p.

---, In Review-c, Ecological requirements for pallid sturgeon reproduction and recruitment in the Missouri River: Annual report 2016: U.S. Geological Survey OFR 201x-xxxx, 171 p.

DeLonay, A.J., Chojnacki, K.A., Jacobson, R.B., Braaten, P.J., Buhl, K.J., Elliott, C.M., Erwin, S.O., Faulkner, J.D.A., Candrl, J.S., Fuller, D.B., Backes, K.M., Haddix, T.M., Rugg, M.L., Wesolek, C.J., Eder, B.L., and Mestl, G.E., 2016b, Ecological requirements for pallid sturgeon reproduction and recruitment in the Missouri River: Annual report 2014: U.S. Geological Survey Open-File Report 2016–1013, 131 p.

DeLonay, A.J., Jacobson, R.B., Chojnacki, K.A., Annis, M.L., Braaten, P.J., Elliott, C.M., Fuller, D.B., Haas, J.D., Haddix, T., Ladd, H.L.A., McElroy, B.J., Mestl, G.E., Papoulias, D.M., Rhoten, J.C., and Wildhaber, M.L., 2014, Ecological requirements for pallid sturgeon reproduction and recruitment in the Missouri River: Annual report 2011: U.S. Geological Survey Open-File Report 2014–1106, 96 p.

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DeLonay, A.J., Jacobson, R.B., Chojnacki, K.A., Braaten, P.J., Buhl, K.J., Eder, B.L., Elliott, C.M., Erwin, S.O., Fuller, D.B., Haddix, T.M., Ladd, H.L.A., Mestl, G.E., Papoulias, D.M., Rhoten, J.C., Wesolek, C.J., and Wildhaber, M.L., 2016c, Ecological requirements for pallid sturgeon reproduction and recruitment in the Missouri River: Annual report 2013: U.S. Geological Survey Open-File Report 2015–1197, 99 p.

Eichelberger, J.S., Braaten, P.J., Fuller, D.B., Krampe, M.S., and Heist, E., 2014, Novel singlenucleotide polymorphism marker confirm successful spawning of endangered pallid sturgeon in the upper Missouri River basin: Transactions of the American Fisheries Society, v. 14, no. 6, p. 1373–1385.

Fischenich, J.C., Marmorek, D.R., Nelitz, M.A., Murray, C.L., Ma, B.O., Buenau, K.E., Long, G., Bonneau, J.L., Fleming, C.A., and Schwarz, C.J., 2018, Science and Adaptive Management Plan: Missouri River Recovery Program, U.S. Army Corps of Engineers ERDC/EL TR-18-XX, 502 p.

Guy, C.S., Treanor, H.B., Kappenman, K.M., Scholl, E.A., Ilgen, J.E., and Webb, M.A.H., 2015, Broadening the regulated-river management paradigm: A case study of the forgotten dead zone hindering pallid sturgeon recovery: Fisheries, v. 40, no. 1, p. 6–14.

Kynard, B., Henyey, E., and Horgan, M., 2002, Ontogenetic behavior, migration, and social behavior of pallid sturgeon (Scaphirhynchus albus) and shovelnose sturgeon (S. platorynchus) with notes on the adaptive significance of body color: Environmental Biology of Fishes, v. 63, no. 4, p. 389–403.

Kynard, B., Parker, E., Pugh, D., and Parker, T., 2007, Use of laboratory studies to develop a dispersal model for Missouri River pallid sturgeon early life intervals: Journal of Applied Ichthyology, v. 23, no. 4, p. 365–374.

Marotz, B.L., and Lorang, M.S., 2017, Pallid sturgeon larvae: The drift dispersion hypothesis: Journal of Applied Ichthyology.

Snyder, D.E., 2002, Pallid and shovelnose sturgeon larvae-morphological description and identification: Journal of Applied Ichthyology, v. 18, p. 240–265.

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Braaten_Table 1. Age groups (days post-hatch; dph), source (wild, captive brood), parental crosses (identified by passive integrated transponder number), and numbers of free embryos produced for the 2019 drift and dispersal study.

Free embryo age group Parental Parental Number of free (dph) Source female male embryos

1 wild 1F48421542 1F4A3E1445 131913

1 wild 4315327C7B 1F4A3E1445 52564

1 wild 4443250A24 1F4A3E1445 113526

1 wild 4A49070E13 1F4A3E1445 110320

1 wild 7F7D7C2E4B 1F4A3E1445 21888

1 captive 4624127119 462616257F 9234

1 captive 4624485842 6C00096524 39312

1 captive 4628391106 4625703115 21735

1 captive 425708072B 4625537512 33579

1 captive 444267561D 425669693F 43326

1 captive 44433E701A 46273A082B 33840

1 captive 46237D0338 4627764F65 17568

1 captive 46242E2A6F 462663187F 40014

1 captive 46254E3E19 487F24652B 27306

1 captive 46255B170B 423F2F737D 27360

1 captive 6C00090926 46256E7712 48222

5 captive 4624276027 0A14082E10 7560

5 captive 4627787556 6C00090633 4064

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5 captive 1F4A125B2A 462446145B 10517

5 captive 4256437C5F 423A2C5022 0

5 captive 42570A387A 4626226048 39744

5 captive 444175202F 423A314D05 32400

5 captive 4624380D66 424F2F3D60 23220

5 captive 462546534B 4626214B69 19008

5 captive 46257D6F35 423A314D00 33840

5 captive 4627716D2D 4628382929 15246

5 captive 6C00090924 423C0C464E 0

5 captive 6C00093506 471C52622E 2227

5 captive 6C00096611 471C52622E 12960

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Braaten_Table 2. Sampling duration (hours post-release) by date and Missouri River river mile (RM) for six sites. Distance (D) downstream from the release site is expressed in river miles. Hours postrelease are based on free embryos released at 2:00 pm on July 1, 2019.

Site, River Mile, Distance from release site

Site 1 Site 2 Site 3 Site 4 Site 5 Site 6

RM 1691.6 RM 1679.7 RM 1620.0 RM 1587.0 RM 1577.0 RM 1551.0

Date D=9.7 D=21.6 D=81.3 D=110.3a D=120.3a D=146.3a

July 1 2.7-7.1 7.5-10.0

July 2 10.1-33.8 24.0-33.8

July 3 34.3-39.8 34.4-57.9 42.7-53.7

July 4 58.0-82.0 66.7-81.9 68.2-77.2

July 5 82.5-85.8 82.3-99.8 91.3-99.7

July 6 115.5-125.8 114.9-124.7 116.2- 122.5

July 7 141.4-148.2 142.2- 147.2

July 8 163.8-168.2 164.6- 167.8

Footnote a excludes a 4.0 river mile oxbow located near Bainville, Montana

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Braaten_Table 3. Low-velocity and backwater habitat complexes sampled in 2019, and corresponding river mile (RM) locations and coordinates.

Complex RM Sample Latitude Longitude Area Hours Volume Free embryos Free embryos lengths method sampled sampled sampled (m3) sampled (mm) (m2)

1 1692.0 Inlet net 1 48.08997 -105.37368 19.72 897.1 6 9.4, 9.5, 9.8, 9.8, 11.9, 14.9

Inlet net 2 48.08997 -105.37368 19.75 669.8 6 8.1, 8.8, 9.5, 9.7, 10.0, 13.2

Seine 1 48.08982 -105.37379 20.9 0

Seine 2 48.08985 -105.37360 26.4 0

2 1689.6 Inlet net 1 48.09177 -105.32877 20.80 840.6 0

Inlet net 2 48.09159 -105.32858 20.75 714.7 0

3 1689.5 Inlet net 1 48.09095 -105.32712 21.25 1023.9 8 7.2, 8.3, 9.3, 9.4, 9.6, 9.6, 10.0, 10.8

Inlet net 2 48.09101 -105.32704 21.20 943.6 1 12.6

6 1686.9 Inlet net 1 48.08350 -105.28599 21.47 3564.1 0

Inlet net 2 48.08421 -105.28597 21.37 2290.0 0

7 1684.5 Inlet net 1 48.08910 -105.23627 21.85 670.0 0

Inlet net 2 48.08914 -105.27627 21.82 739.8 0

Seine 1 48.08913 -105.23623 51.6 0

Seine 2 48.08904 -105.23709 72.0 0

9 1681.6 Inlet net 1 48.06338 -105.21940 22.87 890.2 1 9.6

Seine 1 48.06322 -105.21920 51.6 0

Seine 2 48.06349 -105.21947 48.0 0

10 1680.9 Inlet net 1 48.06652 -105.20387 23.52 581.5 0

Inlet net 2 48.06647 -105.20386 23.52 249.2 0

11 1679.7 Inlet net 1 48.07411 -105.18568 22.65 4608.6 0

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Braaten_Table 4. Larval pallid sturgeon captured in the Missouri River upstream (UTC) and downstream (DTC) from the Yellowstone River confluence across weekly sampling dates and river miles (RM). Free embryo age (days post-hatch, dph) refers to age when released at RM 1701.3 on July 1, 2019.

Free Number of Larvae capture Larvae Larvae Sampling pallid sturgeon mean minimum and embryo Week Sampling Dates location larvae captured RM length maximum release age (mm) length (mm) (dph) 1 July 16-19 UTC 15 1584.1-1587.8 29 25-33 5

DTC 0

2 July 22-23 UTC 2 1586.7-1589.1 44 42-45 5

DTC 1 1556.3 35 5

3 July 29-31 UTC 39 1584.0-1591.8 47 38-52 5

DTC 1 1581.1 47 5

4 August 5-7 UTC 5 1591.8 57 55-61 5

DTC 2 1580.9 59 54-64 5

5 August 12-14 UTC 4 1589.1 68 60-73 5

DTC 6 1552.2-1581.2 71 54-79 5

6 August 19-21 UTC 3 1589.1 84 80-93 5

DTC 0

7 August 26-28 UTC 0

DTC 2 1579.2-1581.1 100 90-110 5

8 September 3-5 UTC 1 1589.1 94 5

DTC 0

9 September 9-11 UTC 1 1591.8 97 5

DTC 1 1574 105 5

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Braaten_Figure 1. Simulation Reach extending from river mile (RM) 1692.5 to RM 1679.0.

Numbered labels refer to the 11 low-velocity and backwater complexes identified for sampling. Water velocity is quantified in meters per second.

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Time, in hours post-release Braaten_Figure 2. Site 1 (RM 1691.6) catch of presumed 1- and 5-day post-hatch (dph) pallid sturgeon free embryos released in the Missouri River by hours post-release.

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Free embryo length, in mm

Braaten_Figure 3. Length-frequency distributions of Scaphirhynchus spp. free embryos sampled at six sites in the Missouri River, July 1-8, 2019. The dashed line separates the length distributions into two groups, where lengths to the left of the dashed line contain presumed 1- day post-hatch (dph) pallid sturgeon free embryos, and lengths to the right of the dashed line

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contain presumed 5-dph pallid sturgeon. Both age groups may also contain lengths from wild- produced shovelnose sturgeon free embryos. Time, in hours post-release Braaten_Figure 4. Site 2 (RM 1679.7) catch of presumed 1- and 5-day post-hatch (dph) pallid sturgeon free embryos released in the Missouri River by hours post-release.

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Time, in hours post-release Braaten_Figure 5. Site 3 (RM 1620.0) catch of presumed 1- and 5-day post-hatch (dph) pallid sturgeon free embryos released in the Missouri River by hours post-release.

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Time, in hours post-release Braaten_Figure 6. Site 4 (RM 1587.0) catch of presumed 1- and 5-day post-hatch (dph) pallid sturgeon free embryos released in the Missouri River by hours post-release.

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Time, in hours post-release Braaten_Figure 7. Site 5 (RM 1577.0) catch of presumed 1- and 5-day post-hatch (dph) pallid sturgeon free embryos released in the Missouri River by hours post-release.

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Time, in hours post-release

Braaten_Figure 8. Site 6 (RM 1551.0) catch of presumed 1- and 5-day post-hatch (dph) pallid sturgeon free embryos released in the Missouri River by hours post-release.

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Braaten_Figure 9. Trawl sampling locations (A) and pallid sturgeon capture locations (B) during July 1619, 2019. Blue circles corresponding to pallid sturgeon captures represent one to many captures at a single location.

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Braaten_Figure 10. Trawl sampling locations (A), and pallid sturgeon capture locations during July 22-

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23 (B), July 29-31 (C), August 5-7 (D), August 12-14 (E), August 19-21 (F), August 26-28 (G), September 3-5 (H), and September 9-11 (I) in 2019. Blue circles corresponding to pallid sturgeon captures represent one to many captures at a single location.

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Migrations, Aggregations, and Spawning of Pallid Surgeon in the Yellowstone River, 2019

Pat Braaten, U. S. Geological Survey, Columbia Environmental Research Center, Fort Peck, Montana, 59223; email: [email protected]

Colt Holley, U. S. Geological Survey, Columbia Environmental Research Center, Fort Peck, Montana, 59223

Background

The reproductive ecology of pallid sturgeon Scaphirhynchus albus has been a focal area of research in the Missouri and Yellowstone river systems. Studies in the early 1990s examined habitat use and seasonal migrations of pallid sturgeon in the Missouri and Yellowstone rivers, and identified potential aggregations of fish in the lower Yellowstone River (Bramblett and White, 2001). Research in 2007 provided additional information on migrations and aggregations of pallid sturgeon, but importantly, this research provided the first verification of spawning by pallid sturgeon in the Yellowstone River (Fuller and others, 2008). These earlier studies have been complemented since 2011 with detailed investigations. For example, recent studies have examined spatial and temporal aspects of pre–spawn migrations for males and females in the Missouri and Yellowstone rivers, assessed male aggregations as early indicators of spawning patches, verified that spawning occurs, evaluated repeat use of spawning patches through time, and quantified hydraulic elements at spawning patches (DeLonay and others, 2014; Braaten and others, 2015; DeLonay and others, 2016a; DeLonay and others, 2016b; DeLonay and others, 2016c; DeLonay and others, In Review-a; DeLonay and others, In Review-b, c). Moreover, recent work has aimed to verify functionality of spawning patches to support fertilization, incubation, hatch, and drift-entry for dispersing free embryos. Since 2011, spawning patches have been verified to be at least partially functional in some years based on collections of dispersing free embryos; however, the number of free embryos sampled has been low across years (for example, 0-4 individuals).

Studies examining drift and dispersal of pallid sturgeon early life stages have been a second focal area of research in the upper Missouri River basin (Braaten and others, 2008; Braaten and others, 2012). For these studies, pallid sturgeon free embryos produced from wild broodstock–and in some cases captive broodstock–are released and serially sampled downstream to discern multiple attributes of early life stage dispersal. Although research on reproductive ecology and early life stage dispersal provide information critical towards management and restoration programs, implementation of both lines of research can be

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conflicting. For example, wild telemetered females are targeted as the production source of free embryos for drift and dispersal studies. Thus, in years when drift and dispersal studies are prioritized, telemetered reproductive females are unavailable as research subjects. This situation occurred in 2019 when all known telemetered reproductive females were captured from the river, and used to produce free embryos for the 2019 drift and dispersal studies (see accompanying report).

Research on pallid sturgeon reproductive ecology during 2019 followed highly collaborative work from previous years where several agencies utilized the same research population of telemetered wild pallid sturgeon and hatchery-origin pallid sturgeon (HOPS) to address different study objectives. For example, as part of the Pallid Sturgeon Population Assessment Program, crews from the U.S. Fish and Wildlife Service (USFWS) and Montana Fish, Wildlife and Parks (MFWP) evaluated pallid sturgeon occupancy of river bends and quantified catches in river bends of the Missouri River. The potential existed for pallid sturgeon in these bends to eventually become spawning migrants and members of spawning aggregations in the Yellowstone and Missouri rivers. For fish that migrated to Intake Dam, crews from the U.S. Bureau of Reclamation (Reclamation) were positioned to translocate upriver migrants past the dam. In addition, crews from all agencies collaborated to capture wild pallid sturgeon and HOPS throughout the Missouri and Yellowstone rivers, and implant/reimplant transmitters.

Progress summarized in this report focuses on pallid sturgeon in the Yellowstone River as progress on the Missouri River is summarized under a separate report by MFWP. In contrast to earlier investigative years, the specific timing and location of spawning events in 2019 could not be ascertained from female behavior and associations with male aggregations as all known telemetered reproductive females had been removed from the river to support free embryo production for the 2019 drift and dispersal study. Lacking telemetered reproductive females, objectives for 2019 included: 1) assessing movements and migrations of wild pallid sturgeon and HOPS to the reach affected by Intake Dam, 2) identifying the location(s) of male aggregations as potential indicators of spawning patches, and 3) verifying functionality of potential spawning patches based on captures of dispersing pallid sturgeon free embryos and larvae.

Methods

Radio telemetry including manual tracking by boat and deployment of automated telemetry ground stations was initiated in April 2019. Pallid sturgeon relocation points and environmental attributes obtained during manual tracking were manually recorded or recorded

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on a highly customized mobile mapping and electronic data collection application. Automated telemetry ground stations were deployed by USGS and MFWP at multiple locations in the Yellowstone River including river mile (RM) 1.0 (near the Missouri River confluence), RM 4.1 (new station in 2019, linked to internet), RM 7.8, RM 39.8, RM 62.9, RM 72.9 (Intake Dam), in the high-flow side channel (HFSC) around Intake Dam (RM 71.4, 75.0), RM 84.6, RM 115.5, RM 149.1 (Powder River confluence, and RM 185.0 (near Miles City). A logging station was also positioned in the Powder River at RM 30.9. Telemetry ground stations were deployed in the Missouri River and Milk River as described in the Upper Missouri and Milk River report. Ground station detections complemented with manual tracking relocations provided a continuous assessment of migrations and movements within and among rivers.

Lacking telemetered reproductive females in 2019, inferences on spawning locations and timing could be obtained only from male aggregations and collections of dispersing pallid sturgeon free embryos and larvae. Sampling for pallid sturgeon free embryos and larvae in the Yellowstone River was conducted by USGS personnel downstream from identified male aggregations (see Progress section below). Lacking a known aggregation in the Powder River, MFWP personnel sampled for dispersing free embryos and larvae primarily in the lower 1.0 mile of the river to quantify cumulative outputs from that river. Sampling for pallid sturgeon free embryos and larvae was conducted using 3.0-m long tapered rectangular nets (1.0 mm mesh) affixed to 0.75-m wide by 0.5-m height rectangular frames (Braaten and others, 2010). Paired nets fitted with flow meters were simultaneously deployed from the port and starboard sides of the boat bow, and fished in the lower 0.5-m of the water column adjacent to the river bed. A sounding weight or paired down rigger weights were attached to the net frame to maintain net contact with the river bed. Sample contents were flushed from the net and terminal collecting cup, transferred to black pans, and Acipenseriformes free embryos and larvae (for example, pallid sturgeon, shovelnose sturgeon Scaphirhynchus platorynchus, paddlefish Polyodon spathula) were live-extracted from the detritus. Specimens were preserved in vials containing 95 percent non-denatured ethanol. In the laboratory, preserved specimens were tentatively identified as Scaphirhynchus spp., paddlefish, or unknown (that is, damaged beyond definitive recognition because of smashed bodies or missing body parts) based on morphometric and meristic characters, measured, and photographed. Preserved Acipenseriformes were sent to Dr. Ed Heist (Southern Illinois University; SIU) for genetic analysis (Eichelberger and others, 2014) to differentiate specimens as shovelnose sturgeon, pallid sturgeon, or paddlefish.

Progress

Research Population of Telemetered Pallid Sturgeon

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Crews from USGS, MFWP, and USFWS worked to capture and implant/re-implant wild pallid sturgeon and HOPS during the 2019 field season. Collective efforts from April through October resulted in about 50 wild pallid sturgeon and about 150 HOPS carrying transmitters. Whereas sex and reproductive status for most wild fish are known, these attributes are unknown for the majority of HOPS. During late April through early May, ultrasound and blood samples (for hormone analysis) were obtained from 11 HOPS in the lower Yellowstone River and Missouri River (Braaten_Table 1). The fish varied from 720 mm to 1,063 mm and 1.3 kg to 5.0 kg. Ultrasound suggested eight HOPS were early stage females (Stage 1, 2) based on indications of ovarian folds; sex could not be determined from ultrasound for the remaining three fish. Blood-hormone analysis is currently in progress, and results may provide additional information on sex and reproductive status.

Hydrologic Conditions

The Yellowstone River and Powder River exhibited highly variable flow and water temperature conditions (Braaten_Figure 1). Discharge in the Yellowstone River was 10,70016,500 ft3/s from April 1 through May 14, increased to 36,600 ft3/s on May 21, then increased to the annual maximum of 58,200 ft3/s on June 11. Discharge in the Yellowstone River remained greater than 30,000 ft3/s until July 19, thereafter diminishing steadily towards late summer low flows. Discharge in the Powder River was 648-1,980 ft3/s from April 1-May 21, then markedly increased to maximum flows on June 2 (7,040 ft3/s). Powder River discharge diminished through early to mid-June, and remained fairly steady (1,740-2,450 ft3/s) during June 15-July 5 prior to increasing in early to mid-July. Water temperature in the Yellowstone River fluctuated between periods of elevated and diminished conditions from mid-April through early July, and did not remain steady until mid-July. The annual maximum temperature of 26.2 oC occurred on August 4.

Pallid Sturgeon Activity at Intake Dam

A total of 22 telemetered pallid sturgeon were detected in the reach affected by Intake Dam during 2019 (Braaten_Table 2). Fourteen pallid sturgeon (HOPS codes 69, 95, 142, 146, 147, 167 on 149.620 MHz, 167 on 149.760 MHz, 173, 175, 176, 177, 181, 192; wild code 11) had initial detections or manual relocations at Intake Dam between April 21 to October 7. Initial detections for other pallid sturgeon (for example, HOPS codes 66, 156, 160) did not result from volitional swimming upstream to the dam, but resulted from upstream to downstream passage over Intake Dam following translocation (see below).

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Twelve pallid sturgeon were translocated by Reclamation personnel between May 4 and May 30. The translocated fish included 10 HOPS of unknown sexual state at time of capture (codes 66, 69, 112, 133, 147, 155, 156, 160, 167, 177) and two wild male pallid sturgeon (codes 11, 131; Braaten_Table 2). Following translocation, pallid sturgeon moved upstream but upstream distances were highly variable among the fish. For the HOPS, the maximum detected upstream location varied from RM 84.6 to RM 232.8. Three translocated HOPS (codes 156, 160, 167) had fairly similar terminal locations spanning RM 103.1-105.1 during early to mid-June. A fourth HOP (code 158) that resides upstream from Intake Dam was also in the area of RM 101.8105.0 during early and mid-June.

Wild male pallid sturgeon codes 11 and 131 exhibited extensive upstream movements after being translocated on May 31 and May 27, respectively. Both fish moved to the Powder River confluence (RM 149.1), then progressed into the Powder River (Braaten_Table 2). The most upstream detection location for code 11 in the Powder River was RM 48.4 on June 15. Tracking efforts by MFWP on June 11 did not detect code 131 up to Powder River RM 81.0, suggesting that this fish was farther upstream. Male code 11 was detected passing downstream over Intake Dam on June 26, and by June 27, code 11 was detected in the lower Yellowstone River at RM 7.8. After exiting the Powder River, code 131 remained in the upper Yellowstone River where the last detection occurred at RM 147.0 on October 24.

Male Aggregations and Potential Spawning Patches

Earlier work in the Yellowstone River has identified that the formation of male aggregations and persistence of aggregations through time provide early indications of where spawning will occur (DeLonay and others, In Review-b). In a typical scenario, males will initially aggregate in early June and the aggregation will persist for a couple of weeks. During this timeframe, crews verify spawning activity by evaluating female entry into the aggregation and performing post-spawn reproductive assessments. The removal of all known telemetered reproductive female pallid sturgeon in 2019 for the free embryo dispersal study prevented us from definitively identifying spawning activity based on female entry into the male aggregation. Lacking telemetered reproductive females in 2019, spawning activity could only be inferred from male aggregations and collections of free embryos (see below).

During 2019, the initial indication of a potential spawning patch occurred on June 6 when an aggregation of 9-10 pallid sturgeon was detected at RM 6.2-6.3 (Braaten_Figure 2). Environmental conditions on June 6 included a mean daily temperature of 20.3 oC, discharge of 46,800 ft3/s, and elevated turbidity (>1,000 NTU). Prior to June 6, pallid sturgeon were scattered individually or as small groups throughout the lower 12 miles of the river. Detections of pallid sturgeon diminished at RM 6.2-6.3 during June 7-12 as flow increased to the seasonal

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maximum and water temperature declined nearly 5.0 oC. From June 13-26, the area from RM 5.9 to 6.3 was the primary aggregation site for pallid sturgeon. A maximum of 12 pallid sturgeon was detected at RM 6.3 on June 13. Elevated numbers of pallid sturgeon were also detected at RM 5.9 (7 fish on June 18 and 22), RM 6.0 (11 fish on June 19), RM 6.1 (8 fish on June 19), and RM 6.2 (8 fish on June 26). The aggregation was comprised almost exclusively of telemetered wild male pallid sturgeon and few HOPS were detected. The location and persistence of the aggregation suggest that the spawning patch was between RM 5.9-6.3, and that spawning activities involving one or more non-transmittered females most likely occurred during June 13-26. During these dates, mean daily discharge varied from 36,200-50,700 ft3/s, mean daily water temperature varied from 16.5 to 19.4 oC, and instantaneous measurements of turbidity varied from 220-486 NTU.

Verification of Hatch and Drift Entry of Acipenseriformes Free Embryos and Larvae

Sampling for Acipenseriformes eggs, embryos, free embryos, and larvae to verify spawning by pallid sturgeon was conducted by USGS crews during June 10-27 in the lower Yellowstone River and by MFWP crews during June 17-27 in the Powder River (Braaten_Table 3). Sampling in the Yellowstone River resulted in the collection of 937 eggs and developing embryos, and 1,334 free embryos and larvae. Sampling in the Powder River yielded nine eggs and embryos, and 140 free embryos and larvae. Genetic analysis is in progress to differentiate developing embryos (Kashiwagi and others, 2019) and free embryos and larvae (Eichelberger and others, 2014) as pallid sturgeon, shovelnose sturgeon, or paddlefish.

Discussion

The pallid sturgeon population in the Missouri and Yellowstone Rivers included a large number of wild fish and HOPS implanted with transmitters during 2012 and 2013. These transmitters were expired or nearly expired during the 2019 season, and overall the number of pallid sturgeon in the research population was reduced compared to earlier study years. Crews from all agencies pursued implanting/re-implanting both wild pallid sturgeon and HOPS to enhance numbers of telemetered fish in the research population, and to maintain a reproductive and movement chronology of individual fish. Whereas sex and reproductive state is known for most wild pallid sturgeon in the research population, less reproductive information is known for the telemetered population of HOPS. Ultrasound in 2019 categorized several HOPS as early stage females (for example, Stage 1, 2), but none were identified as reproductive. The ultrasounds and blood-hormone analyses (currently in progress) provide sex and reproductive baselines for these fish. As early stage females, the timeframe for

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reproductive maturity is uncertain; however, recaptures and reproductive assessments in coming years will track reproductive progression in these and other HOPS.

The specific timing and location of spawning events could not be directly verified in 2019 owing that all telemetered reproductive females were removed from the river to support the 2019 free embryo drift and dispersal project. Despite the lack telemetered reproductive females, additional sources of information lend inferences towards potential spawning events. First, earlier work in the lower Yellowstone River has indicated that the establishment and persistence of a male aggregation typically at one location (and in some cases two locations) identifies the eventual spawning patch (DeLonay and others, 2014; Braaten and others, 2015; DeLonay and others, 2016a; DeLonay and others, 2016b; DeLonay and others, 2016c; DeLonay and others, In Review-a; DeLonay and others, In Review-b, c). The 2019 aggregation area spanning RM 5.96.3 of the Yellowstone River was persistent and was positioned within or in close proximity to aggregation areas and spawning locations verified in earlier years. For example, aggregations and spawning were verified at RM 5.9-6.7 in 2018 and RM 5.4-6.1 in 2017. In other years, aggregations and spawning have occurred at RM 5.8- 5.9 (2015), RM 5.3-5.8 (2014), RM 5.7-5.9 (2013), and RM 6.7-7.1 (2012). Similar to 2019, telemetered reproductive females in 2016 were removed to support the 2016 drift and dispersal study. Males aggregated at RM 2.2 – 2.9 of the Yellowstone River for several days in 2016, denoting the most downstream location of a male aggregation recorded during research on the Yellowstone River. Interestingly, only a few HOPS were noted to occupy the area utilized by the wild male aggregation in 2019. Greater numbers of known HOPS males and other HOPS have been in the area of wild male aggregations in previous years (DeLonay and others, In Review-a).

Genetic confirmation of a pallid sturgeon free embryo collected downstream from the 2019 aggregation area would provide a second line of evidence supporting the male aggregation area as a spawning location. A substantial number of free embryos and larvae (for example, about 950) was sampled below the aggregation during June 18-27. If spawning commenced on June 13 in association with persistent formation of the male aggregation, samples collected on June 18-19 would verify hatch of eggs initially deposited on June 13 (assuming a 5-6 day incubation period). Subsequent samples would quantify free-embryo emergence from spawning events that occurred during June 19-22. Genetic analyses on free embryos and larvae is inprogress. A confirmed pallid sturgeon free embryo and parental linkage to a known telemetered male pallid sturgeon in the aggregation would provide verification of the spawning area, and provide an estimate of spawn and hatch dates.

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The lower Yellowstone River was a focal area for pallid sturgeon as evidenced from the male aggregations, but wild pallid sturgeon and HOPS were also found in other areas of the Missouri and Yellowstone rivers. Within the Yellowstone River, several HOPS and two wild pallid sturgeon were in the vicinity of the reach affected by Intake Dam. No fish naturally passed upstream or around Intake Dam, but passage for 12 pallid sturgeon was facilitated by Reclamation translocation activities. The translocated HOPS exhibited variable upstream movements and terminal detections following translocation (for example, maximum RM 84.4 for HOPS code 69, maximum RM 232.8 for HOPS code 112). Terminal detections for three translocated HOPS (codes 156, 160, 167) occurred at RM 103.1-105.1 during early to mid- June, and a fourth HOPS (code 158) was similarly found in this area of the river during the same timeframe. Although the more broadly spaced locations of the four HOPS differ from dense spawning aggregations of wild male pallid sturgeon in the lower Yellowstone River, the proximity of the HOPS (and potentially other non-telemetered HOPS) presents the possibility of group-attraction to this area for some reason. Reproductive status of these fish was unknown at time of capture, and hormone analyses are in-progress. Thus, it is uncertain if the fish were reproductive. If reproductive, results suggest the possibility of a loosely spaced spawning aggregation. If not reproductive, this localized area of the upper Yellowstone River may provide other beneficial resources (for example, food). Repeat use of this area in 2020 may lend additional information.

The two translocated wild male pallid sturgeon (codes 11, 131) exhibited upstreammigrations that persisted into the Powder River. For code 11, 2019 was the third consecutive year that this fish moved into the Powder River where the detected migration apex occurred at RM 48.4 on June 15. Code 11 had a Powder River migration apex at RM 87.7 in 2018 (June 1) and at RM 88.1 in 2017 (June 1; DeLonay and others, In Review-a). Code 131 was detected at Intake Dam in 2018. In 2019, this fish was captured prior to potentially arriving at the dam and was translocated on May 27. Although the exact Powder River apex location and date are not known, code 131 was most likely upstream of RM 81.0 on July 11. Lacking a telemetered reproductive female entering the Powder River in 2019, it is difficult to discern if spawning occurred. Genetic results from free embryos and larvae collected in the Powder River may confirm spawning in this river system if a pallid sturgeon is identified. Spawning in the Powder River was documented in 2014 (DeLonay and others, 2016b). The Powder River has emerged as a target migration location for some pallid sturgeon in the upper Yellowstone River system. Continued work is needed to verify additional spawning events in the Powder River, identify spawning locations, and quantify reproductive output.

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References

Braaten, P.J., Elliott, C.M., Rhoten, J.C., Fuller, D.B., and McElroy, B.J., 2015, Migrations and swimming capabilities of endangered pallid sturgeon (Scaphirhynchus albus) to guide passage designs in the fragmented Yellowstone River: Restoration Ecology, v. 23, no. 2, p. 186-195.

Braaten, P.J., Fuller, D.B., Lott, R.D., Ruggles, M.P., Brandt, T.F., Legare, R.G., and Holm, R.J., 2012, An experimental test and models of drift and dispersal processes of pallid sturgeon (Scaphirhynchus albus) free embryos in the Missouri River: Environmental Biology of Fishes, v. 93, no. 3, p. 377–392.

Braaten, P.J., Fuller, D.B., Lott, R.D., Ruggles, M.P., and Holm, R.J., 2010, Spatial distribution of drifting pallid sturgeon larvae in the Missouri River inferred from two net designs and multiple sampling locations: North American Journal of Fisheries Management, v. 30, no. 4, p. 1062– 1074.

Bramblett, R.G., and White, R.G., 2001, Habitat use and movements of pallid and shovelnose sturgeon in the Yellowstone and Missouri Rivers in Montana and North Dakota: Transactions of the American Fisheries Society, v. 130, no. 6, p. 1006–1025. DeLonay, A.J., Chojnacki, K.A., Jacobson, R.B., Albers, J.L., Braaten, P.J., Bulliner, E.A., Elliott, C.M., Erwin, S.O., Fuller, D.B., Haas, J.D., Ladd, H.L.A., Mestl, G.E., Papoulias, D.M., and Wildhaber, M.L., 2016a, Ecological requirements for pallid sturgeon reproduction and recruitment in the Missouri River: A synthesis of science, 2005-2012: U.S. Geological Survey Scientific Investigations Report 2015-5145, 224 p.

DeLonay, A.J., Chojnacki, K.A., Jacobson, R.B., Braaten, P.J., Buhl, K.J., Elliott, C.M., Erwin, S.O., Bulliner, E.A., Candrl, J.S., Schaeffer, D.B., Fuller, D.B., Rugg, M.L., Haddix, T.M., Hunziker, J., Best, E., Haas, J.D., Mestl, G.E., and Heist, E.J., In Review-a, Ecological requirements for pallid sturgeon reproduction and recruitment in the Missouri River: Annual report 2017: U.S. Geological Survey Open-File Report 201x-xxxx.

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---, In Review-c, Ecological requirements for pallid sturgeon reproduction and recruitment in the Missouri River: Annual report 2016: U.S. Geological Survey OFR 201x-xxxx, 171 p.

Eichelberger, J.S., Braaten, P.J., Fuller, D.B., Krampe, M.S., and Heist, E., 2014, Novel single-nucleotide polymorphism marker confirm successful spawning of endangered pallid sturgeon in the upper Missouri River basin: Transactions of the American Fisheries Society, v. 14, no. 6, p. 1373–1385.

Fuller, D.B., Jaeger, M.E., and Webb, M., 2008, Spawning and associated movement patterns of pallid sturgeon in the lower Yellowstone River. Report submitted to the Western Area Power Administration, upper basin pallid sturgeon work group.: Montana Fish, Wildlife and Parks, 22 p.

Kashiwagi, T., DeLonay, A.J., Braaten, P.J., Chojnacki, K.A., Gocker, R.M., and Heist, E.J., 2019, Improved genetic identification of Acipenseriform embryos with application to the endangered pallid sturgeon Scaphirhynchus albus: Journal of Fish Biology.

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Braaten_Table 1. Hatchery-origin pallid sturgeon (HOPS) captured in 2019, and assessed by ultrasound for reproductive status.

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Braaten_Table 2. Hatchery-origin pallid sturgeon (HOPS) and wild pallid sturgeon (Wild) detected at or within 0.3 river miles (RM) of Intake Dam, translocated above the dam, and detected in or passed through the high-flow side channel (HFSC) in 2019. Maximum river mile, date, and river are listed for fish that were translocated or naturally passed upstream around Intake Dam. Dates identified in bold and italics denote the date when a fish moved from upstream to downstream over Intake Dam.

a Fish on frequency 149.620 MHz. b Fish on frequency 149.760 MHz. This fish had a few detections on May 1, then was not detected again. c Montana Fish, Wildlife and Parks crews tracked up to about RM 81.0 and did not locate the fish, suggesting that the fish was farther upstream. 141

Braaten_Table 3. Samples and numbers of Acipenseriformes eggs, embryos, free embryos, and larvae sampled in the Yellowstone River and Powder River by date during 2019.

Acipenseriform es Acipenseriform es free Date Location eggs and embryos embryos and larvae 6/10/2019 Yellowstone 6 2 6/11/2019 Yellowstone 20 6/12/2019 Yellowstone 41 7 6/13/2019 Yellowstone 32 14 6/14/2019 Yellowstone 64 41 6/15/2019 Yellowstone 61 75 6/16/2019 Yellowstone 44 110 6/17/2019 Yellowstone 72 120 6/18/2019 Yellowstone 44 165 6/19/2019 Yellowstone 36 189 6/20/2019 Yellowstone 18 61 6/21/2019 Yellowstone 121 118 6/22/2019 Yellowstone 84 67 6/24/2019 Yellowstone 30 19 6/25/2019 Yellowstone 69 67 6/26/2019 Yellowstone 80 114 6/27/2019 Yellowstone 115 148 6/17/2019 Powder 1 52 6/24/2019 Powder 0 8 6/25/2019 Powder 0 8 6/26/2019 Powder 4 19 6/27/2019 Powder 4 53

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3 7 /1/19 Number of wild male pallid sturgeon and HOPS and sturgeon pallid male wild of Number 2 6 /24/19 6 /17/19 6 /10/19 1 6 /3/19 11 10 9 5 /27/19 8 7 5 /20/19 Date Yellowstone River location,6 5 4 in river miles 3 2 5 /13/19 1 0

EXPLANATION Number of pallid sturgeon Braaten_Figure 2. Aggregations of telemetered wild male pallid sturgeon and hatchery-origin pallid sturgeon (HOPS) by river mile location and date in the Yellowstone River, May-June 2019.

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Genotypic Analyses and Parental Identifications of Juvenile and

Sub-adult Pallid Sturgeon in the Missouri River

FY 2019 Report

February 24, 2020

Meredith Bartron and Jeff Kalie [email protected], [email protected] U.S. Fish and Wildlife Service Northeast Fishery Center 227 Washington Ave., PO. Box 75 Lamar, PA 16848 (570) 726-4995

Submitted to:

Steven Krentz [email protected] U.S. Fish and Wildlife Service Missouri River Fish and Wildlife Conservation Office 3425 Miriam Ave Bismarck, ND 58501 (701) 250-4419 And

Wayne Nelson-Stastny [email protected] Pallid Sturgeon Recovery Coordinator 55245 NE HWY 121 Crofton, NE 68730 (402)-667-2884

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Introduction

Pallid sturgeon (Scaphirhynchus albus) are endangered throughout their range and were listed under the Endangered Species Act in 1990. The primary limiting factor for pallid sturgeon recovery in the Missouri River appears to be a lack of natural recruitment over the past 25-30 years. Because of this lack (or low level) of natural recruitment, stocking of hatchery produced pallid sturgeon juveniles has been used to supplement and maintain pallid sturgeon. Adult pallid sturgeon are captured annually in the Missouri River and taken to fish hatcheries for spawning, after which they are returned to the capture area. However, the pallid sturgeon recovery plan requires that all hatchery-produced pallid sturgeon are identifiable by some type of tag. In the past, offspring were reared in hatcheries until they were large enough to be marked with PIT tags or other physical tags.

Typically juveniles remain in hatcheries for nearly a year before they reach the size at which they can be tagged, approximately 140-220mm. Pallid sturgeon are highly fecund and will produce several thousand offspring in a single year, resulting in large numbers of offspring necessary to maintain separately until tagging size. Hatchery space constraints often limit the number of juvenile pallid sturgeon that can be retained, resulting in culling of large numbers of offspring each year. In hatcheries, juvenile pallid sturgeon are also highly susceptible to outbreaks of iridovirus that can severely limit survival. The use of bi-parentally inherited genetic markers is a viable alternative to physical tags (DeHaan et al. 2005; DeHaan et al. 2008), and allows tracking of each unique family group to learn about family-specific distribution and survival, in addition to providing genetic data for management to track genetic diversity over time.

The geographic focus of this project was the upper and middle Missouri River. The upper Missouri River is comprised of RPMAs 1 and 2. RPMA 1 is the Missouri River from the headwaters of Fort Peck Reservoir to the confluence of the Marias River in Montana. RPMA 2 is defined as the Missouri River below Fort Peck Dam to the headwaters of Lake Sakakawea and the Yellowstone River and its tributaries up to the confluence of the Tongue River. The middle Missouri River is comprised of RPMAs 3 and 4. RPMA 3 is the Missouri

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River below Fort Randall Dam to Gavins Point Dam. RPMA 4 is the Missouri River downstream of Gavins Point Dam down to the confluence with the Mississippi River.

There were three main objectives completed during FY2019. These objectives include:

1) Estimate the degree of relatedness among adult pallid sturgeon at federal (national) and state fish hatcheries (NFH and SFH, respectively) to be used as hatchery broodstock for the Missouri River and provide mating plans for hatchery spawning (Miles City State Fish Hatchery and Garrison Dam National Fish Hatchery, with Gavins Point National Fish Hatchery as needed) 2) Add adult pallid sturgeon to the genetic baseline dataset used to distinguish hatchery and natural origin juveniles 3) Use the baseline dataset to conduct species ID for any unmarked juvenile sturgeon collected in the Missouri River and use parentage analysis to determine if unmarked juveniles identified as pallid sturgeon are hatchery or natural origin fish

Methods

Fin clips preserved in 95% non-denatured ethanol were collected by biologists participating in monitoring efforts for juvenile and adult putative pallid sturgeon. A total of 1247 samples were returned to the U.S. Fish and Wildlife Service (USFWS) Northeast Fishery Center Conservation Genetics Lab with biological information including PIT tag, length, weight, sampling location, and sampling date.

DNA was extracted using the Purgene method (Qiagen, Valencia, CA). DNA concentrations were obtained and concentrations were standardized for polymerase chain reaction (PCR). DeHaan et al. (2005) identified 17 microsatellite loci that can be used for parentage analysis in pallid sturgeon as well as for differentiating pallid and shovelnose sturgeon: Spl15, Spl18, Spl19, Spl26, Spl30, Spl34, Spl35, Spl36, Spl40, Spl56, Spl60, Spl101, Spl105, Spl106, Spl119, Spl158, Spl173 (McQuown et al. 2000). Two additional loci (Spl12

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and Spl53 (McQuown et al. 2000)) have been added to the original suite of 17 for standardization purposes with Southern Illinois University, and they will be used for species identification in the Central Lowlands Management Unit (CLMU) and Interior Highlands Management Unit (IHMU) baselines.

Multiplex reactions were created to streamline the amplification process; four pre-PCR multiplex reactions were created, with 3 to 5 loci within each reaction. Locus Spl26, Spl40, Spl53, and Spl105 were amplified separately. Locus Spl 26, Spl40, and Spl105 were added to one of the multiplexes post-PCR. Locus Spl53 was not added to a multiplex reaction and run individually. For the multiplex reactions, reagent concentrations were the same. Each 20 uL PCR reaction consisted of 1.5 l of genomic DNA extract, 1.5 X PCR buffer (10 mM Tris-

HCl, pH 8.3; 50 mM KCl), 3.75 mM MgCl2, 0.3175 mM each dNTP, 0.12-0.80 M of each primer (forward primer fluorescently labeled; Applied Biosystems, Foster City, CA), 0.06 units of Taq polymerase (Promega Corporation, Madison, WI), and deionized water added to achieve the final volume. Single PCR reactions, Spl40 and Spl105, were 10ul PCR reactions, and each consisted of 1.5 l of genomic DNA extract, 1.5 X PCR buffer (10 mM Tris-HCl, pH

8.3; 50 mM KCl), 3.75 mM MgCl2, 0.3175 mM each dNTP, 0.12-0.24 M of each primer (forward primer fluorescently labeled; Applied Biosystems, Foster City, CA), 0.06 units of Taq polymerase (Promega Corporation, Madison, WI), and deionized water added to achieve the final volume. The amplification cycle for all loci, except Spl105 which had a different annealing temperature, consisted of an initial denaturing at 94 °C for 2 min; 35 cycles of 94 °C denaturing for 45 sec, 56 °C annealing for 45 sec, 72 °C extension for 2 min; and a 30 min extension at 72 °C. Locus Spl105 had an annealing temperature of 50 °C. Genotypes were visualized using an ABI 3130 (Applied Biosystems, Foster City, CA). Genemapper (ver. 6) software from Applied Biosystems (Foster City, CA) was used to identify alleles at each of the 19 loci.

Species identification

Tranah et al. (2004) used nine microsatellite loci to discriminate pallid and shovelnose sturgeon in the upper Missouri River with an 82 to 95% probability of correct assignment. The 17 loci used in these analyses included the loci identified by Tranah et al. (2004) and

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additional loci that allowed the discrimination between pallid and shovelnose sturgeon with a high degree of confidence (DeHaan et al. 2005).

Genetic based species assignments and detection of hybridization were accomplished using the computer program NewHybrids (Anderson and Thompson 2002). This Bayesian- based method uses markers that differ in allele frequency but do not have fixed differences between known baseline groups, (such as with pallid and shovelnose sturgeon in the upper and middle Missouri River), and the model computes the posterior probability that each individual belongs to one of the two species or one of four classes of hybrids (F1’s, F2’s, and backcrosses). Individuals were classified as a pallid sturgeon if the probability of assignment to known pallid sturgeon was 95% or greater, based on methods developed by USFWS Abernathy Fish Technology Center and in conjunction with Dr. Ed Heist (Southern Illinois University). However, for individuals sampled in the lower portion of the basin (RPMA 3&4), if the probability of assignment is 90% or greater, origin determination is also conducted due to the slightly reduced ability to distinguish pallid and shovelnose sturgeon based on allele-frequency differences between both species.

Hatchery versus unknown origin identification

Following species identification, genetic parentage analysis was used to determine if a pallid sturgeon originated from the hatchery program or was naturally produced within the Missouri River. Multi-locus genotypes for almost every hatchery-spawned adult since 2000 have been obtained at 17 microsatellite loci (see objectives 1&2), and genotype data are stored in a Microsoft Access database developed and maintained by NEFC. A tagging database developed by USFWS Missouri River FWMAO maintains additional information such as spawning and stocking information, and this database is used to reference genetic parentage assignments to known spawning pairs and stocking locations.

Parentage assignments were conducted using Cervus (ver 3.0; Kalinowski et al. 2007). Genetic parentage assignments occur using modified exclusion based method allowing for a single mismatch in the offspring-parent-parent triplet. If a juvenile fish is not compatible at two or more loci with a particular hatchery-spawned pair, then that pair is excluded as potential parents of that juvenile sturgeon. DeHaan et al. (2005) determined that by using 17

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highly variable loci and allowing for a single mismatch to accommodate for genotype or lab errors, the probability of an incorrect match was very low. Parentage assignments were compared to the spawning database maintained by USFWS Missouri River Fish and Wildlife Conservation Office to confirm if a genetically assigned spawning pair represented a known spawning pair. If the juvenile fish was not assigned to any hatchery parents, then the individual was identified as unknown origin. Additionally, a small number of broodstock have not been genotyped and therefore are not available for parentage assignment, and some spawning records are incomplete for individual spawning pairs (one or both parents listed as “unknown” or “mixture”). Thus, if not assigned to a documented spawning pair, results cannot be confirmed if a fish is not of hatchery origin or wild simply because they are not assigned to hatchery parents. Therefore, individuals not assigned to hatchery families are identified as “unknown” origin.

Results

Objective 1 – Estimate the degree of relatedness among Missouri River broodstock

Due to the limited number of pallid sturgeon broodstock available during a spawning season, genetic information was used to help reduce the potential for inbreeding and to maintain genetic diversity during hatchery production. Relatedness estimates of adult pallid sturgeon broodstock are used to identify spawning pairs to avoid; such as crosses between full and half siblings, and to allow for the minimum degree of relatedness among families created.

Pairwise relatedness values (Rxy) are determined between all individuals using multi-locus genotypes for all pairs of broodstock using the computer program SPAGeDi (Hardy and Vekemans 2002), based on relatedness algorithm of Queller and Goodnight (1989).

Potential broodstock are genotyped at 17 microsatellite loci. For potential broodstock, the species (pallid, shovelnose, or hybrid) is determined, and for individuals identified as pallid sturgeon, origin (hatchery or unknown) was assessed. Mating plans are developed for broodstock of reproductive maturity at Garrison Dam National Fish Hatchery (GDNFH) and Miles City State Fish Hatchery (MCSFH). Quality control is also done with Dr. Ed Heist at Southern Illinois University on all broodstock being held at Gavins Point National Fish

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hatchery (GPNFH), Neosho National Fish Hatchery (NNFH), and Blind Pony State Fish Hatchery (BPSFH) to insure no hybrid individuals are included in the mating plan.

Cryopreserved milt from males previously captured for spawning is also considered in the spawning matrices. There are currently 114 males in the cryo repository. Fourteen of those males are captive offspring.

One broodstock report was provided during FY 2019 for GDNFH as part of the pallid sturgeon larval drift and dispersal study. Pallid sturgeon broodstock were captured in the upper Missouri River and transferred to Garrison Dam NFH (GDNFH) for spawning. The report made spawning recommendations for six reproductive pallid sturgeon females and three reproductive pallid sturgeon males at GDNFH (Kalie and Bartron 2019). All nine individuals were previously spawned or attempted to spawn in previous years. Also, a potential of 97 pallid sturgeon males that had been previously cryopreserved at Garrison Dam NFH were also included in the report. All potential broodstock, including the cryopreserved milt, were determined genetically to be pallid sturgeon (Table 1). Dr. Ed Heist at Southern Illinois University along with NEFC also provided spawning guidance to Gavins Point NFH for all potential captive offspring (67) being used in the larval drift study. Thirty three females and thirty four males were analyzed and compared between labs for the project.

Additionally, priority scores were also assessed to aid in field collection efforts of broodstock that had not previously been spawned or to avoid spawning individuals that had already contributed offspring to multiple spawning events. These priority scores were distributed during broodstock collection efforts, and incorporated updated retention targets of captive individuals maintained at Gavins Point National Fish Hatchery and updated spawning and capture records from Ryan Wilson (USFWS). Priority scores are updated yearly based on recapture rates and retention targets of captive broodstock determined by GPNFH.

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Table 1. Summary results of the 2019 broodstock analysis by hatchery. Species results included: PD (Pallid), SH (Shovelnose), HY (Hybrid), and origin results included Hatchery (HA) or unknown (UN).

Results-species ID Results-Origin Hatchery PD SH HY HA UN GDNFH 106*** 106 *** Includes 97 cryopreserved males

Objective 2 – Continue to add adult pallid sturgeon to the genetic baseline dataset

Broodstock collection efforts generally capture more adults than are eventually spawned because not all adults are reproductively viable in a given spawn year, reproductive viability can not necessarily be determined at time of collection, or adults were collected for broodstock use in future years. These additional adults collected from the Missouri River are also genotyped and characterized for species and origin determination. Because these additional adults are returned to the river, they do have the potential to reproduce in the wild in subsequent years. Inclusion of these adults into the baseline dataset is useful for future analysis of natural reproduction.

A total of 54 additional adults or broodstock were genotyped and included into the genetic baseline following species and origin determination (Table 2: Adults (AD) and Broodstock (BR)). Samples are considered adults if the fork length of the individual is 1000 mm or more (Ryan Wilson, USFWS, pers. comm.).

No individuals were sampled from RPMA 1, 12 individuals from RPMA 2, no individuals from RPMA 3, 30 individuals were sampled from RPMA 4, and 12 individuals from captive offspring (CO) being held at GPNFH used for the 2019 larval drift project. Species identification was first performed to determine if samples received were from pallid sturgeon, shovelnose sturgeon, or a hybrid between pallid and shovelnose. Of the 54 samples analyzed, 51 were identified to be pallid sturgeon, zero individuals were identified as shovelnose, and three samples were identified to be a hybrid. Of the 49 pallid sturgeon identified, 32 were of hatchery origin and 19 were of unknown origin (Table 2). All hybrid individuals were from RPMA 4.

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Table 2. Summary of the samples received during FY 2019 for genetic analysis, by RPMA and captive offspring (CO), life stage, species, and origin for the adult (AD) and broodstock (BR) samples. Species results include pallid (PA), shovelnose (SH), hybrid (HY), and unknown (UN). Origin was calculated for individuals identified as pallid sturgeon, and was either hatchery (HA) or unknown (UN).

Life stage Species Origin RPMA AD BR PA SH HY HA UN 1 0 0 0 0 0 0 0 2 10 2 12 0 0 8 4 3 0 0 0 0 0 0 0 4 4 26 27 0 3 12 15 CO 0 12 12 0 0 12 0 Total 14 40 51 0 3 32 19

Objective 3 – Conduct genetic parentage analysis for unknown juvenile pallid sturgeon to determine hatchery vs. natural origin

A total of 290 putative pallid sturgeon samples were provided from larvae, young-of- year, or juveniles for species and origin determination (Table 3). This does not include Project 3.7 samples or captive offspring. Zero individuals of this size class were sampled from RPMA 1, 41 individuals were sampled from RPMA 2, 40 individuals were sampled from RPMA 3, and 209 samples were from RPMA 4. Genetic species identification was first performed to determine if samples received were from pallid sturgeon, shovelnose sturgeon, or a hybrid between pallid and shovelnose. Parentage analysis was also determined for all pallid sturgeon. Of the 290 samples provided, a total of 254 pallid sturgeon were identified, eight were determined to be a shovelnose sturgeon, 26 were determined to be hybrids, and two individuals were triploid. All shovelnose, hybrid, and triploid individuals were from RPMA 4. Of the 254 samples identified to be pallid sturgeon, 241 were of hatchery origin and 13 were of unknown origin. Results from all samples received were provided to the biologist(s) who collected and sent the sample.

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Table 3. Summary of the samples received during FY 2019 for genetic analysis, by RPMA, life stage, species, and origin for the larvae (Lar), young-of-year (YOY), and juvenile (JU) samples. Species results include pallid (PA), shovelnose (SH), hybrid (HY), and unknown (UN). Origin was calculated for individuals identified as pallid sturgeon, and was either hatchery (HA) or unknown (UN).

Life stage Species Origin RPMA Lar YOY JU PA SH HY Triploid/No Amp. HA UN 1 0 0 0 0 0 0 0 0 0 2 0 0 41 41 0 0 0 41 0 3 0 0 40 40 0 0 0 40 0 4 0 0 209 173 8 26 2 160 13 Total 0 0 290 254 8 26 2 241 13

In continuing with acquiring genetic information for the complete inventory of captive offspring at GPNFH, 731 pallid sturgeon were also analyzed in 2019 (Table 4). Sixty five different hatchery crosses were confirmed from four different year classes. Nine individuals (1.2 %) were triploid and could not be analyzed. The triploid samples were from the 2005, 2008, and 2009 year classes.

Table 4. Summary of captive offspring samples received during FY 2019 for genetic analysis by GPNFH by year class, number of individuals per year class, confirmed hatchery crosses, and triploid individuals.

Year Class # Individuals # Confirmed hatchery crosses # Triploid individuals 2005 131 127 4 2007 190 190 0 2008 194 190 4 2009 216 215 1

Genetic analysis of unmarked pallid sturgeon in RPMA 1 and 2 (Project 3.7) was also conducted. One hundred and seventy two individuals were analyzed for species identification and hatchery vs unknown origin identification (Table 5). Eighty four samples were collected in RPMA 1, and 88 samples were collected in RPMA 2. All 172 samples were determined to be pallid sturgeon and hatchery origin. There were 78 different families and 17 different year

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classes represented from the 172 hatchery origin individuals collected in RPMA 1 and 2 in 2019 (Table 6).

Table 5. Summary of the Project 3.7 samples received during FY 2019 for genetic analysis, by RPMA, life stage, species, and origin for juvenile (JU) samples. Species results include pallid (PA), shovelnose (SH), hybrid (HY). Origin was calculated for individuals identified as pallid sturgeon, and was either hatchery (HA) or unknown (UN). Life stage Species ____ Origin RPMA JU AD PA SH HY Triploid HA UN 1 60 24 84 0 0 0 84 0 2 88 0 88 0 0 0 88 0 ___ Total 148 24 172 0 0 0 172 0

Editor’s note: Table 6. 2019 Project 3.7 samples broke down by RPMA, family female, family male, year class, and number of individuals per year class has not been included in this report for brevity. Please contact Jeff for more detailed information.

In 2019, standardization and updating of baselines for the Central Lowlands Management Unit and Interior Highlands Management Unit continued in collaboration with Dr. Ed Heist from Southern Illinois University. Genotypes were compared as a quality control measure for all individuals added to these baselines.

Also in 2019, a phased transition from 125 kHz tags to 134.2 kHz tags began March 1. All crews are supposed to have dual-frequency readers to have the capability to read the old 125 and new 134.2 kHz PIT tags. Twenty two samples collected in 2019 from RPMA 1-4 were repeat recaptures with new PIT tags. This high occurrence of repeat recaptures might indicate the new PIT Tag readers aren’t working properly to read the existing 125 kHz tags already in the pallid sturgeon.

References

Anderson, E.C. and E. A. Thompson. 2002. A Model-Based method for identifying species hybrids using

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multilocus genetic data. Genetics 160: 1217–1229.

Banks, M.A. and Eichert, W. 2000. WHICHRUN (version 3.2): A computer program for population assignment of individuals based on multilocus genotype data. Journal of Heredity 91:87-89.

DeHaan P.W., D.E. Campton, and W.R. Ardren. 2005. Genotypic analysis and parental identification of hatchery-origin pallid sturgeon in the Upper Missouri River: Phase I Inheritance of Microsatellite, Nuclear DNA Markers. June 23rd, 2005. 35pp. USFWS Abernathy Fish Technology Center Final Report.

DeHaan P.W., G.R. Jordan, and W.R. Ardren. 2008. Use of genetic tags to identify captive-bred pallid sturgeon (Scaphirhynchus albus) in the wild: improving abundance estimates for an endangered species. Conservation Genetics 9: 691-697.

Goodknight K.F. and Queller D.C. 1999. Computer software for performing likelihood tests of pedigree relationship using genetic markers. Molecular Ecology 8:1231-1234.

Kalie, J. and M. L. Bartron. 2019. 2019 Mating Plan for Upper Missouri River Pallid Sturgeon Larval Drift and Dispersal Stujdy: Garrison Dam National Fish Hatchery. June 25, 2019, provided to Garrison Dam National Fish Hatchery

McQuown, E.C., Sloss, B.L., Sheehan, R.J., Rodzen, J. Tranah, G.J. and May, B. 2000. Microsatellite analysis of genetic variation in sturgeon: new primer sequences for scaphirhynchus and acipenser. Transactions of the American Fisheries Society 129:130-1388.

Queller D.C. and Goodnight K.F. 1989. Estimating relatedness using genetic markers. Evolution 43:258–275.

Snyder, D.E. 2002. Pallid and shovelnose sturgeon larvae – morphological description and identification. Journal of Applied Ichthyology 18:240-265.

Tranah, G., D. E. Campton, and B. May. 2004. Genetic evidence of hybridization of pallid and shovelnose sturgeon. Journal of Heredity 95:474-480.

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Determination of Reproductive Indices in Hatchery-origin Pallid Sturgeon in the Missouri and Yellowstone Rivers

2019 Annual Report

Molly A.H. Webb

USFWS, Bozeman Fish Technology Center

Hilary B. Treanor

Sandhill Crane Consulting

Executive Summary: This project will determine the age at onset of puberty and first maturity and spawning periodicity of hatchery-origin (HO) pallid sturgeon in the Missouri and Yellowstone rivers using known-aged juvenile pallid sturgeon. Millions of dollars and considerable effort for endangered pallid sturgeon in the upper basin of the Missouri River has been placed in the conservation propagation program to prevent extirpation of the species. Hundreds of thousands of pallid sturgeon young-of-year have been released into the upper Missouri River since 1998. Those fish are now reaching sexual maturity as determined by circulating sex steroid concentrations and monitoring efforts (Holmquist et al. 2019a and 2019b). As the heritage fish age and die, we will be reliant on the HO pallid sturgeon to maintain the species. Determining whether the HO pallid sturgeon are capable of reestablishment through natural spawning is vital to the recovery program.

As well, understanding the reproductive dynamics of the HO pallid sturgeon is important for successful management, and incorporating sex and stage of maturity information into research and monitoring programs is valuable as migratory behavior, habitat use, and sampling gear bias have been found to differ between sexes or among different classes of sex and stage of maturity in some sturgeon species (McKinley et al. 1998; Erickson and Webb 2007; Shaw et al 2012; Shaw et al. 2013; Richards et al. 2014; Holmquist et al. 2019b). To describe the reproductive dynamics of a population, there are several reproductive indices that may be used, including but not limited to reproductive structure (the proportion of the population in each stage of the reproductive cycle), age and size at puberty or first spawning, sex ratio, fecundity, and spawning periodicity. These reproductive indices are not yet well known for the HO pallid sturgeon.

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Since 2009, 1,271 plasma samples from HO pallid sturgeon (this represents fish that have been resampled over time) captured in the Missouri and Yellowstone rivers have been analyzed to determine age-at-maturity. The 1997 and older year classes provide an opportunity to determine age and size at onset of puberty and spawning periodicity in pallid sturgeon as these fish are now over 20 years old.

The pattern of steroid production in sturgeons allows for discrimination of sex and stage of maturity less-invasively with blood collection (Webb and Doroshov, 2011; Webb et al. 2018). The objective of this project is to analyze blood plasma sex steroid concentrations in HO pallid sturgeon in the upper Missouri and Yellowstone rivers to assign sex and stage of maturity and determine the age and size at sexual differentiation and first maturity as well as spawning periodicity.

Project Status/Anticipated/Expected Date of Completion: This project was completed in March 2020. Analysis of 1,271 HO pallid sturgeon plasma samples is reported here.

Accomplishments/Recommendations/Results:

Methods Blood plasma samples from HO pallid sturgeon that have been analyzed to date for testosterone (T) and estradiol-17β (E2) concentrations were collected from: RPMA 1 by Montana Fish Wildlife and Parks (MTFWP) personnel in the Spring and Fall of 2011, 2012, 2013, 2014, 2015, 2016, 2017, 2018, and 2019; RPMA 2 on the Missouri River by USFWS personnel in Spring of 2010, 2011, 2015, 2016, 2017, 2018, and 2019 and Fall 2011, 2012, 2014, 2015, 2016, 2017, 2018, and 2019; RPMA 2 on the Missouri River by MTFWP in Spring and Fall of 2012, 2014, 2015, 2016, 2017, 2018, and 2019; RPMA 2 on the Yellowstone by MTFWP in Spring 2014, 2015, 2016, 2017, 2018, and 2019 and Fall 2011, 2013, 2014, 2015, 2016, 2017, 2018, and 2019; RPMA 3 by USFWS personnel in Spring 2012, 2013, 2014, 2016, 2017, 2018, and 2019. Capture location on the Missouri River varied between river mile (RM) 1880 and 2050 for RPMA 1, between RM 1533 and 1762 in RPMA 2, and between RM 828 and 863 in RPMA 3. Capture location on the Yellowstone River varied between RM 5 and 70.

Blood samples were collected from the caudal vasculature, centrifuged to separate plasma, frozen, and sent to the Bozeman Fish Technology Center (BFTC) for analysis of plasma T and E2 concentrations. Blood plasma steroid concentrations were measured by radioimmunoassay

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following the methods described in Fitzpatrick et al. (1986) and modified by Feist et al. (1990).

Sex and stage of maturity was assigned to fish based on the following steroid concentrations:

T<10 ng/ml and E2=Non-detect (ND) is unknown at this time or not assigned

T>10 ng/ml and E2=ND is a potential male but not assigned at this time

T>38 ng/ml and E2=ND is a reproductive male

T>8 ng/ml and E2>0.30 ng/ml is a reproductive female.

The concentrations used to assign sex and stage of maturity are shifting as we learn more from recaptures every year. This will increase our accuracy in using plasma sex steroids to assign sex and stage of maturity non-invasively and will allow us to eventually determine the most accurate concentrations of T that may be used to differentiate non-reproductive females and non-reproductive males. Because of the low accuracy in assigning sex to individuals that have not reached puberty, these fish were classified as non-reproductive, and sex was not assigned.

Results and Discussion

Blood plasma T and E2 concentrations have been analyzed in a total of 1,271 samples to date. The youngest year class to be sampled was 2011 (age 8 at time of capture) and the oldest was 1997 (age 22 at time of capture). Sixty-two of the 111 HO individuals captured in 2019 (55.8%) had been previously caught in years past. Nine of the 111 HO individuals caught in 2019 (8.1%) were assigned as reproductive based on plasma sex steroids, and six of these 9 individuals had been previously captured (Table 1).

In the 2019 dataset, six reproductive HO males were identified. These males were from the 1997 and 2009-year classes. Of the six males, four were captured in RPMA 1, and two were captured in RPMA 2. Fish ages were 22 years for the 1997-year class and 10 years for the 2009-year class. Body size ranged from 1018-1236 cm fork length and 4.33-8.98 kg body weight. Three of the six males had been previously captured at least once. Individual 5870 was captured in 2015, 2016 and 2019. This male was assigned as a ripe male each time using plasma sex steroids. Individual 2A09 was captured in 2011 and 2012 and was assigned as non-reproductive. This fish was captured in 2014, 2015, 2016, and 2019 and was assigned as

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reproductive using plasma sex steroids each time. Individual 644D was captured twice and was non-reproductive in 2016 and reproductive in 2018.

Two HO ripe females were identified in 2019. Two females were captured in the spring and were from the 1997-year class (22 years old at capture). Individual 5A67 was previously captured in 2016 and was assigned non-reproductive by plasma sex steroids. She was 1233 cm FL and 8.76 kg in the spring of 2019 and was assigned as reproductive. Individual 0E09 was previously captured in 2015 and was classified as ripe. She was recapture in March 2016 and subsequently in June 2016. Her circulating plasma E2 was not elevated in March but was elevated in June indicating that she was initiating vitellogenesis following a spawn in 2015. She was 1149 cm FL and 8.14 kg in the spring of 2019 and was assigned as reproductive indicating that she may have a biennial reproductive cycle.

The youngest HO males to show signs of reproductive maturity have been documented at the age of 10 as seen by plasma T concentrations above 38 ng/ml. All other mature HO males have been 13 years or older.

Using data from Holmquist et al. (2019a) and this study, we have been able to determine the spawning periodicity in 8 HO males. Six males have had an annual reproductive cycle, and two males have had binennial cycles. More information needs to be collected over time to determine spawning periodicity in females, but preliminary data suggests that a biennial cycle in HO females does exist (Cox, Guy, and Webb, unpublished; this study).

Dummy runs, in which fish produce ovarian follicles but do not ovulate and oviposit during their first cycle, have been described in other long-lived species such as rockfish and cod. Mass follicular atresia is the result. The dummy run may 'condition' gonads and other organs through the elevation of circulating hormones, which would, upon subsequent hormone exposure, decrease the cellular response time, rate of target receptor upregulation and binding protein synthesis. This process is similar to the immune response, where physiological “learning” primes the system for a subsequent response following first exposure to the physiological event. Many of the HO ripe females followed in this study to date have undergone follicular atresia during their first known reproductive cycle indicating a potential of a dummy run in pallid sturgeon females (Holmquist 2019a; Cox, Guy, Webb, unpublished). Several of the HO females that underwent follicular atresia during their first reproductive cycle have successfully ovulated during their second reproductive cycle (Tanner, Guy, Webb,

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unpublished). There is great value in following the reproductive fate of HO females in their first and subsequent cycles to determine if follicular atresia may be attributed to a dummy run or lack of environmental conditions suitable for spawning.

References

Erickson, D.L. and M.A.H. Webb. 2007. Spawning periodicity, spawning migration, and size at maturity of green Sturgeon, Acipenser medirostris, in the Rogue River, Oregon. Environmental Biology of Fishes 79:255-268.

Feist, G., C.B. Schreck, M.S. Fitzpatrick & J.M. Redding. 1990. Sex steroid profiles of coho salmon (Oncorhynchus kisutch) during early development and sexual differentiation. General and Comparative Endocrinology 80:299 - 313.

Feist, G., J.P. Van Eenennaam, S.I. Doroshov, C.B. Schreck, R.P. Schneider, & M.S. Fitzpatrick. 2004. Early identification of sex in cultured white sturgeon, Acipenser transmontanus, using plasma steroid levels. Aquaculture 232:58 -590.

Fitzpatrick, M.S., G. Van Der Kraak & C.B. Schreck. 1986. Profiles of plasma sex steroids and gonadotropin in coho salmon, Oncorhynchus kisutch, during final ovarian maturation. General and Comparative Endocrinology 62:437-451.

Holmquist, L.M., Guy, C.S., Webb, M.A.H., and A. Tews. 2019a. First maturity and spawning periodicity of hatchery-origin pallid sturgeon in the upper Missouri River above Fort Peck Reservoir, Montana. Journal of Applied Ichthyology 35:138-148.

Holmquist, L.M., Guy, C.S., Tews, A., Trimpe, D.J., and M.A.H. Webb. 2019b. Reproductive ecology and movement of pallid sturgeon in the upper Missouri River, Montana. Journal of Applied Ichthyology 35:1069-1083.

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McKinley, S., Van Der Kraak, G., and G. Power. 1998. Seasonal migrations and reproductive patterns in the lake sturgeon, Acipenser fulvescens, in the vicinity of hydroelectric stations in northern Ontario. Environmental Biology of Fishes 51:245-256.

Richards, R. R., Guy, C. S., Webb. M. A., Gardner, and Jensen, C. B. 2014. Spawning related movement of shovelnose sturgeon in the Missouri River above Fort Peck Reservior, Montana. Journal of Applied Ichthyology 30:1-13.

Shaw, S. L., Chipps, S. R., Windels, S. K., Webb, M. A. H., McLeod, D. T., and Willis, D. W. 2012. Lake sturgeon population attributes and reproductive structure in the Namakan Reservoir, Minnesota and Ontario. Journal of Applied Ichthyology 28:168-175.

Shaw, S. L., Chipps, S. R., Windels, S. K., Webb, M. A. H., and McLeod, D. T. 2013. Influence of Sex and Reproductive Status on Seasonal Movement of Lake Sturgeon in Namakan Reservoir, Minnesota-Ontario. Transactions of the American Fisheries Society 142:10-20.

Webb, M.A.H. & S.I. Doroshov. 2011. Importance of environmental endocrinology in fisheries management and aquaculture of sturgeons. General and Comparative Endocrinology 170:313-321.

Webb, M.A.H, Van Eenennaam, J.P., Crossman, J.A., & Chapman, F.A. 2018. A practical guide for assigning sex and stage of maturity in sturgeons and paddlefish. Journal of Applied Ichthyology 35:169-186.

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Table 1. Reproductively active Pallid Sturgeon that were captured in 2019 that had been captured in previous years. Sex and stage of maturity was assigned by plasma sex steroids. Fish are identified by the last four digits of their PIT tag. NA=not assinged, NR=not reproductive, R=reproductive, ND=non-detectable steroid concentration.

PIT Tag Dates Management T E2 Sex Stage Number Caught Area (ng/mL) (ng/mL) Prediction Prediction

Males

2A09 9/19/2011 1 25.92 ND NA NR

10/9/2012 1 2.64 ND NA NR

6/16/2014 1 82.71 ND M R

6/24/2015 1 39.1 ND M R

4/25/2016 1 105.05 ND M R

5/24/2019 1 112.14 ND M R

5870 9/2/2015 1 220.38 0.18 M R

4/26/2016 1 121.28 ND M R

5/10/2019 1 93.35 ND M R

644D 8/25/2016 1 6.55 ND NA NR

5/11/2019 1 103.55 ND M R

Females

0E09 5/19/2015 1 62.18 0.67 F R

3/16/2016 1 29.73 ND NA NR

6/27/2016 1 25.45 1.51 F R

4/23/2019 1 33.71 4.32 F R

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7/9/2019 1 0.2 ND NA NR

5A67 8/29/2016 1 8.65 0.25 NA NR

5/6/2019 1 65.05 0.43 F R Pallid Sturgeon Propagation at Gavins Point NFH

2019

Introduction

In 1993 The Pallid Sturgeon Recovery Plan provided direction for propagation, research needs, collection of wild fish and reintroduction of pallid sturgeon to augment recovery efforts. Production of wild and captive held pallid sturgeon was successful in 2019 producing 1,197,179 eggs from 33 captive females and 6 wild brood fish. A total of 8 wild adults of middle basin origin were transferred to Gavins Point NFH from October to April 2018, with the help of multiple agencies.

Captive Brood Stock Production

Gavins Point NFH is currently rearing 1,976 captive pallid sturgeon brood fish that represent 20 year classes as a backup genetic pool for use in supplementing wild populations in the upper basin and research needs. These 1,976 progeny held in refuge represent 148 wild individuals from upper basin spawns. These 148 individuals represent 157 unique family crosses.

Captive Brood Stock Spawning

In 2019 there were 4 spawning events involving 33 females and 28 males. Four different studies were conducted using the eggs from these fish. The first spawning event took place on May 15th. For this spawn 2 females and 3 males were utilized. 29,400 fish from one cross were transferred to the Bozeman Fish Tech Center for a diet trial. The second round of spawning produced 223,400 eggs utilized 6 females and 12 males. Of the 12 males spawned 8 of these fish were cryopreserved. The eggs from this study helped fill research needs dealing with fin curl at Miles City State Fish Hatchery and Neosho National Fish Hatchery. In mid- May Pat Braaten with USGS in Montana asked if the hatchery could produce captive pallid eggs for a larval drift study taking place in the upper basin. There were two different spawning events that took place in late June utilizing 24 females and an additional 23 males. From these two spawns a total of 837,714 eggs were produced and shipped to Rob Holm at Garrison Dam National Fish Hatchery.

Middle Basin Spawning

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Gavins Point NFH currently serves as the only fish hatchery that’s currently producing pallid sturgeon for recovery in the middle and lower Missouri River. As such the hatchery has adopted a hold over program to successfully generate reproductive cycling of brood fish. Through this process our goal is to increase the effective population size of stocked fish in the Missouri River. The middle basin has developed a 1x2 spawning regime thus males are represented at 280 fish/individual and females at 560 fish/individual.

Several spawning events occurred at Gavins Point National Fish Hatchery in 2019. There were a total of 6 females and 5 males spawned. On April 16th one female with 2 cryopreserved males. On April 22nd an additional 2 females was spawned with four males housed at the hatchery. On May 2nd the third spawn took place with 1 female and 2 cryopreserved males. The last and final spawn of the year took place on May 12th with 1 female being paired with 2 cryopreserved males. In all there was a total of 106,667 middle basin eggs taken resulting in the survival of 9 family cross’s that will be stocked in the spring and summer of 2020. There were 5 wild males spawned and all were added to the cryo repository.

Captive Brood Sampling

For many years there has been a growing problem with the number of fish being retained and the amount of space in the brood stock building. Thus we have started phase 1 of the captive reduction plan which entails us to genotype all the fish of upper basin origin at the hatchery. The last year over 1000 fin clips were taken to access parentage of captive brood fish and a new database was created using Microsoft access. Ultrasound images were taken on all fish as well. The captive reduction plan is now in place and phase 1 will be starting in early 2020.

Findings

Pond water was brought in and put into the new re-use systems. Systems were filled with Missouri River water from pond 4. This was one of the first ponds filled and as a result was thought to have the most stable water quality parameters. This was however not the case as it was found to be high in un-ionized ammonia levels. Fry that were hatched on the water experienced lethargy which was later correlated to the high levels of un-ionized ammonia. With the first two spawns of the year being so close in timing adjustments weren’t made to the second system filled and fish exhibited the same type of behavior. However, fish were moved at varying times to heated lake water and some of the effects were reversed. Egg numbers were also lower than expected and diet changes have been made to the brood fish to ensure adequate forage.

2019 Research

Early life stage mortality evaluations, collaborated with the USGS on water chemistry parameters. This study was a continuum of a 2018 early life stage mortality study. Comparing

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survival on lake water vs pond water. SPMD test (Pesticides, Insecticides, Industrial waste, fossil fuels, etc.) were utilized and no noticeable distinctions between water sources were observed. Due to un-ionized ammonia (flooding effect) the study was cancelled.

Middle Basin Spawning Events

April 16th Spawning Event 4627463568 was spawned on 4.16.19 with a P.I of .0910 on 4.8.19. Fish ovulated slow releasing 2 eggs at 48hrs, 5 eggs at 50hrs and then 50mls at 52hrs. She gave 15,300 eggs Take Female Male Time mls of eggs Nueralization 4627463568 434C1B2F2E 2:00 50 55.7 4627463568 413C485208 4:00 80 39 4627463568 434C1B2F2E 6:00 75 44.5 4627463568 413C485208 8:00 75 58.9 4627463568 434C1B2F2E 10:00 20 55.7

Fry hatched 4627463568 x 434C1B2F2E = 3691 4627463568 x 434C458208 = 3844

Observations Fish were lethargic in the tanks. Slow mortality throughout first month. All surviving fish from the first spawned were euthanized at 120 days old as they experienced high un-ionized ammonia levels which created abnormalities within the fish.

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April 23rd Spawning Event 6C000112047 was spawned on 4.23.2019 with a P.I of .1067 on 4.8.19. Fish began ovulating after 43 hours. She gave 23,290 eggs Nueralization Female Male Take Time mls of eggs (%) 6C00112047 47032D1576 6:20 55 90 6C00112047 470A676363 8:00 100 92 6C00112047 47032D1576 10:00 85 90 6C00112047 470A676363 12:00 100 88 6C00112047 47032D1576 2:00 90 95 6C00112047 470A676363 4:00 30 91

Fry hatched 6C00112047 X 47032D1576 = 5280 6C00112047 X 470A676363 = 5262

Remaining fish to be stocked: 6C00112047 X 47032D1576 = 280 6C00112047 X 470A676363 = 280

Observations Eggs from 6C00112047 were split between two different systems. One system utilized UV pond water where the other system utilized pond water without the UV. All of the eggs on the pond water without UV started hatching on day 10. Pond water was then tested and found to be high in un-ionized ammonia.

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April 23rd Spawning Event 46277B6F29 was spawned on 4.23.2019 with a P.I of .1079 on 4.8.19. The Fish began ovulated 43 hours after the priming dose. She gave 22,431 eggs nueralization Female Male Take Time mls of eggs (%) 46277B6F29 46273E0255 6:20 4 85 46277B6F29 46273E0255 8:00 85 88 46277B6F29 4627156C74 10:00 118 90 46277B6F29 46273E0255 12:00 115 92 46277B6F29 4627156C74 2:00 100 96 46277B6F29 46273E0255 4:00 55 95

Fry Hatched 46277B6F29 x 46273E0255 = 6724 46277B6F29 x 4627156C74 = 3794

Remaining fish to be stocked: 46277B6F29 x 46273E0255 = 280 46277B6F29 x 4627156C74 = 200

Observations Eggs from 46277B6F29 were split between three different systems. One system utilized UV pond water, another system utilized pond water without the UV and the last system utilized heated lake water. All of the eggs on the non UV Pond water system never hatched. Pond water was then tested and found to be high in un-ionized ammonia which affected the survival of the fry. The family that utilized heated lake water had the best survival.

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April 26th Spawning Event 4704214268 was spawned on 5.02.2019 with a P.I of .1120 on 4.8.2019. The fish began to ovulate 42 hours after the priming dose. She gave 16,750 eggs Female Male Take Time mls of eggs Nueralization 4704214268 116216091A 4:00 30 58.3 4704214268 116216091A 6:00 85 58.3 4704214268 4716365143 8:00 60 31.2 4704214268 4716365143 10:00 45 31.2 4704214268 4716365143 12:00 80 29.5 4704214268 116216091A 2:00 35 25.6

Fry hatched 4704214268 x 116216091A =3968 4704214268 x 4716365143 =2818

Remaining fish to be stocked 4704214268 x 116216091A = 140 4704214268 x 4716365143 = 280

Observations Eggs form Female 4704214268 were subpar. As water sources were still being tested it was decided to hatch eggs on heated well and pond water with UV disinfection.

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May 2nd Spawning Event 471A2F6D1B was spawned on 5.02.2019 with a P.I of .1200 on 4.08.19. The fish ovulated slowly and never really gave a substantial amount of eggs. She gave 7,600 eggs Nueralization Female Male Take Time mls of eggs (%) 471A2F6D1B 412C2A286F 8:00 50eggs <1 471A2F6D1B 412C2A286F 10:00 250eggs <1 471A2F6D1B 48685E0F60 12:00 60 5 471A2F6D1B 48685E0F60 2:00 45 2 471A2F6D1B 412C2A286F 4:00 35 <1 471A2F6D1B 412C2A286F 6:00 12 <1

Fry hatched 471A2F6D1B x 48685E0F60 = 512 471A2F6D1B x 412C2A286F = 53

Remaining fish to be stocked 471A2F6D1B x 48685E0F60 = 19 Observations Female 471A2F6D1B spawned very poorly. This fish had trouble ovulating and produced very few high quality eggs. Her eggs were split among heated well water and pond water. In both cases hatched fry did very poorly and resulted in only 50 fry alive after the first two weeks.

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May 13th Spawning Event Female 4627D2971 was spawned on 05.12.2019 with a P.I of .1413 on 04.08.19 and .1088 on 4.29.19. The fish ovulated after 40 hours. She gave 21,385 eggs Nueralization Female Male Take Time mls of eggs (%) 4627D2971 4348434520 10:00 80 66.00% 4627D2971 48685D1608 12:00 110 79.00% 4627D2971 4348434520 2:00 130 81.00% 4627D2971 48685D1608 4:00 95 69.00% 4627D2971 48685D1608 6:00 40 74.00%

Fry hatched 4627D2971 x 4348434520 = 9870 4627D2971 x 48685D1608 = 11515

Remaining fish to be stocked 4627D2971 x 4348434520 = 280 4627D2971 x 48685D1608 = 280

Observations Eggs were split among three different water sources to test survival on all three water sources. Heated well had the lowest survival, heated lake and pond water had very similar survival among fry.

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172

Middle Basin Brood Fish housed at the hatchery during 2019

Date PITTAG Arrival Arrival Weight Weight Origin Sex Boat Ramp Length(mm) Weight(g) (May) Dec

4/11/2015 46280F5C1C 954 2708 5180 - Wild Female BARTLETT

10/22/2015 46267D2971 860 1686 3220 - Wild Female MONDAWIN

10/6/2016 4715591B05 960 3242 4800 - Wild Male REMINGTON

10/11/2016 47032D1576 1000 3872 5120 - Wild Male BROWNVILLE

10/13/2016 471A2F6D1B 975 3230 3740 - Wild Female BROWNVILLE

10/18/2016 46273E0255 975 3160 5300 - Wild Male THURNAU

11/17/2016 46277B6F29 900 2444 3720 - Wild Female INDIAN CAVE

3/30/2017 4704214268 820 2100 3000 - Wild Female FRENCH BOTTOMS

4/8/2017 4627463568 895 2630 3100 - Wild Female NODAWAY ISLAND

4/11/2017 4627156C74 940 2768 3500 - Wild Male BARTLETT

4/20/2017 470A676363 890 2220 3260 - Wild Male ST.HELENA

10/25/2018 6C00112047 801 1896 1800 - Wild Female PLATTSMOUTH

4/16/2019 46270E6D7B 924 3436 3360 - Wild Female BLAIR

4/11/2016 4626564564 975 3254 4700 5300 Wild Female PLATTSMOUTH

10/11/2016 47160E450B 1053 4156 6040 6600 Wild Female BROWNVILLE

4/4/2017 6C00112742 1052 4540 6200 7300 Wild Female JENTELL BREES

4/6/2017 4627017870 1035 4055 6220 6960 Wild Female LOUSVILLE

4/8/2017 462764734F 850 2190 2400 2640 Wild Female NODAWAY ISLAND

10/18/2017 4713167929 961 3220 3700 4260 Wild Female MONDAMIN

4/18/2019 48685B0B62 1096 5106 4740 5540 Wild Male BLAIR

4/2/2019 4704713261 1056 5138 5200 7660 Hatchery Male SIOUX CITY

4/3/2019 4348465D00 923 3456 3780 5280 Hatchery Male S. SIOUX CITY

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4/3/2019 47155A172A 800 2198 2420 4260 Hatchery Female S. SIOUX CITY

4/3/2019 4627753443 900 2806 3240 4160 Hatchery Female S. SIOUX CITY

4/9/2019 4348472475 902 3370 3620 4340 Hatchery Female BLENCOE

11/15/2019 470B111065 917 3174 - 3320 Wild Male Nebraska City

10/18/2017 4715674971 852 2450 2820 3460 Wild Female MONDAMIN

3/27/2018 4368022F2E 874 2260 3200 4320 Wild Female LANGDON

3/28/2019 4717595D25 980 3668 3800 3880 Wild Female Decatar

4/14/2017 412C37665D 954 3170 4920 5240 Wild Male PLATTSMOUTH

3/29/2018 434A4C0328 901 2497 3300 3900 Wild Male DAKOTA CITY

10/22/2018 470C244359 915 2935 3300 3800 Wild Male MYRON GROVE

Female Spawning Summary Captive Brood 2019 Spawn PI Female Spawning Fish Total Yearclass Female Date Score Weight Temp Conditon eggs 2002 4628143464 5/18/20191 .09773(5.18) 4.9 57.1 Good 29400 2004 46276C5969 5/18/20191 .0961(5.18) 4.02 57.1 Good Few eggs 1997 424F2A5B2C 6/9/20192 .0782(6.3.19) 4.5 61.2 Good 56875 1997 1F4B211065 6/9/20192 .0841(6.3.19) 9.74 61.2 Good 51240 1997 1F4A132460 6/9/20192 .08014(6.3.19) 11.4 61.2 Good 75635 2002 4256237F3E 6/9/20192 .0866(6.3.19) 8.4 61.2 Good 29400 2001 425536071B 6/9/20192 .0843(6.3.19) 6 61.2 Good 39600 2003 4442050D5A 6/9/20192 .0842(6.3.19) 5.08 61.2 Good 18720 1997 1F4A125B2A 6/20/2019 .0978(6.11.19 4.54 61 Good 30525 1997 462546534B 6/20/2019 .079(6.11.19) 4.32 61 Good 33060 2004 4627716D2D 6/20/2019 .0929(6.11.19) 4.24 61 Good 27178 2004 46257D6F35 6/20/2019 .09879(6.11.19) 4.45 61 Good 33904 2004 4627787556 6/20/2019 .0935(6.11) 5.34 61 Good 44440 2003 6C00096611 6/20/2019 .1018(6.11) 4.2 61 Good 36750 2003 6C00093506 6/20/2019 .0877(6.11) 3.25 61 Good 23478

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2003 444175202F 6/20/2019 .0874(6.11) 7.98 61 Good 49062 2004 6C00090924 6/20/2019 .0884(6.11) 5.56 61 Good 39312 2002 4256437C5F 6/20/2019 .0919(6.3) 14.58 61 Good 46830 2003 4624380D66 6/20/2019 .0839(6.3) 4.9 61 Good 33488 1997 42570A387A 6/20/2019 .0947(6.3) 4.2 61 Good 28841 2003 4624276027 6/20/2019 .0919(6.3) 2.6 61 Good 22464 2004 46275A3252 6/20/2019 .081(6.11) 4.2 61 Good 28350 2004 4627632E48 6/20/2019 .079(6.11) 5.85 61 Good 36225 2004 462838516B 6/20/2019 .0933(6.11) 4.2 61 Good few eggs 2003 46242E2A6F 6/25/2019 .1076(6.11) 5.9 61 Good 50600 2003 444267561D 6/25/2019 .0953(6.11) 6.34 61 Good 40356 2003 4624485842 6/25/2019 .10(6.11) 4.2 61 Good 44872 2004 46254D0546 6/25/2019 .1026(6.11) 4.94 61 Good 16080 2004 46237D0338 6/25/2019 .1011(6.11) 3.4 61 Good 27060 2004 46255B170B 6/25/2019 .0967(6.11) 3.3 61 Good 31521 2004 6C00090926 6/25/2019 .1038(6.11) 3.66 61 Good 29607 2001 425708072B 6/25/2019 .1139(6.11) 5.5 61 Good 46655 2004 46254E3E19 6/25/2019 .1041(6.11) 4.8 61 Good 21948 2003 44433E701A 6/25/2019 .0953(6.11) 5.7 61 Good 31590 2004 4628391106 6/25/2019 .112(6.11) 4.74 61 Good 30282 2004 4624127119 6/25/2019 .1054(6.11) 5.1 61 Good 33891 1Spawning event that occurred on May 15th for Bozeman Fish Tech Center-29,400 eggs transferred. 2Spawning event occurring on June 6th for Blind Pony and Neosho fin curl issues-223,400 eggs transferred.

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Male Spawning Summary Captive Brood 2019 Amount Number Number Male

Spawn Motility Collected of Times of Times Weight

Yearclass Male Date Score (mls) Collected Produced (kg) 2005 461E0E543C 5.17.2019 50-80 60 2 2 2.46

2005 4626386C48 5.17.2019 75-80 50 2 2 4.3

2005 No Tag 5.17.2019 0 25 1 0 5.3

2006 4718627A31 6.10.2019 50 55 1 1 4.8 2005 4626214B69 6.10.2019 90 55 2 2 3.4 2005 46255F612D 6.10.2019 60-80 95 2 2 3.1 2004 6C00096308 6.10.2019 0 0 2 0 3.5 2004 4627611E52 6.10.2019 60-80 100 2 2 3.7 2007 4A5B143666 6.10.2019 0 0 1 0 4 2007 47186E347B 6.10.2019 70 40 1 1 3.5 2004 4625766179 6.10.2019 110 60-80 2 2 3.5 2005 46274B1E75 6.10.2019 63 60-80 2 2 3 2005 4626187467 6.10.2019 75 80 2 2 2.5 2007 472E30694C 6.10.2019 60 85 1 1 3.38 2005 0A14082E10 6.19.2019 95-100 85 2 2 4.9

2005 4626080970 6.19.2019 clear 0 0 0 2.4

2005 4626214B69 6.19.2019 75-100 115 2 2 3.4

2005 6C00090633 6.19.2019 90-100 97 2 2 2.7

2005 4626226048 6.19.2019 90-100 125 2 2 3.6

2005 4626404757 6.19.2019 0 0 0 0 0

2005 4628382929 6.19.2019 90-100 50 2 2 3.1

2005 462446145B 6.19.2019 80-100 80 2 2 3.1

2005 4626187467 6.19.2019 0 0 0 0 2.3

1999 423A314D00 6.19.2019 50-100 90 2 2 6.2

1999 423A2C5022 6.19.2019 90-100 95 2 2 6.64

2003 424F2F3D60 6.19.2019 50-90 95 2 2 7

1999 471C52622E 6.19.2019 90-100 90 2 2 7.56

1999 423A314D05 6.19.2019 90-100 95 2 2 5.6

1999 423C0C464E 6.19.2019 90-100 100 2 2 5.22

2002 46273A082B 6.24.2019 30-100 105 2 2 6.4

2004 4625537512 6.24.2019 95-100 85 2 2 3.7

1998 423F2F73D 6.24.2019 75-80 88 2 2 4

2002 462616257F 6.24.2019 95-100 60 2 1 6.8

2002 42561F2E3A 6.24.2019 75-80 55 1 1 6.1

2002 462663187F 6.24.2019 75-100 100 2 2 6.21

2004 4627764F65 6.24.2019 95-100 75 2 2 3

2004 46256C0D03 6.24.2019 95-100 85 2 2 3.8

1998 424E680B49 6.24.2019 0 0 1 0 6.1

2004 4627563D42 6.24.2019 5-10% 65 1 1 4.5

2002 425669693F 6.24.2019 95-100 70 2 2 4.3

2004 4623767415 6.24.2019 0 0 1 0 4.3

2002 6C00096524 6.24.2019 75-100 75 2 2 5.9

2004 4625703115 6.24.2019 75-100 105 2 2 4.6

2002 46276E1F17 6.24.2019 25-30 60 1 1 4.5

2004 46256E7712 6.24.2019 25-80 63 2 2 2.9

2002 4255326D1F 6.24.2019 0 55 1 0 6.6

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2004 487F24652B 6.24.2019 75-100 75 2 2 3.4

Spawning event 6/20/2019- For Larval Drift and BFTC

Female Male Eggs per ml 1st 2nd 3rd 4th 5th 6th 7th Total mls Total eggs

462546534B 4626214B69 38 50 175 130 100 125 170 120 870 33060

4627716D2D 4628382929 42.8 60 120 175 175 105 0 635 27178

46257D6F35 423A314D00 41.6 60 105 140 150 150 160 50 815 33904 4627787556 6C00090633 44 0 175 175 200 200 210 50 1010 44440 1F4A125B2A 462446145B 37 90 125 155 120 185 150 825 30525

6C00096611 471C52622E 50 175 130 140 120 100 70 735 36750

6C00093506 471C52622E 51.6 25 70 100 65 120 75 455 23478

444175202F 423A314D05 44.4 160 175 200 230 230 110 1105 49062

6C00090924 423C0C464E 50.4 25 120 140 160 175 160 780 39312

4256437C5F 423A2C5022 42 0 150 150 240 240 185 150 1115 46830 4624380D66 424F2F3D60 36.4 125 150 150 175 200 120 920 33488 42570A387A 4626226048 38.2 145 125 150 175 160 0 755 28841

4624276027 0A14082E10 41.6 75 65 90 100 135 75 540 22464

Total 449332

Spawning Event 6/20/2019 for BFTC Female Male eggs per ml 1st 2nd 3rd 4th 5th Total mls Total eggs 46275A3252 01A14082E10 45 80 115 150 110 175 630 28350 4627632E48 01A14082E10 41.4 125 175 180 180 215 875 36225

Neuralization Female Male (%) 462546534B 4626214B69 94.1 4627716D2D 4628382929 92.6 46257D6F35 423A314D00 97.3 4627787556 6C00090633 97.7 1F4A125B2A 462446145B 96.4 6C00096611 471C52622E 94.9 6C00093506 471C52622E 97.7 444175202F 423A314D05 95.9 6C00090924 423C0C464E 22.9 4256437C5F 423A2C5022 82.9 4624380D66 424F2F3D60 95.3 42570A387A 4626226048 92.5 4624276027 0A14082E10 90.8

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Spawning event 6/25/2019- For larval drift study Total Female Male Eggs per ml 1st 2nd 3rd 4th 5th 6th 7th 8th mls Total eggs 46242E2A6F 462663187F 50.6 110 200 230 270 190 0 1000 50600

444267561D 425669693F 34.2 130 150 180 180 180 190 110 60 1180 40356 4624485842 6C00096524 56.8 70 110 175 160 160 115 790 44872

46254D0546 46256C0D03 48 75 130 130 335 0

46237D0338 4627764F65 49.2 10 70 100 110 110 100 50 550 27060

46255B170B 423F2F73D 47.4 130 190 150 170 25 0 665 31521

6C00090926 46256E7712 42.6 75 90 115 125 140 110 40 695 29607

425708072B 4625537512 43.4 75 135 150 160 150 170 175 60 1075 46655 46254E3E19 487F24652B 37.2 60 110 160 160 100 0 590 21948

44433E701A 46273A082B 54 0 80 130 150 140 70 15 585 31590

4628391106 4625703115 41.2 0 35 60 130 140 140 130 100 735 30282 4624127119 462616257F 47.4 0 0 0 130 140 145 150 150 715 33891 Total 388382

Female Male Nueralization (%) 46242E2A6F 462663187F 90.1 444267561D 425669693F 92.4 4624485842 6C00096524 93.7 46237D0338 4627764F65 89 46255B170B 423F2F73D 92 6C00090926 46256E7712 88.1 425708072B 4625537512 95.8 46254E3E19 487F24652B 94.3 44433E701A 46273A082B 87.6 4628391106 4625703115 85.7 4624127119 462616257F 89.1

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PALLID STURGEON PROPAGATION 2019 Garrison Dam NFH

Rob Holm

US Fish and Wildlife Service Garrison Dam NFH, Riverdale ND

Background/Introduction The Pallid Sturgeon Recovery Plan (1993) established guidance for collection of wild brood fish, propagation, research needs, and reintroduction of progeny to accomplish recovery goals. This hatchery’s role in the recovery effort focuses on the development of techniques for spawning and rearing of pallids and propagation for augmentation. Pallid Sturgeon propagation at Garrison Dam NFH began in 1997. Successful spawning has occurred annually since 1998. Stockings from Garrison Dam NFH have occurred annually since 2002 in the Missouri and Yellowstone Rivers from Missouri to Montana. As of the spring larval drift stocking in 2019, a total of 285,379 fish and 2,473,416 fry had been directly stocked from Garrison Dam NFH to supplement recovery. Additional eggs, fry and fish from Garrison Dam NFH have been shipped to other participating recovery hatcheries and research facilities for further growth prior to stocking. Cryopreservation of milt also is a priority recovery action with Garrison Dam NFH being one of three hatcheries maintaining the pallid sturgeon milt repository. As of the fall 2019, 100 unique wild males have been cryopreserved for the Upper Basin Recovery. Four have no progeny stocked in any RPA and an additional 16 are not represented in all three RPAs.

Objectives Garrison Dam NFH will be the spawning site for all wild brood collected that have not contributed progeny into the three upper RPA’s. Pairing for family lots will be based on the results from the Lamar FTC for broodstock selection. Milt will be shipped overnight to be cryopreserved at Warm Springs FTC. We will use a 1X3 mating strategy making use of wild collected brood fish that have not contributed progeny to the recovery effort. The cryo repository will serve as a backup in the event we are short ‘new’ males.

The stocking request for 2019 includes fry for a larval drift study as well as propagation augmentation to increase the effective population size. For the larval drift study 550,000 day old and 150,000 five day old larvae are requested. HRPS yearling stocking numbers will depend upon the number of adults captured that have not contributed progeny. Our stocking target will approximate the mean number of offspring released for the male families in each RPA to increase the effective population.

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RPA1 - 270 spring yearlings/family RPA2 - 400 spring yearlings/family RPA3 - 30 spring yearlings/family up to a maximum of 180 fish

Approximately five thousand eggs per family (100 mls) will be sent to Gavins Point NFH for broodstock development provided the eggs represent new family lots. Gavins Point NFH will be spawning wild (RPA 4) and captive broodstock again this year. Progeny from the captive brood program will be used for research.

Further studies will be conducted this year to increase larval survival. Since Bio-Oregon’s Biodiet larval feed was discontinued in 2007, our survival success has been poor. While we are typically able to meet the stocking targets, there is concern that we are selecting for individual fish that will accept and/or assimilate the Otohime diet that is used today. Many studies of larval fish have shown that the digestive tracts of larval fishes lack enzymes needed to break down the more complex proteins found in processed fish feeds. This year we will be experimenting with a diet, KilliFeast, produced by Aquanix whose primary ingredients are marine fish hydrolysate, followed by zooplankton and mysis. These ingredients prepared by low-temperature drying suggest that there will be better retention of vitamins, lipids and amino acids. The hydrolysis of fish byproducts provides more readily available simple proteins for the larval fish to digest which should improve survival.

The following two charts help illustrate the survival issues we have see. In 2004 we were feeding Biodiet for the first few months, in 2017 Otohime. Although both charts show an increase in mortality around the typical day 18 starvation period, the overall survival rates are much lower in 2017 than in 2004. Also note the similarities in mortality spikes based on female lot and that not all mortality spikes are associated with starvation.

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Chart 1. Larval Mortality by Family Lot in 2004 Feeding Biodiet Diets.

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Chart 2. Larval Mortality by Family Lot in 2017 Feeding Otohime Diet.

Spring Capture 2019 April 17 - Female code 124 (4A49070E13) was located by radio 5 miles upstream of the HWY 85 bridge near Williston captured and taken to the hatchery. Black eggs were biopsied at capture and the PI reading was 0.2.

April 22- Female code 39 (7F7D7C2E4B) was captured at the confluence - first day of targeted brood capture. This fish was taken to the hatchery. Both 0E13 and 2E4B were spawned at Garrison Dam NFH in 2017 and will be used in the larval drift study. Seven boats fishing this week.

April 24 - Two females captured, code 127 (4443250A24) and code 87 (1F48421542). Water in the river warmed to 55F today. Tank temperatures were increased slightly to 53F.

April 29 - Water temp in river back down to 47F as a result of the snow storm that passes over the weekend. Six boats fishing. Four at the confluence, one in the Yellowstone and one upstream on the Missouri. No fish captured.

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April 30 - An additional boat from USGS and a representative from Lotech onsite to evaluate the radio transmitters. Air temperature overnight in the low 20's with day highs in the low 40's. Three fish captured today and taken to the hatchery, one female (4315327C7B) and two males, code 23 (1F4A31445) and code 179 (4718447879).

May 21 - Three boats fishing half day - no captures

May 22- Three boats fishing in the Missouri River with two fish collected, female, code 43(220F01755C) and male code 15(220F0F7677). Temps in the river had dropped to 50F and flows are coming up.

Table 1. RPA 2 Pallid Broodstock Captures No additional broodstock were collected.

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Pallids Females Males Unknowns Collected Collected Collected

1988 3 1 1 1

1989 5 1 0 4

1990 5 1 1 3

1991 11 1 4 6

1992 38 4 16 18

1993 27 4 5 18

1994 66 6 23 37

1995 22 5 12 5

1996 20 5 12 3

1997 24 6 14 4

1998 14 3 9 2

1999 17 5 9 3

2000 26 4 17 5

2001 22 5 16 1

2002 30 8 21 1

2003 53 7 39 7

2004 65 16 48 1

2005 44 11 28 5

2006 66 14 49 3

2007 44 12 32 0

2008 62 15 47 0

2009 51 8 43 0

2010 40 5 35 0

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2011 10 3 6 1

2012 43 9 34 0

2013 31 7 23 1

2014 25 5 20 0

2015 27 3 24 0

2016 17 8 25 1

2017 37 8 28 1

2018 3 0 3 0

2019 9 6 3 0

Total 963 196 647 131

% of individuals captured this 9% 3% 0% year

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Table 2. Adults Captured in 2019 2019 Pallid Broodstock Captures

Last Stocked Progeny

PIT Tag PIT Tag 2 Code Capture Sex Spawn Cryo RPA1 RPA2 RPA3 Comments Weight Date lbs Year Year

4A49070E13 124 4/17/19 F 603 696 46 Spawned in 2017 at GAD

7F7D7C2E4B 39 4/22/19 37.4 F 611 441 22 Spawned in 2017 at GAD

4443250A24 127 4/24/19 59.4 F 1176 1532 138 Spawned in 2006 at MC

1F48421542 87 4/24/19 48.4 F 2722 1488 180 Spawned in 1997, 2006, 2015 at GAD, radioed in 2015

4315327C7B 66 4/30/19 33 F 2009 827 1170 70 Spawned in 2009 at GAD, radioed in 2008

4718447879 179 4/30/19 61.6 M 2009 2009 283 436 24 Spawned in 2009 at GAD, new radio in 2013

1F4A3E1445 1F4A2F3A2E 23 4/30/19 35.2 M 2007 2007 970 2696 394 Spawned in 2002 & 2004 at GAD, and at MC 2006 & 2007

220F01755C 2204657963 43 5/22/19 52.3 F 2009 12099 20408 264 Spawned in 1999 at GAD, and at MC 2009, radio implanted in 2017

220F0F7677 15 5/22/19 35.2 M 2004 2004 13 1277 0 21,288 fry released in RPA2, Spawned at GAD in 2004

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Spawning and Propagation June 3 - Oocytes were taken from all females collected. Female 220F0177C had immature eggs. The other four showed no response to the progesterone assay and PI ranged from 0.09 to 0.16. Female 4443250A24, with a PI of 0.09 was moved to the south tank to slow her PI progression while those in the north tank had the water temperature increased to speed up the oocyte migration.

June 17 - Oocytes taken from female 2E4B and evaluated with the progesterone assay. No response to the progesterone. The PI was at 0.115 with very low standard deviation. The rate of migration was determined to be 0.0032/day putting her at 0.088 on June 25th.

June 23 - Hormone injections were administered to initiate spawning at 10:00 pm

June 24 - Resolving dose administered at 10:30 am. Neurulated eggs from Gavins Point captive brood spawn arrived at 5:30 pm. Thirteen family lots received for the larval drift study.

June 25 - Ovulation noted at the midnight check with female 4443250A24 releasing eggs. She was spawned approximately every 30 minutes until 4:30 when a second female, 4A49070E13 initiated ovulation. See spawning results on Table 5 through Table 10. Eggs from Gavins Point NFH are hatching beginning at 2:30 am and progressing throughout the day.

June 27 - Eggs from the 6/20 captive spawn at Gavins Point NFH completed hatch in the morning hours. Half of the family lots are behaving a little unusual with the larvae hanging low and toward the center of the tank rather than the typical random dispersal throughout the tank. Female broodstock are fully stripped to expel all remaining ovulated eggs.

June 28 - Significant losses of larvae from the Gavins Point NFH lots - what we have termed the ‘early life stage mortality syndrome’ and have seen periodically throughout pallid propagation (see mortality records, Table 15 and Chart 4). Oxygen levels were checked and at 7.5 -8 ppm. Nitrogen readings were up to 105% with a delta P of a minus 10. A storm was occurring overnight causing a change to the barometric pressure accounting for the rise in nitrogen. Eggs from the second captive spawn (June 25) at Gavins Point NFH transferred by FWS FWCO offices today and put in incubation jars.

June 29- Losses continue from the 6/20 spawn. Temperature is increased a couple degrees to speed up hatch at 9:00 pm.

June 30 - Hatch of the 6/25 spawns occurring at ~10:00 am. Temperature again increased to 68

July 1 - Preparations for packaging begins at 4:00 am with sample counts taken using the Xpertcount larval counter. Additional staff arrive at 5:00 to begin bagging larvae. Water

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temperature was turned down to 60< F for the trip and to match up to release temperatures. By 6:00 am ten staff are assisting with netting, enumerating, and bagging larvae at 200 mls per bag. 500 ml graduated cylinders were used to measure out larvae in 100 ml increments to avoid having larvae in the cylinder for an extended time. The trailer with larvae pulled out of the hatchery at 8:45 am headed for the Culbertson, MT release site. See Table 12 & Table 13 for larval release information.

July 2 - Received fish health report from Bozeman FHC on the wild broodstock. Male 1445 was positive for MRSIV via standard and qPCR. The others were clear. Male 1445 was also the only male that produced milt this year and so is represented in all family lots produced at Garrison Dam NFH this year.

July 3 - Captive spawn from Gavins Point NFH (6/20/2019) 30% settled to bottom

July 5 - Captive spawn from Gavins Point NFH (6/20/2019) 70% settled to bottom

July 6 - Larvae from 6/20 spawn losing plugs - initiated feed with 10 mls Otohime B2 diet/tank

July 8 - Larvae from June 25th spawn 50% settled to bottom. Moved captive brood larvae from FT tanks to production tanks and redistributed wild spawn larvae to the 30 FT tanks for the feed study at 300 larvae per tank. Surplus fish inventoried and moved to tank D7.

July 22 - Mortality has spiked in several family lots from the captive brood adults. The mortality counts are based on volume samples rather than counts at this time.

July 31 - Fish from each of the captive progeny are sampled for initial month’s growth. Fish are averaging 1.6 inches in length and 0.26 grams/fish. Feed was primarily Otohime B2 feeding out 2423 grams over the month and an additional 143 grams C1 the last two days of July. The Feed Study is showing similar results for the Portman and Otohime diets - with 15% and 16% survival to feed respectively. The Razerback diet has 24% survival to date. The results don’t account for growth which appear to favor the Otohime and Portman’s diets. Although not part of the Feed Study, an alternate feed we are providing a single tank appears to be much superior to all the others - Golden Pearl Reef and Larval Fish diet 200-300 micron size. The feed is available through www.brineshrimpdirect.comwith an ingredients list of marine fish, krill (23%) and fish roe as the top three. 55% protein, 15% Lipids, 12% ash and 8% moisture (see results pp. 98-99).

August 2 - Female 7F7D7C2E4B found dead in the tank. Necropsy didn’t provide any clues as to a cause of death. No internal or external hemorrhaging except on the gill tissue.

August 3 - Two additional brood fish found dead in the brood tanks - on in the south (male 1F4A3E1445) and one in the north 20 foot tank (female 4443250A24 ). The forage fish (rainbow trout) were also dead. The remaining six brood fish were returned to the Missouri River Confluence where they were relocated in October by radio. 189

Sept - Noted that several siblings from the family 7119 X 257F were ‘proportionally short’ as compared to their siblings or other in the cohort.

Sept 9 - Call from Recovery Team decision not to stock any progeny from the captive broodstock. The concerns for stocking the progeny were: 1) the potential over-representation from lower priority males, 2) confounding results from drift study, 3) if there were higher survival of larval from the drift study the additional stockings could exacerbate the effect (see Appendix 17. p100).

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Table 3. Broodstock Data

2019 PALLID STURGEON BROODSTOCK DATA

Tag Number Sex Tank 2019 Spawning Result Previous Spawn Results Date Wt 5 mgLH-RH/10 ml Last Collected (Kg) saline PI

Dose (mls) (mg ) Cry Progeny (RPA) (mg/kg) Year 0.05 or

0.02 1 2 3

4A49070E13 4/17/19 F 15 1.50 0.75 0.05 0.13 N Larvae released 603 696 46

7F7D7C2E4B 4/22/19 F 17 1.70 0.85 0.05 0.115 N Larvae released, died PS 611 441 22

4443250A24 4/24/19 F 27 2.70 1.35 0.05 0.091 S Larvae released, died PS 1176 1532 138

1F48421542 4/24/19 F 22 2.20 1.10 0.05 0.125 N Larvae released 2722 1488 180

4315327C7B 4/30/19 F 15 1.50 0.75 0.05 0.135 N Larvae released 827 1170 70

4718447879 4/30/19 M 28 1.12 0.56 0.02 N Did not spermiate 2009 283 436 24

1F4A3E1445 4/30/19 M 16 0.64 0.32 0.02 N Larvae released, died PS 2007 970 2696 394

220F0F7677 5/22/19 M 16 0.64 0.32 0.02 Did not spermiate 2004 13 1277 0

TOTAL 12.00 6.00

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Table 4. Milt Collections

Pit Tag # Inj Inject Take Time Amount Motility Characteristics Last Milt Motility 5 ml 0.5 ml Date Time Date (mls) (fresh) cryo Cryo (at freeze) straws straws year 2019

1F4A3E1445 6/23 10:00p 6/25 XX 2007

220F0F7677 no milt

4718447879 no milt

100 wild males now represented in the repository. No straws were frozen in 2019.

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Table 5. Stocking Targets by RPA to Approximate RPA1 RPA2 RPA3 maximize Ne

Female Median YE 560 734 102

Male Median YE 352 661 63

Overall Median YE 695 1277 70

Table 6. Proposed Stocking Targets for Progeny of Captive Broodstock

PIT Tag PIT Tag Wild Parent Cryo Current YE Stocked Recaptures (to 2019) Target stocking #

Female Male F/M/F/M Yr RPA1 RPA2 RPA3 RPA1 RPA2 RPA3 RPA1 RPA2 RPA3 TOTAL

4624276027 0A14082E10 7F7B016070 556 0 224 4 0 34

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1F4A363031 2007 702 395 68 10 17 9 0 0 0 0

7F7B026102 1787 4454 155 48 115 15

7F7B023253 2005 1187 1719 165 55 33 18

6C00090924 423C0C464E 114476216A 25 3174 170 1 35 18

220F107A6F 2016 575 2173 144 5 171 22 0 0 0 0

7F7B021573 0 478 181 0 29 17

7F7D441774 X 0 159 66 0 6 7

42570A387A 4626226048 220E345E09 0 734 140 0 39 36

1F4A27214F 2001 3 378 195 0 23 42 270 0 0 270

115676635A 245 1822 436 23 54 27

7F7D2D723D 2008 1196 7611 352 49 89 26

6C00093506 471C52622E 115679394A 0 0 0 0 0 0

1F47760123 2007 1654 450 141 18 13 16

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7F7B021573 0 478 181 0 29 17 0 250 0 250

7F7D441774 0 159 66 0 6 7

1F4A301354 290 103 76 134 31 41

1F4A4A1439 X 295 314 156 81 11 22

1F4A125B2A 462446145B 0 300 0 300

132213574A 2002 1422 39 99 25 8

7F7B023253 2005 1187 1719 165 45 31 17

462546534B 4626214B69 114476216A 25 1343 170 1 35 18

430E452777 2010 0 1343 20 0 15 2 0 0 0 0

4443240458 1200 4427 96 80 118 8

444334021A 2008 935 2268 63 91 70 2

4627716D2D 4628382929 114476216A 25 3174 170 1 35 18

220F107A6F 2017 575 2173 144 5 171 22 0 0 0 0

115557165A 8603 12130 448 189 279 44

7F7D2D723D 2008 1196 7611 352 49 89 26

46257D6F35 423A314D00 114476216A 25 3174 170 1 35 18

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220F107A6F 2016 575 2173 144 5 171 22 0 0 0 0

7F7B021573 0 478 181 0 29 17

7F7F06583D 2016 151 315 129 35 24 16

4627787556 6C00090633 114476216A 25 3574 170 1 35 18

220F107A6F 2016 575 2173 144 5 171 22 0 0 0 0

7F7B026102 1787 4454 155 44 115 14

7F7B023253 2005 1187 1719 165 45 31 17

4624380D66 424F2F3D60 115679394A 0 0 0 0 0 0

1F47760123 2007 1654 450 141 18 13 16 270 400 30 700

7F7B021573 0 478 181 0 29 17

7F7F06583D 2016 151 315 129 35 24 16

6C00096611 471C52622E 7F7B016070 566 0 224 4 0 34

1F4A363031 2007 702 395 68 6 16 7 270 250 0 520

7F7B021573 0 488 181 0 29 17

7F7D441774 X 0 159 66 0 6 7

196

4256437C5F 423A2C5022 116224546A 0 3986 601 0 236 90

1F477B3A65 2002 404 1541 188 14 61 29 270 240 0 510

7F7B021573 0 478 181 0 29 17

113719262A X 0 160 65 0 11 7

444175202F 423A314D00 7F7B016070 556 0 224 4 0 34

7F7D365422 2009 727 1144 159 32 40 26 270 0 0 270

7F7B021573 0 478 181 0 29 17

7F7F06583D 2016 151 315 129 45 31 17

46237D0338 4627764F65 115679394A 0 0 0 0 0 0

1F47760123 2007 1654 450 141 18 13 6 270 0 0 270

114476216A 25 3174 170 1 35 18

430E452777 2010 0 1343 20 0 12 3

197

PIT Tag PIT Tag Wild Parent Cryo Current YE Stocked Recaptures (to 2019) Target stocking #

46255B170B 423F2F737D 454910202B 147 2281 41 1 12 4

220F0F7677 2004 13 655 0 0 2 0 270 400 30 700

7F7F056171 0 200 98 0 15 15

1F47760123 2007 1654 450 141 18 13 16

4628391106 4625703115 132211792A 195 186 513 0 1 61

1F4A312640 2004 25 489 67 0 2 8 245 0 0 245

115551683A 185 379 44 2 7 7

115552116A 2004 1702 871 65 7 13 6

46254E3E19 487F24652B 115555495A 0 0 26 0 0 4

431565767B 2007 1884 527 65 18 11 6 270 400 0 670

454B380D60 789 2734 127 16 24 15

7F7D376F73 2015 473 553 80 27 15 7

198

44433E701A 46273A082B 132256586A 538 581 202 8 26 25

1F47760123 2007 1654 450 141 18 13 16 0 0 0 0

116224546A 0 3986 601 0 236 90

1F477B3A65 2002 404 1541 188 14 61 29

425708072B 4625537512 411D262C1F 2247 1781 280 11 146 59

411D0B4E09 2016 588 414 10 3 33 0 270 215 20 505

115555495A 0 0 26 0 0 4

431565767B 2007 1884 527 65 18 11 6

46242E2A6F 462663187F 7F7F054855 1841 1886 89 16 55 9

132313521A 2016 327 368 76 8 27 0 0 0 0 0

116224546A 0 3986 601 0 236 90

1F477B3A65 2002 404 1541 188 14 61 29

4624485842 6C00096524 7F7B016070 566 0 224 4 0 34

199

1F4A363031 2007 702 395 68 6 16 7 0 0 0 0

116224546A 0 3986 601 0 236 90

1F4A27214F 2001 3 378 195 0 23 42

444267561D 425669693F 7F7B016070 556 0 224 4 0 34

7F7D365422 2009 727 1144 159 32 40 26 270 400 0 670

116224546A 0 3986 601 0 236 90

220F107A6F 2017 575 2173 144 5 171 22

6C00090926 46256E7712 115555495A 0 0 26 0 0 4

431565767B 2007 1884 527 65 18 11 6 220 215 0 435

132211792A 195 186 513 0 1 55

7F7D487531 2004 50 246 83 19 39 12

4624127119 462616257F 7F7F054855 1841 1886 89 16 65 9

132313521A 2016 327 368 76 8 27 0 270 0 0 270

200

116224546A 0 3986 601 0 236 90

116167123A 2008 4 661 144 0 35 12

201

Table 7. Female # 4443250A24 - Spawning Results FEMALE # 4443250A24 Spawn Date - 6/25/19 Hatch - 6/30/2019

Time MALE # Milt Data Inventory % neur Larvae Larval Survival from Tank at hatch mls Drift hatch to: released Releases Milt mls Date Motility mls at Total eggs 1st 4th / H20 GDNFH @ 38.2/ml week week mls

10:00 initial injection p

10:00 resolving dose a

12:35 a 1F4A3E1445 205 7831

12:50 a 1F4A3E1445 205 7831 32881 95% 383 26010 94% FT1

1:15 a 1F4A3E1445 400 15280

1:48 a 1F4A3E1445 545 20819 20819 97% 185 12546 85% FT3

2:25 a 1F4A3E1445 590 22538 24944 106% 261 17748 90% FT4

2:55 a 1F4A3E1445 490 18718 18997 98% 99 9732 79% FT5

3:45 a 1F4A3E1445 700 26740 26197 103% 243 16524 87% FT6

4:28 a 1F4A3E1445 620 23684 23302 101% 180 12240 83% FT7

202

5:42 a 1F4A3E1445 530 20246 18812 92% 140 9486 83% FT8

6:28 a 1F4A3E1445 310 11842

20078 94% 180 12240 91% FT2

7:40 a 1F4A3E1445 220 8404

TOTAL 4815 183933 186030 101% 1671 116526

Table 8. Female # 4A49070E13 - Spawning Results FEMALE # 4A49070E13 Spawn Date - 6/25/19 Hatch - 6/30/2019

Time MALE # Milt Data Inventory % neur Larvae Larval Survival from Tank at hatch mls Drift hatch to: released Releases Milt mls Date Motility mls at Total eggs 1st 4th / H20 mls GDNFH @ 50/ml week week

10:00 p initial injection

10:00 a resolving dose

4:35 a 1F4A3E1445 820 41000 43762 107% 455 38633 6% FT9

5:50 a 1F4A3E1445 570 28500 29665 104% 324 28188 3% FT10

6:45 a 1F4A3E1445 360 18000

27293 91% 293 25155 5% FT12

203

7:42 a 1F4A3E1445 240 12000

8:36 a 1F4A3E1445 125 6250

19258 135% 213 18344 2% FT21

9:12 a 1F4A3E1445 160 8000

TOTAL 2275 113750 119978 105% 1285 110320

Table 9. Female # 4315327C7B - Spawning Results FEMALE # 4315327C7B Spawn Date - 6/25/19 Hatch - 6/30/2019

Time MALE # Milt Data Inventory % neur Larvae Larval Survival from Tank at hatch mls Drift hatch to: released Releases Milt mls Date Motility mls at Total eggs 1st 4th / H20 GDNFH @ 43/ml week week mls

10:00 initial injection p

10:00 resolving dose a

6:00 a 1F4A3E1445 280 12040

24592 79% 137 10944 92% FT16

7:10 a 1F4A3E1445 440 18920

204

9:00 a 1F4A3E1445 340 14620

35194 117% 311 28877 91% FT22

9:55 a 1F4A3E1445 350 15050

8:22 a 1F4A3E1445 500 21500

10:25 1F4A3E1445 100 4300 22835 71% 159 12744 91% FT15 a

11:00 a 1F4A3E1445 150 6450

TOTAL 2160 92880 82621 89% 607 52565

Table 10. Female # 1F48421542 - Spawning Results FEMALE # 1F48421542 Spawn Date - 6/25/19 Hatch - 6/30/2019

10:00 p initial injection

10:00 a resolving dose

6:52 a 1F4A3E1445 470 20657 20472 99% 185 15129 92% FT13

205

7:50 a 1F4A3E1445 670 29447 29099 100% 254 20304 96% FT14

8:25 a 1F4A3E1445 290 12746

31442 86% 367 29376 96% FT19

9:48 a 1F4A3E1445 540 23733

9:05 a 1F4A3E1445 420 18459

31970 117% 383 30600 96% FT23

10:30 a 1F4A3E1445 200 8790

6:12 a 1F4A3E1445 450 19778

39992 102% 468 36504 97% FT11

10:58 a 1F4A3E1445 440 19338

TOTAL 3480 153120 152975 100% 1657 131913

Table 11. Female # 7F7D7C2E4B - Spawning Results FEMALE # 7F7D7C2E4B Spawn Date - 6/25/19 Hatch - 6/30/2019

Time MALE # Milt Data Inventory % neur Larvae Larval Survival from Tank at hatch mls Drift hatch to:

206

Milt mls Date Motility mls at Total eggs released Releases 1st 4th / H20 mls GDNFH @ 44.3/ml week week

10:00 initial injection p

10:00 resolving dose a

6:10 a 1F4A3E1445 70 3100

6:55 a 1F4A3E1445 200 8856 4366 16% 30 2400 94% FT17

9:52 a 1F4A3E1445 340 15055

10:27 a 1F4A3E1445 230 10184

10:59 a 1F4A3E1445 350 15498 5753 14% 50 4000 90% FT18

7:45 a 1F4A3E1445 320 14170

8:30 a 1F4A3E1445 290 12841

19505 77% 104 8320 99% FT20

9:10 a 1F4A3E1445 280 12398

TOTAL 2080 92144 29624 32% 184 14720

207

Table 12. Gavins Point NFH Captive Broodstock Larval Releases and Survival

Gavins Point NFH Captive Broodstock Larval Releases and Survival

Female # Male # Hatched *Larval Age of Inventory Tank Inventory 1 month Larvae Releases Larvae post LD # at 31 dph % dph releases Survival

4624276027 0A14082E10 27,576 7,560 5 392 C1 43 0.2%

6C00090924 423C0C464E 14,328 0 - 0 - 0 0.0%

42570A387A 4626226048 48,364 39,744 5 3,085 C3 664 8.8%

6C00093506 471C52622E 23,743 2,228 5 741 C4 163 0.8%

1F4A125B2A 462446145B 34,543 10,517 5 2,361 C5 165 0.7%

462546534B 4626214B69 26,109 19,008 5 2,630 C6 219 3.2%

4627716D2D 4628382929 30,366 15,246 5 1,514 C7 236 1.6%

46257D6F35 423A314D00 44,825 33,840 5 469 C9 80 0.7%

4627787556 6C00090633 49,816 4,064 5 1,709 C10 117 0.3%

4624380D66 424F2F3D60 46,358 23,220 5 4,238 A6 1817 8.1%

6C00096611 471C52622E 31,383 12,960 5 3,332 A7 259 1.5%

4256437C5F 423A2C5022 33,715 0 5 627 A8 256 0.8%

444175202F 423A314D05 50,687 32,400 5 844 B7 215 1.2%

46237D0338 4627764F65 16,444 14,112 1 2,332 C8 202 8.7%

208

46255B170B 423F2F737D 35,932 27,360 1 8,554 B4 201 2.3%

4628391106 4625703115 23,217 21,735 1 1,457 C2 185 12.5%

46254E3E19 487F24652B 32,203 27,306 1 4,897 B6 438 8.9%

44433E701A 46273A082B 36,580 33,840 1 2,740 B8 212 7.7%

425708072B 4625537512 38,934 33,579 1 5,355 A1 653 12.2%

46242E2A6F 462663187F 43,995 40,014 1 3,981 A2 779 19.6%

4624485842 6C00096524 40,941 39,312 1 1,629 A3 10 0.6%

444267561D 425669693F 48,897 43,326 1 5,571 A4 1226 22.0%

6C00090926 46256E7712 50,206 48,222 1 1,984 A5 307 15.5%

4624127119 462616257F 27,039 9,234 1 17,805 B5 1797 10.1%

Total 856,201 538,827 78,247 10,244 3.5%

* larval release numbers are estimates and are adjusted down 10% to offset water weight from netting larvae 5 dph larvae = 200,787 1 dph larvae = 338,040

209

Table 13. Egg Hatch and Fry Survival

Egg Hatch and Fry Survival Summary - Garrison Dam NFH

FEMALE # MALE # Egg Egg size Number % Hatch Inventory Mortality to % fry Larval # Larval Drift end of first week 1 Drift larvae/ volume (#/ml) week Survival for ml Release (mls) week 1 Release (7/7/2019) mls

4443250A24 1F4A3E1445 4815 38.2 183,933 114% 72,504 23,677 67% 1670 68 113,526

4A49070E13 1F4A3E1445 2275 50.0 113,750 111% 9,963 5,430 45% 1284 86 110,319

4315327C7B 1F4A3E1445 2160 43.0 92,880 97% 30,071 7,202 76% 607 87 52,565

1F48421542 1F4A3E1445 3480 44.0 153,120 104% 22,336 5,325 76% 1656 80 131,913

7F7D7C2E4B 1F4A3E1445 2080 44.3 92,144 33% 7,736 1,023 87% 274 80 21,888

TOTAL 14,810 635,827 97% 142,610 42,657 70% 5491 78 430,211

210

Table 14. Survival Summary Editor’s note: Table 14 has not been included in this report for brevity. Please contact Rob for more detailed information.

Table 15. Mortality Records to 31 Days Post Hatch Editor’s note: Table 15 has not been included in this report for brevity. Please contact Rob for more detailed information.

Chart 3. Daily Mortality - Captive Brood Progeny to 31 DPH

211

Table 16. Initial Growth and Survival Editor’s note: Table 16 has not been included in this report for brevity. Please contact Rob for more detailed information.

Table 17. Feed Amounts and Cost Editor’s note: table 17 has not been included in this report for brevity. Please contact Rob for more detailed information.

212

Chart 4. Broodstock Tank Temperature - North 20 Foot Tank

213

Chart 5. Broodstock Tank Temperature - South 20 Foot Tank

214

Chart 6. Production Temperatures

215

Table 18. Proposed Spawning Strategy Using Relatedness Analysis

Proposed 2019 Family Crosses

Stocking Targets Lamar Priority Current # Stocked Progeny Female PIT Male PIT Rxy RPA1 RPA2 RPA3 RPA1 RPA2 RPA3 RPA1 RPA2 RPA3

1F4A3E1445 0 0 0 5 5 5 955 1676 394

220F0F7677 352 661 63 2 2 2 13 655 0

4718447879 0 661 63 4 2 2 204 318 0

4A49070E13 1F4A3E1445 0 0 0 2 2 2 603 696 46

7F7D7C2E4B 1F4A3E1445 0 0 0 2 2 2 620 441 22

4443250A24 1F4A3E1445 0 0 0 4 4 2 1176 1288 138

1F48421542 1F4A3E1445 0 0 0 5 5 3 1365 1485 180

4315327C7B 1F4A3E1445 0 0 0 4 2 2 649 928 47

Famil y cross by highlight made indicated yellow

Table 19. Oocyte Maturation Assay

Editor’s note: Table 19 has not been included in this report for brevity. Please contact Rob for more detailed information.

Table 23. Oocyte Maturation Assay

Editor’s note: Table 23 has not been included in this report for brevity. Please contact Rob for more detailed information.

216

Appendix 1. Milt Repository at Garrison Dam NFH

Editor’s note: Appendix 1 has not been included in this report for brevity. Please contact Rob for more detailed information.

Appendix 2. Cryopreserved Milt Repository - Upper Basin Wild

114 males are represented in the repository, 100 from the wild and 14 from the broodstock program. 45 males have been cryopreserved on more than one occasion (repeats). Four wild males are not yet represented as progeny in the river and 16 are needed on one of the three RPA’s. Milt prior to 2008 is of poorer quality due to cryoprotectant used (HBSS pre-2008, AKOS from 2008 to present)and 0.5 ml straws are generally higher quality.

217

Appendix 3. RPA 2 Missouri River Stockings from Garrison Dam NFH Editor’s note: Appendix 3 has not been included in this report for brevity. Please contact Rob for more detailed information.

Appendix 4. RPA 2 Yellowstone River Stockings from Garrison Dam NFH Editor’s note: Appendix 4 has not been included in this report for brevity. Please contact Rob for more detailed information.

Appendix 5. RPA 1 Stockings from Garrison Dam NFH Editor’s note: Appendix 5 has not been included in this report for brevity. Please contact Rob for more detailed information.

Appendix 6. RPA 3 Stockings from Garrison Dam NFH Editor’s note: Appendix 6 has not been included in this report for brevity. Please contact Rob for more detailed information.

Appendix 7. RPA 4 Stockings from Garrison Dam NFH Editor’s note: Appendix 7 has not been included in this report for brevity. Please contact Rob for more detailed information.

Appendix 8. Females Used in Upper Basin Recovery Effort Editor’s note: Appendix 8 has not been included in this report for brevity. Please contact Rob for more detailed information

Appendix 9. Males Used in Upper Basin Recovery Effort Editor’s note: Appendix 9 has not been included in this report for brevity. Please contact Rob for more detailed information

Appendix 10. RPA2 Unknown Sex Wild Broodstock Editor’s note: Appendix 10 has not been included in this report for brevity. Please contact Rob for more detailed information.

218

Appendix 11. Garrison Dam NFH Hatchery Capacity

Tank Capacity at a Density of 0.5 Pounds per Square Foot

Tank # Square Total Capacity Capacity Capacity Capacity Capacity Capacity of 2" of 4" of 6" of 8" of 9" of 10" Size(ft) Ft (ea) Sqft fish/tank fish/tank fish/tank fish/tank fish/tank fish/tank

892.86 111.61 33.07 13.95 9.80 7.14

4 Circular 10 12.6 126 5625 703 208 88 62 45

5 Circular 38 19.6 744.8 8750 1094 324 137 96 70

6 Circular 0 28.2 0 12589 1574 466 197 138 101

8 Circular 7 50.2 351.4 22411 2801 830 350 246 179

20 Circular 2 314 628 140179 17522 5192 2190 1538 1121

3 X 29 Tanks 8 87 696 38839 4855 1438 607 426 311

Total 65 2546 1136696 142087 42100 17761 12474 9094

Normal Hatchery Capacity 55 545625 68203 20208 8525 5988 4365

Overwinter Tank Capacity at a Density of 0.70 Pounds per Square Foot

Tank # Square Total Capacity Capacity Capacity Capacity Capacity Capacity of 10" of 11" of 12" of 13" of 14" of 15" Size(ft) Ft (ea) Sqft fish/tank fish/tank fish/tank fish/tank fish/tank fish/tank

7.14 5.37 4.13 3.25 2.60 2.12

4 10 12.6 126 63 47 36 29 23 19 Circular

219

5 38 19.6 744.8 98 74 57 45 36 29 Circular

6 0 28.2 0 141 106 82 64 51 42 Circular

8 7 50.2 351.4 251 189 145 114 91 74 Circular

20 Circular 2 314 628 1570 1180 909 715 572 465

3 X 29 Tanks 8 87 696 435 327 252 198 159 129

Total 65 2546 12731 9565 7367 5795 4640 3772

Normal Hatchery 55 6111 4591 3536 2782 2227 1811 Capacity

220

Appendix 13. Fish Health Inspection Report

221

Appendix 14. RPA1 Variance Effective Population

The median number for males in RPA1 is 352 and for females 560.

222

Appendix 15. RPA2 Variance Effective Population

Median number for males in RPA2 is 661 and for females 734.

223

Appendix 16. RPA3 Variance Effective Population

The median number for males in RPA3 is 63 and for females 102.

224

Table 24. Feed Study

2019 Feed Study

Female Male Diet Tank Initial Number % tank g/fish on July weight Number survival

31 (g)

4A49070E13 1F4A3E1445 Portman's 9 300 20 7% 14 0.70

4A49070E13 1F4A3E1445 Portman's 20 300 14 5% 4 0.29

4A49070E13 1F4A3E1445 Portman's 26 300 14 5% 3 0.21

4A49070E13 1F4A3E1445 Otohime 22 300 0 0%

4A49070E13 1F4A3E1445 Otohime 15 300 27 9% 19 0.70

4A49070E13 1F4A3E1445 Otohime 14 300 17 6% 12 0.71

4A49070E13 1F4A3E1445 Razorback 4 300 7 2% 1 0.14

4A49070E13 1F4A3E1445 Razorback 28 300 0 0%

4A49070E13 1F4A3E1445 Razorback 21 300 10 3% 6 0.60

4315327C7B 1F4A3E1445 Portman's 17 300 79 26% 41 0.52

4315327C7B 1F4A3E1445 Portman's 6 300 73 24% 37 0.51

4315327C7B 1F4A3E1445 Portman's 12 300 48 16% 23 0.48

4315327C7B 1F4A3E1445 Otohime 23 300 19 6% 10 0.53

4315327C7B 1F4A3E1445 Otohime 1 300 51 17% 24 0.47

4315327C7B 1F4A3E1445 Otohime 7 300 52 17% 27 0.52

4315327C7B 1F4A3E1445 Razorback 16 300 72 24% 19 0.26

4315327C7B 1F4A3E1445 Razorback 18 300 12 4% 3 0.25

225

4315327C7B 1F4A3E1445 Razorback 8 300 43 14% 20 0.47

1F48421542 1F4A3E1445 Portman's 25 300 104 35% 70 0.67

1F48421542 1F4A3E1445 Portman's 13 300 77 26% 42 0.55

1F48421542 1F4A3E1445 Portman's 3 300 99 33% 60 0.61

1F48421542 1F4A3E1445 Otohime 10 300 106 35% 66 0.62

1F48421542 1F4A3E1445 Otohime 24 300 20 7% 11 0.55

1F48421542 1F4A3E1445 Otohime 5 300 84 28% 41 0.49

1F48421542 1F4A3E1445 Razorback 11 300 48 16% 21 0.44

1F48421542 1F4A3E1445 Razorback 19 300 61 20% 27 0.44

1F48421542 1F4A3E1445 Razorback 2 300 58 19% 26 0.45

1F48421542 1F4A3E1445 Artemia 27 300 21 7% 3 0.14

1F48421542 1F4A3E1445 GP 29 300 0 0% Advanced

1F48421542 1F4A3E1445 Gold Pearl 30 300 116 39% 82 0.71 Re

226

Chart 7. Feed Study

227

Table 25. Feed Study Evaluation Feed Study Evaluation

Female 7C7B 7C7B 7C7B 1542 1542 1542 0E13 0E13 0E13

Male 1445 1445 1445 1445 1445 1445 1445 1445 1445

Diet Otohime Razerback Portman's Otohime Razerback Portman's Otohime Razerback Portman's

Total Mortality 778 773 700 690 733 620 856 883 852

% Mortality 86% 86% 78% 77% 81% 69% 95% 98% 95%

Family Mortality 83% 76% 96%

Fish on Feed 122 127 200 210 167 280 44 17 48

Fish Weight (g) 61 42 101 118 74 172 31 7 21

g/fish 0.50 0.33 0.51 0.56 0.44 0.61 0.70 0.41 0.44

Diet Mortality Otohime 86% Razerback 88% Portman's 80%

g/fish Otohime 0.59 Razerback 0.40 Portman's 0.52

Diet Survival g/fish

Otohime 14% 0.59

Razerback 12% 0.40

Portman's 20% 0.52

GP Adv 0% 0.00

Artemia 7% 0.14

Gold Pearl 39% 0.71

Female Survival g/fish

228

7C7B 17% 0.45

1542 24% 0.54

0E13 4% 0.52

229

230

231

232