Aaron J. DeLonay

Hatchery supplementation is vital for conserving dwindling fish populations. Effective augmentation requires distinguishing hatchery‐origin from wild individuals and accurately identifying species, particularly in systems where closely related species coexist. Genetic monitoring is key to quantifying genetic differences, but conventional markers do not distinguish hybrids, especially backcrosses. Misidentifying hybrids in hatchery programs compromises wild gene pools because hatchery broodstock contributes to numerous offspring being released into the wild. Here, we present a workflow for developing and evaluating the Genotyping‐in‐Thousands by sequencing (GT‐seq) single nucleotide polymorphism (SNP) panel for North American river sturgeons ( Scaphirhynchus spp.). This panel is designed to detect complex hybrid classes and to determine parent‐offspring relationships. Our species identification panel (S‐loci) contains 155 SNPs selected for high genetic differentiation (F _ST ) between Pallid Sturgeon ( S. albus ) and Shovelnose Sturgeon ( S. platorynchus ), and the parentage assignment panel (P‐loci) includes 112 SNPs with high heterozygosity within Pallid Sturgeon. Simulation analyses demonstrated that our GT‐seq S‐loci panel reliably classifies pure species, F1, F2 and backcross hybrids, even with up to 70% missing data. The P‐loci panel achieves high‐confidence parentage assignment with ≥ 80% typed loci, with performance influenced by the proportion of sampled parents. Overall, the novel Scaphirhynchus GT‐seq panel developed in this study represents a robust and efficient tool for detecting hybridisation, assigning parentage and providing critical information for management decisions in ongoing Pallid Sturgeon conservation.

The shovelnose sturgeon (Scaphirhynchus platorynchus) and endangered pallid sturgeon (S. albus) deposit demersal and adhesive eggs in swift currents, near or over coarse substrate. Hydrographic surveys have demonstrated the dynamic nature of spawning habitats and that coarse substrates may episodically be buried (partially or completely) by fine sediments. To evaluate embryo survival of both species in various substrate conditions, laboratory trials were conducted with substrates of clean glass, gravel, medium-coarse sand (MCS), and fine sand-silt (FSS). Embryos in MCS and FSS were tested three ways: unburied, partially buried, and fully buried (1–2-mm depth). Embryos were exposed to trial conditions for 10 days from the day of fertilization (5 days beyond expected hatching). For both species, mean hatch of normally developed free embryos was highest in unburied treatments where embryos were incubated on substrates and not covered with sediments and ranged from 81.0 to 87.1% for shovelnose sturgeon and 55.2–80.0% for pallid sturgeon. Mean hatch of normal free embryos was lowest where incubating embryos were fully buried by MCS or FSS and ranged from 2.4 to 11.6% for shovelnose sturgeon and 4.8–15.2% for pallid sturgeon. We observed free embryos with physical abnormalities in all treatments; however, the occurrence was most variable in treatments fully and partially buried by MCS. Hatch of both species was also delayed in treatments where embryos were incubated fully and partially buried by MCS. Our results may be useful to estimate the relative suitability of spawning substrates in relevant river reaches.

Acipenseriformes (sturgeons and paddlefishes) are of substantial conservation concern, and development of genomic resources for these species is difficult due to past whole genome duplication. Development of disomic markers for polyploid organisms can be challenging due to difficulty in resolving alleles at a single locus from those among duplicated loci. In this study, we detail the development of disomic markers for the endangered pallid sturgeon (Scaphirhynchus albus) found in North America. One of the strategies for pallid sturgeon conservation is to stock U.S. rivers with offspring of pure pallid sturgeon, but introgression with the sympatric shovelnose sturgeon (S. platorynchus) threatens pallid sturgeon genetic integrity. Currently, 19 microsatellite loci are used to differentiate between both species and their hybrids, but the markers are insufficient to robustly identify backcrosses. We performed double digest restriction site‐associated DNA sequencing (ddRADseq) on shovelnose sturgeon haploid gynogens to produce a reduced‐representation genomic reference. Contiguous sequences that were heterozygous within a haploid individual were flagged as potentially encompassing multiple loci. Approximately 60 individuals of each species from two management units were sequenced, and reads were mapped to the haploid reference to identify single nucleotide polymorphisms (SNPs) at individual loci. The final data set contained 11,082 microhaplotyped loci which offer at least an order of magnitude greater resolution for species discrimination than the current panel of 19 microsatellites. These markers will be used to examine a larger sample of Scaphirhynchus individuals throughout their ranges to determine the extent and trajectory of hybridization.

The imperiled pallid sturgeon (Scaphirhynchus albus) and closely related, but more common, shovelnose sturgeon (S. platorynchus) are believed to broadcast adhesive, demersal eggs in the current and over coarse substrate in turbid rivers of the North American midcontinent. It has been hypothesized that eggs settle immediately following fertilization, but field conditions preclude direct observation. We conducted laboratory studies to characterize the diameter, shape, settling velocity, and specific gravity of pallid sturgeon and shovelnose sturgeon eggs. Based on these laboratory measurements, we then modeled the potential fate of pallid sturgeon eggs by considering these physical properties in the context of two field sites where spawning has occurred. Although eggs of pallid sturgeon and shovelnose sturgeon were of a similar size and shape, shovelnose sturgeon eggs had slightly higher specific gravity and settling velocity. For representative hydraulic conditions at documented spawning sites in the mainstem Missouri and Lower Yellowstone Rivers, eggs of both species will likely be concentrated near the bed although some eggs may be distributed throughout the water column. Simulations of egg transport indicate that eggs may be transported up to several hundred meters downstream from spawning locations in the approximate length of time required for eggs to become adhesive. Estimates of egg transport presented here rely on simplifying assumptions about river hydraulics and limited understanding of how eggs interact with the complex substrates and flow near the bed; results highlight the need for additional studies to evaluate the fate of eggs and fertilized embryos of both species.

We produced pallid sturgeon Scaphirhynchus albus embryos at five pre‐hatch developmental stages and isolated and quantified genomic DNA from four of the stages using four commercial DNA isolation kits. Genomic DNA prepared using the kit that produced the largest yields and concentrations were used for microsatellite DNA analyses of 10–20 embryos at each of the five developmental stages. We attempted to genotype the hatchery‐produced embryos at 19 microsatellite loci and confirmed reliable genotyping by comparing the microsatellite genotypes to those of known parents. Embryos at stages 5 and 8 did not produce reliable genotyping while those at stages 14, 24 and 33 did. We used the same DNA isolation method on 262 wild‐caught acipenseriform embryos collected from the lower Yellowstone River. A total of 200 of the wild embryos were successfully identified to stages 8 to 34 and the rest could not be staged. Using a combination of single nucleotide polymorphism and microsatellite markers, 249 of the wild‐caught embryos were genetically identified as paddlefish Polyodon spathula, five were identified as shovelnose sturgeon Scaphirhynchus platorynchus and eight failed to amplify. None were identified as pallid sturgeon. This study demonstrates that early‐stage wild‐spawned acipenseriform embryos can be genetically identified less than 24 h post‐spawn. This methodology will be useful for recovery efforts for endangered pallid sturgeon and can be applied to other acipenseriform species.

In the Upper Missouri River, Fort Peck and Garrison Dams limit the length of free‐flowing river available to the endangered pallid sturgeon. These barriers restrict the upstream migration of adults and downstream larval dispersal. A one‐dimensional (1D) modelling framework is currently in use to evaluate reservoir operation alternatives and to simulate drift of dispersing free embryos for different flow regimes and reservoir stages. This paper presents the results of a large‐scale tracer experiment conducted in 2016 and associated modelling performed to evaluate flow management scenarios that might aid species recovery. Breakthrough curves from the tracer experiment were used to infer the 1D longitudinal dispersion coefficient from a parameter optimization procedure. Simulations generated using the calibrated 1D advection–dispersion model were compared with field observations of the passive tracer and with larval fish collected during a previous experiment in 2007. When used with the appropriate range of dispersion coefficients, the 1D modelling framework agrees well with the available direct measurements of larval drift distances. Although we cannot unequivocally state whether insufficient length of free‐flowing river alone is causing recruitment failure, given the current thermal regime and our understanding of pallid sturgeon development, the time required for pallid sturgeon to transition to the benthos and initiate feeding might exceed the duration of drift available given constraints of reservoir operations.