Stock structure of shovelnose sturgeon analyzed with microsatellite DNA and morphological characters

Shovelnose sturgeon (Scaphirhynchus platorhynchus) caviar fisheries exist in several states throughout the Mississippi River drainage. Management of these fisheries may benefit from information about genetic stock structure. Sixteen microsatellite loci and morphological analysis were used to examine geographic stock structure of shovelnose sturgeon among seven geographic locations: five within continuous shovelnose sturgeon habitat, and two isolated by artificial barriers. Tissue samples were collected from 1999 to 2006 from the upper Missouri, Platte, lower Missouri, middle Mississippi, Ohio, Wabash, and Atchafalaya rivers. Geographic samples of shovelnose sturgeon samples could be separated into three groups with discriminate function analysis of four morphological characters. The microsatellite loci were highly variable (allelic richness range 5.65–13, observed heterozygosity range 0.64–0.89). Bayesian clustering did not identify multiple groups in the genetic data. However, significant genetic differentiation (θ_ST = 0.017, P < 0.0001) was observed among a priori defined geographic samples and all pairwise estimates of θ_ST were significant. Assignment testing among a priori defined groups indicated that the sturgeon from the upper Missouri, Platte, and Atchafalaya rivers had the highest assignment scores and thus were most distinct, while the lower Missouri and the middle Mississippi were less distinct and a larger fraction of the sturgeon from these rivers was genetically assigned to other rivers. The Ohio and Wabash rivers were genetically most similar. A Mantel test revealed a positive relationship between genetic and geographic distance (r = 0.464, P = 0.055) that was not statistically significant. The level of genetic differentiation observed at both molecular and morphological characters suggests that multiple shovelnose sturgeon populations may exist within the studied area, yet demographic factors and possible gene flow may have minimized the amount of genetic differentiation among locations.     

Hybridization between pallid sturgeon Scaphirhynchus albus and shovelnose sturgeon Scaphirhynchus platorynchus

This study found that introgressive hybridization of the pallid sturgeon Scaphirhynchus albus with the common shovelnose sturgeon Scaphirhynchus platorynchus has probably occurred across the range of S. albus. Bayesian clustering found evidence of hybridization in all management units of S. albus. Some individuals were intermediate at both genetic and morphological characters, and some had discordant results. The results support introgressive hybridization throughout much of the range of S. albus, yet individuals consistent with being pure members of each species were detected in all management units. Simulations demonstrated that it would be very difficult to distinguish introgressed individuals from pure specimens after multiple generations of backcrossing with these microsatellite markers. Using hybrid or backcross fish as broodstock could artificially accelerate the loss of unique genetic variation in S. albus. Additional microsatellite loci or additional genetic markers, along with morphological data may be required to ensure that hybrid or backcross fish are not used. Introgressive hybridization requires at least two generations and generation lengths of S. albus are long, perhaps as long as 30 years. The proportion of individuals consistent with introgressive hybrid origins indicates that hybridization between S. albus and S. platorynchus probably has occurred for several generations and is not a recent phenomenon.     

A note on the fecundity of pallid sturgeon

Fecundity was estimated for two pallid sturgeon, Scaphirhynchus albus, from the Lower Mississippi River (LMR). Sturgeon measured 827 and 886 mm fork length, weighed <3.2 kilograms and had 16 237 and 17 810 ova per kilogram of body mass (based on mean ovum density) with total fecundity estimates ranging from 43 357 to 58 913. Ovum size in the two sturgeon was 2.5–3.0 mm diameter. Pectoral fin ray sections showed distinct annuli and spawning bands. The smaller sturgeon was aged at 10 and the larger fish at 12 years. Spawning bands were present and suggest the smaller sturgeon first spawned at age 8 and the larger fish at age 9. When compared to published results, the data representing specimens from more northern populations suggest that pallid sturgeon from the LMR mature at comparable ages but at smaller sizes and have greater gonadal mass. These features and greater fecundity per mass unit (number of eggs per kilogram body weight) suggest that LMR pallid sturgeon populations exhibit greater potential for population growth and recovery than other populations with slower growth and lower fecundity.     

Diet of shovelnose sturgeon and pallid sturgeon in the free-flowing Mississippi River

Gut contents of shovelnose and pallid sturgeon from the lower and middle Mississippi River were obtained by colonic flushing, a safe and easily implemented alternative to gastric lavage. Diets of both species were dominated numerically by immature Trichoptera, Ephemeroptera, and Diptera. Primary prey, based on volume, for shovelnose sturgeon were Trichoptera, and for pallid sturgeon were various fishes. Geographic and seasonal nuances in diet were observed for both species, but the general dichotomy of shovelnose sturgeon as browser on invertebrates and pallid sturgeon as predator on fishes did not change. Data indicate that both species require hard substrates for feeding. Data demonstrate that colonic flushing is an effective technique for describing diet and inferring ecological and behavioral information about sturgeon.     

Inheritance of microsatellite loci in the white sturgeon (Acipenser transmontanus).

Nine tetramer motif (GATA)n microsatellite systems were developed for use in the white sturgeon, Acipenser transmontanus. We report inheritance patterns for these nine systems, which range from one possible disomic system to tetrasomy and octosomy, with some systems containing null alleles. Because of the complex modes of inheritance underlying these systems and the highly duplicated nature of the genome, we propose each allele be scored as its own dominant marker, similar to AFLPs or RAPDs. The utility of this method is validated by the observation that individual alleles within a microsatellite system generally fit the expectation for independent transmission and fit the expected transmission frequency for single copy nuclear markers.     

Population viability analysis of Lower Missouri River shovelnose sturgeon with initial application to the pallid sturgeon

Demographic models for the shovelnose (Scaphirhynchus platorynchus) and pallid (S. albus) sturgeons in the Lower Missouri River were developed to conduct sensitivity analyses for both populations. Potential effects of increased fishing mortality on the shovelnose sturgeon were also evaluated. Populations of shovelnose and pallid sturgeon were most sensitive to age‐0 mortality rates as well as mortality rates of juveniles and young adults. Overall, fecundity was a less sensitive parameter. However, increased fecundity effectively balanced higher mortality among sensitive age classes in both populations. Management that increases population‐level fecundity and improves survival of age‐0, juveniles, and young adults should most effectively benefit both populations. Evaluation of reproductive values indicated that populations of pallid sturgeon dominated by ages ≥35 could rapidly lose their potential for growth, particularly if recruitment remains low. Under the initial parameter values portraying current conditions the population of shovelnose sturgeon was predicted to decline by 1.65% annually, causing the commercial yield to also decline. Modeling indicated that the commercial yield could increase substantially if exploitation of females in ages ≤12 was highly restricted.     

Genetic evidence for hybridization of pallid and shovelnose sturgeon

To determine the genetic origin of individual sturgeon that are morphologically intermediate to pallid (Scaphirhynchus albus) and shovelnose (Scaphirhynchus platorhynchus) sturgeon, we combined previously published mitochondrial DNA (mtDNA) and microsatellite data with additional microsatellite data. Two sympatric populations of pallid and shovelnose sturgeon from the upper Missouri River and a sympatric population containing pallid, shovelnose, and putative pallid-shovelnose hybrids from the Atchafalaya River were analyzed using an index of hybridization and a principle components analysis of individual relatedness scores. The addition of new microsatellite data improved our ability to genetically differentiate individual pallid and shovelnose sturgeon collected in both areas. Our methods distinguished morphologically intermediate Atchafalaya River sturgeon, which appear to be genetically intermediate between pallid and shovelnose sturgeon. The results support a hybrid origin for morphologically intermediate individuals, although it is unclear whether they are all first-generation hybrids or if some are the result of subsequent backcrossing with the more common shovelnose sturgeon.     

Inheritance of microsatellite loci in the polyploid lake sturgeon (Acipenser fulvescens).

Inheritance in the expression of amplicons for four microsatellite primer pairs was determined using 10 families created from gametes of wild lake sturgeon (Acipenser fulvescens). Loci Afu34 and Afu68 expressed a maximum of two even-intensity bands per individual and had progeny genotype ratios that fit disomic inheritance (P > 0.05). Some variation exhibited at Afu34 and Afu68 was attributable to a null allele. Genotype expression at both loci also indicated that one female parent had transmitted unreduced gametes. Primer Afu39 amplified products that exhibited four gene doses, where genotype counts fit expected ratios for disomic inheritance (P > 0.05) indicating amplification of products from two disomic loci that share alleles. Meiotic drive was evident at the Afu39 loci based on a test for random segregation (P < 0.05). Only the expression of Afu19 gave evidence of tetrasomic inheritance based on a single progeny potentially produced by a double reduction gamete. No evidence for proposed octoploid inheritance was observed.     

Cortisol responses of pallid sturgeon and yellow perch following challenge with lipopolysaccharide

Plasma cortisol responses of pallid sturgeon Scaphirhynchus albus and yellow perch Perca flavescens following injection with equal doses of lipopolysaccharide were compared. Concentrations of cortisol in plasma from pallid sturgeon did not change following injection (6·0–11·0 v. 6·4 ng l⁻¹ pre-stress) while in yellow perch plasma they were shown to increase up to 6 h (117·0 v. 9·8 ng l⁻¹ pre-stress) after the injection. These results are consistent with other reports for pallid sturgeon that illustrate a reduced cortisol response following other applied stressors relative to teleosts and suggest differences in the expression and regulation of their inflammatory responses.     

Genetic discrimination of middle Mississippi River <Emphasis Type="Italic">Scaphirhynchus</Emphasis> sturgeon into pallid,shovelnose, and putative hybrids with multiple microsatellite loci

The pallid sturgeon (Scaphirhynchus albus), which is protected under the US endangered species act, and shovelnose sturgeon (S. platorhynchus), which is legally harvested in some locations, are sympatric throughout the range of pallid sturgeon. There is considerable morphological overlap between the species making discrimination problematic. The inability to reliably differentiate between species across all life stages has hampered pallid sturgeon recovery efforts. Furthermore, the two species are believed to hybridize. This study used allele frequency data at multiple microsatellite loci to perform Bayesian and likelihood-based assignment testing and morphological measures and meristics to discriminate pallid, shovelnose, and putative hybrid sturgeons from the middle Mississippi River. Bayesian model-based clustering of the genetic data indicated that two natural genetic units occur in the region. These units correspond to morphologically identified pallid and shovelnose sturgeon. Some individuals were morphologically intermediate and many of these failed to strongly assign genetically as either pallid or shovelnose sturgeon, suggesting they may be hybrids. These data indicate that pallid sturgeon and shovelnose sturgeon are genetically distinct in the middle Mississippi River (F _ST = 0.036, P < 0.0001) and suggest that hybridization between pallid sturgeon and shovelnose sturgeon has occurred in this region with genetic distance estimates indicating the greatest distance is between pallid and shovelnose sturgeon, while hybrid sturgeon are intermediate but closer to shovelnose. This study demonstrates that assignment testing with multiple microsatellite markers can be successful at discriminating pallid sturgeon and shovelnose sturgeon, providing a valuable resource for pallid sturgeon recovery and conservation.

Identification of microsatellite loci in lake sturgeon,Acipenser fulvescens,and their variability in green sturgeon,A. medirostris

From (CATC)_n, (GATA)_n, (AAAC)_n, and (CA)_n–enriched libraries for the lake sturgeon Acipenser fulvescens, 254 primer pairs were developed. These primer pairs resulted in the identification of 128 microsatellite loci in either A. fulvescens or A. medirostris. Polymorphic loci were identified in both sturgeon species for 48 of the primer pairs and 14 of the primer pairs amplified polymorphic loci only in A. medirostris. Most of the identified loci appear to be tetrasomic (79.1% in A. fulvescens and 64.5% in A. medirostris). These results offer estimates of the degree of diploidization in each of these species.     

Age and growth of pallid sturgeon in the free-flowing Mississippi River

Trotlines were used to capture pallid sturgeon in the free‐flowing Mississippi River, which extends from the Gulf of Mexico to the mouth of the Missouri River. Trotlines were baited with worms, and set overnight usually along the channel border. The pectoral fin rays of 165 pallid sturgeon caught in the Mississippi River were aged; 118 were from the lower Mississippi River (LMR) between the Gulf and mouth of the Ohio River, and 47 were from the middle Mississippi River (MMR) between the mouths of the Ohio and Missouri rivers. Initial agreement within ±1 year between two readers ranged from 53% for the LMR specimens, which were read first, to 84% for the MMR. Final age was agreed upon by both readers. For LMR pallid sturgeon, final age estimates ranged from 3 to 21 years with a mean (±SD) of 11.0 ± 4.7. For MMR pallid sturgeon, final age estimates ranged from 5 to 14 years with a mean of 9.5 ± 2.1. Seven pallid sturgeon marked with coded wire tags (CWT), indicating hatchery origin, were collected in the MMR. Age estimates for CWT fish were 7–8 years representing 1997 stocked fish, and 11–12 years representing 1992 progeny stocked in 1994. Von Bertalanffy growth equations for length indicated that pallid sturgeon in the MMR had higher growth rates for a given age than pallid sturgeon in the LMR. However, there were no significant differences (anova , P > 0.5) in the length–weight relationships between reaches. In the LMR, pallid sturgeon fully recruited to trotlines at age 11 and instantaneous total mortality (Z; slope of catch curve) was estimated at −0.12 (n = 10 year classes, r² = 0.55, P = 0.01). Of the 118 sectioned rays from the LMR, 28 could not be reliably aged (only one section from the MMR could not be aged). Therefore, age was predicted from length using the von Bertalanffy equation. The catch curve was re‐calculated using the predicted ages of the 28 pallid sturgeon in the LMR resulting in Z = −0.07. In the MMR, pallid sturgeon fully recruited to trotlines at age 9 and Z was estimated at −0.36 (n = 6 year classes, r² = 0.67, P = 0.04), which was significantly higher (anova , P = 0.04) than the LMR estimate. Higher mortality in the MMR may be due to habitat limitations compared to a larger, more diverse channel in the LMR, and incidental take of larger, older individuals during commercial harvesting of shovelnose sturgeon. Commercial take of shovelnose does not occur in the LMR except in the northern portion of the reach. Considering the presence of pallid sturgeon with CWT, recruitment of older individuals in the MMR may have been influenced by stocking a decade earlier. Management strategies for this endangered species should consider the differences in mortality rates among reaches, the impacts of commercial fishing on recovery of pallid sturgeon in the MMR, and the long‐term effects of hatchery fish now recruiting into the free‐flowing Mississippi River.     

Vulnerability of age-0 pallid sturgeon Scaphirhynchus albus to fish predation

Stocking is a commonly employed conservation strategy for endangered species such as the pallid sturgeon, Scaphirhynchus albus . However, decisions about when, where and at what size pallid sturgeon should be stocked are hindered because vulnerability of pallid sturgeon to fish predation is not known. The objective of this study was to evaluate the vulnerability of age-0 pallid sturgeon to predation by two Missouri River predators under different flow regimes, and in combination with alternative prey. To document vulnerability, age-0 pallid sturgeon (<100 mm) were offered to channel catfish Ictalurus punctatus and smallmouth bass Micropterus dolomieu in laboratory experiments. Selection of pallid sturgeon by both predators was measured by offering pallid sturgeon and an alternative prey, fathead minnows Pimephales promelas, in varying prey densities. Smallmouth bass consumed more age-0 pallid sturgeon (0.95 h⁻¹) than did channel catfish (0.13 h⁻¹), and predation rates did not differ between water velocities supporting sustained (0 m s⁻¹) or prolonged swimming speeds (0.15 m s⁻¹). Neither predator positively selected pallid sturgeon when alternative prey was available. Both predator species consumed more fathead minnows than pallid sturgeon across all prey density combinations. Results indicate that the vulnerability of age-0 pallid sturgeon to predation by channel catfish and smallmouth bass is low, especially in the presence of an alternative fish prey.     

The use of microsatellite loci for identification of sturgeon species (Acipenseridae) and hybrid forms

The genetic polymorphism of ten sturgeon species that inhabit the territory of the Russian Federation (Russian sturgeon, Siberian sturgeon, Amur sturgeon, Sakhalin sturgeon, Persian sturgeon, ship sturgeon, sterlet, stellate sturgeon, beluga, and kaluga) was examined at five microsatellite loci (Afug41, Afug51, An20, AoxD161, AoxD165) (in total, 3821 individuals). The examined loci were successfully amplified with the same primer set in all species tested and demonstrated a high level of variation. Alleles specific to different species have been identified, which allows them to be used to identify species of sturgeon and their food products. In addition, the possibility of identifying hybrid forms was demonstrated. The assignment test performed in the STRUCTURE software program showed a high probability of correctly assigning each individual to its species based on genotyping with five microsatellite loci examined (96–98%, on average). However, for Russian and Persian sturgeon, the rate of proper species assignment was considerably lower (75 and 84%, respectively).

     

Improved genetic identification of acipenseriform embryos with application to the endangered pallid sturgeon Scaphirhynchus albus

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.     

The clonal structure of Quercus geminata revealed by conserved microsatellite loci

The scrub oak communities of the southeastern USA may have existed at their present locations for thousands of years. These oaks form suckers, and excavations of root systems suggest that clones may occupy very large areas. Resolution of the clonal nature of scrub oaks is important both to manage the tracts of this ecosystem that remain, and in conducting long-term ecological studies, where the study area must substantially exceed the area occupied by any single clone. Microsatellites were used to determine the genetic diversity of a dominant oak species within a 2-ha long-term experimental site on Merritt Island at the Kennedy Space Center. This area contains a long-term study of the effects of elevated CO2 on the ecosystem. Conservation of seven microsatellite loci, previously identified in the sessile oak, Quercus petraea, was tested in two Florida scrub oak species, Q. geminata and Q. myrtifolia. Sequence analysis revealed that all seven microsatellite loci were conserved in Q. geminata and five loci were conserved in Q. myrtifolia. Six microsatellite loci were polymorphic in Q. geminata and these were subsequently used to investigate the clonal structure of the Q. geminata population. Twenty-one unique combinations of microsatellites, or haplotypes, occurred only once, whereas the remaining 26 individuals belonged to a total of seven different haplotypes. Trees with identical haplotypes were in close proximity, supporting the interpretation that they were clones. The results showed that there is significant genetic diversity within the 2-ha experimental site. Microsatellites provided a powerful and noninvasive tool for distinguishing individual genotypes and determining an adequate area for long-term ecosystem studies.     

Gender identification of shovelnose sturgeon using ultrasonic and endoscopic imagery and the application of the method to the pallid sturgeon

Monthly sampling of shovelnose sturgeon Scaphirhynchus platorynchus , a biological surrogate for the endangered pallid sturgeon Scaphirhynchus albus , was conducted to develop a multi‐seasonal profile of reproductive stages. Data collected included histological characteristics of gonads from wild caught fish and laboratory and field ultrasonic and endoscopic images. These data were used to compare effectiveness of ultrasonic and endoscopic techniques at identifying gender of adult shovelnose sturgeon at different reproductive stages. The least invasive method ( i.e . ultrasound) was least effective while the most invasive ( i.e . endoscope through an abdominal incision) was the most effective at identifying shovelnose sturgeon gender. In most cases, success rate for identifying males was greater than females, with success at identifying both genders greater in more advanced reproductive stages. Concomitantly, for most months average reproductive stage was more advanced for males than females. April and May were the months with the most advanced reproductive stage, and were the months when ultrasound was most effective. Methods were also applied in the Upper Missouri River to validate their use on pallid sturgeon Scaphirhynchus albus . Ultrasound was successful at identifying pallid sturgeon gender, however, endoscopic examination through the urogenital duct was only successful at identifying pallid sturgeon gender when the urogenital duct was not opaque.     

Estimation of gonad volume, fecundity, and reproductive stage of shovelnose sturgeon using sonography and endoscopy with application to the endangered pallid sturgeon

Most species of sturgeon are declining in the Mississippi River Basin of North America including pallid (Scaphirhynchus albus F. and R.) and shovelnose sturgeons (S. platorynchus R.). Understanding the reproductive cycle of sturgeon in the Mississippi River Basin is important in evaluating the status and viability of sturgeon populations. We used non‐invasive, non‐lethal methods for examining internal reproductive organs of shovelnose and pallid sturgeon. We used an ultrasound to measure egg diameter, fecundity, and gonad volume; endoscope was used to visually examine the gonad. We found the ultrasound to accurately measure the gonad volume, but it underestimated egg diameter by 52%. After correcting for the measurement error, the ultrasound accurately measured the gonad volume but it was higher than the true gonad volume for stages I and II. The ultrasound underestimated the fecundity of shovelnose sturgeon by 5%. The ultrasound fecundity was lower than the true fecundity for stage III and during August. Using the endoscope, we viewed seven different egg color categories. Using a model selection procedure, the presence of four egg categories correctly predicted the reproductive stage ± one reproductive stage of shovelnose sturgeon 95% of the time. For pallid sturgeon, the ultrasound overestimated the density of eggs by 49% and the endoscope was able to view eggs in 50% of the pallid sturgeon. Individually, the ultrasound and endoscope can be used to assess certain reproductive characteristics in sturgeon. The use of both methods at the same time can be complementary depending on the parameter measured. These methods can be used to track gonad characteristics, including measuring Gonadosomatic Index in individuals and/or populations through time, which can be very useful when associating gonad characteristics with environmental spawning triggers or with repeated examinations of individual fish throughout the reproductive cycle.     

Reproductive ecology and movement of pallid sturgeon in the upper Missouri River,Montana

Successful recruitment of endangered pallid sturgeon has not been documented in the upper Missouri River basin for decades, and research on the reproductive ecology of pallid sturgeon has been hindered by low sample size. A conservation propagation program was initiated in the 1990s, and the oldest age class of hatchery‐origin pallid sturgeon are becoming sexually mature increasing the number of reproductively‐active fish in the system. However, it is currently unknown how the reproductive ecology of hatchery‐origin pallid sturgeon relates to the few remaining wild fish. Following spring reproductive assessments, weekly relocations were recorded for each individual from late‐May to mid‐July to facilitate comparisons of spawning season movements among reproductive classifications and between spring hydrographs (2015 and 2016) for male pallid sturgeon. Mean total movement distances (±SE) were 104.5 km (18.9) for reproductively‐active wild males, 116.0 km (18.1) for reproductively‐active 1997‐year class males, and 20.6 km (3.0) for non‐reproductively‐active fish of unconfirmed sex. Movement characteristics of reproductively‐active males did not differ between 2015 and 2016 despite a difference of eight days in the timing of peak discharge and a difference of 79 m³/s (16.7%) in magnitude. Male aggregations were observed on the descending limb of the hydrograph in 2016 during temperatures suitable for spawning, but female pallid sturgeon underwent follicular atresia, similar to the other years of the study. Hatchery‐origin pallid sturgeon from the conservation propagation program appear to have retained reproductive characteristics from the wild broodstock, a key finding for a population where local extirpation of the wild stock is imminent.