Fish collection

We have treated all animals in accordance with NDSU guidelines on animal care (IACUC protocol A17007).

In the Red River Basin, Bigmouth Buffalo were collected from Otter Tail County, Minnesota, along two tributaries of the main stem of the Red River of the North (henceforth, Red River): the Pelican River and the Otter Tail River. The Pelican River Basin sites included eight lakes: Crystal, Lizzie, Rush, Fish, Pelican, Little Pelican, North Lida, and Prairie. These lakes are along a 26 km reach of the river from which specimens (n = 224) were taken during 2016–2018 via Fyke net, gill net, hook and line, and bowfishing. Otter Tail River Basin individuals (n = 33) came from a single site below Orwell Dam in April of 2018 via hook and line. Specimens were immediately measured to obtain wet mass (±1 g) and total length (±0.1 cm), photographed laterally with a scale bar, and then dissected to obtain gonadal tissue for sex determination and mass (±0.1 g).

In the Mississippi River Basin, Bigmouth Buffalo were collected along two tributaries of the Mississippi River: the Crow River and the Minnesota River. Fish were obtained from a commercial harvest on 4 May 2017 from Artichoke Lake (n = 52) in Big Stone and Swift County, Minnesota (on the Minnesota River); and on 22 Sept 2017 from Lake Minnetaga (n = 66) in Kandiyohi County, Minnesota (on the Crow River). An additional 11 specimens were obtained from a bowfishing take on Tenmile Lake, Otter Tail County, Minnesota (on the Minnesota River) in May of 2018. For Artichoke Lake fish, measurements, photographs, and sex determination were obtained after fish had been frozen and thawed. For Lake Minnetaga and Tenmile Lake, fish data were obtained as previously described for the Red River Basin specimens, except that Lake Minnetaga specimens were dissected for sex determination after being frozen and thawed.

Otolith preparation and age analysis

Otoliths were removed from fish by first exposing the ventral surface of the cranium, through the otic bullae under the operculum. At least one otolith was obtained from every fish dissected (n = 386) and in most cases (77%) the complete set of six otoliths (asterisci, sagittae, and lapilli) was collected. Following extraction, the otoliths were gently removed from the labyrinth organ with forceps and placed in 1.5 ml plastic microvials pre-filled with water to prevent any residual tissue or fluid from drying to the surfaces. All collected otoliths were rinsed and submersed in distilled water to photograph the whole otolith set at 10X with an Olympus® SZH10 dissecting microscope using transmitted light in dark-field mode. The orientation of the nuclear transect to be thin-sectioned from the whole otolith was determined from these images. Otoliths were then dried for 30 min at 55 °C and lapilli were weighed (±0.001 g) using a CAHN Electrobalance®. Only the lapilli were weighed because they produce the most reliable weight measures. Of all Bigmouth Buffalo otoliths, lapilli are the largest, least fragile, and least likely to hold residual endolymph. Sagittae are the smallest otoliths in Bigmouth Buffalo and fracture easily, while asterisci have grooves that are difficult to thoroughly clean of non-otolith material (both factors that led to unreliable weight measures).

Weighed otoliths were embedded in ACE® quick-setting epoxy within 1.5 cm3 compartments (lined with petroleum jelly) in a plastic tray. After the epoxy hardened, the epoxy block was placed in a Buehler IsoMet™ 1000 low-speed saw equipped with diamond-embedded thin-sectioning blades to obtain 300–500 μm sections via the wafer method. A total of 557 otoliths (315 asterisci, 241 lapilli, and 1 sagittal) were thin-sectioned to obtain age estimates for these 386 Bigmouth Buffalo. Sagittae are the most difficult otoliths to section in Bigmouth Buffalo, and thus were rarely used. Sections of asterisci and lapilli from the same individual produce essentially the same age estimate for the entire range of ages (Figs. 1 and 2), proving that either structure can be used. For 165 individuals, only asterisci were thin sectioned, and for 114 specimens only lapilli. The remaining specimens had both asterisci and lapilli (n = 106), or lapilli, asterisci, and sagittae (n = 1) thin sectioned to provide comparison opportunities within individual fish. In addition, both asterisci from a single specimen were sectioned for 25 individuals, and both lapilli for 11 individuals. Although many sections were taken, a small portion (~15%) were too fractured or structurally polymorphic to be readable. Nonetheless, at least one readable section from every Bigmouth Buffalo in this study was obtained.

Thin sections of the otoliths were mounted on a glass microscope slide and immersed in mineral oil to enhance visibility and photographed at 75× under a compound microscope using transmitted light. Multiple images per thin section were required to provide a composite image of the whole otolith section at this magnification. Images were stitched together using Adobe Photoshop software to create the high-resolution composite image of the whole thin section. The composite images were then examined for annuli that could be quantified and were digitally marked (Fig. 1).

The best otolith sections were assigned ages by multiple readers, with consensus readings used to determine the final age assigned to each specimen. First, a primary and secondary reader independently marked annuli on duplicate images of the thin section. Discrepant annuli counts between the primary and secondary reader were identified using a minimum criterion of 1 year per decade of age. For example, reader counts for individuals scored 0–9 years of age were deemed discrepant if the primary and secondary reader scores differed by more than ±1 annulus count. This approach was used for individuals scored up to 110–119 years (deemed discrepant if the primary and secondary reader scores differed by more than ±12 annulus counts). If reader scores fell into separate decades, the younger age group criterion was used. Images of otoliths identified as discrepant based on these criteria were then either independently analyzed by a third reader (n = 29), or another otolith section(s) already available from the same fish was aged by both primary and secondary readers. If consensus scores were still not obtained between readers, then yet another otolith was thin-sectioned from that specimen and again scored independently by the primary and secondary reader, at which point all age estimates were resolved. Otoliths for which annuli counts were not identical between readers, but not identified as discrepant (e.g., scored 12 by the primary and 13 by the secondary), a final determination was made by the primary reader. The overall between-reader precision (primary and secondary) was a coefficient of variation (CV) of ~5.6%. This precision varied with age and was highest in the youngest group of fish, as expected. For individuals across each of the 12 decadal age groups in this study (from 0–9, to 110–119 years) the precision was CV ~10.4, 5.7, 4.0, 4.5, 4.5, 3.6, NA, 3.3, 2.9, 3.4, and 2.7, and 3.9% respectively.

Bomb radiocarbon dating

We selected for radiocarbon analysis 15 lapillus otoliths from Bigmouth Buffalo previously aged via a thin-sectioned asteriscus or lapillus (or both) annulus count(s). These fish spanned the range of chronological dates required for this type of age validation work (Table 1). Typically, a selection of birth years that range from the pre-bomb period (earlier than ~1955) to the post-peak decline period (more recent than the 1970s) is used to trace the bomb-produced 14C signal through the lifespan of the species, and to potentially provide diagnostic ages from birth years associated with the rapid rise of 14C in the 1950s and 1960s. For dating, we chose a lapillus to the matching, thin-sectioned asteriscus or lapillus (or both) used for age determination (Table 1), because lapilli are the largest otoliths by mass for Bigmouth Buffalo and thus were most likely to provide a sufficient amount of calcium carbonate for 14C analyses. The 15 lapillus otoliths selected for bomb 14C dating were sectioned in a similar manner to the previously described thin sectioning, except that they were serially sectioned using a single blade to a thickness of ~1 mm. We selected a section that contained the desired core (the first 1–2 years of growth), with a planar orientation normal to the growth layer structure, such that the growth layers were not tilted and the micromilled material would include only the targeted growth years. A section thickness of 1 mm was necessary to provide greater material depth for micromilling and sufficient mass for 14C analysis.

Otoliths were micromilled using a New Wave Research micromilling machine to a depth of ~600–800 μm providing ~0.5–1.3 mg of carbonate per sample (Table 1). A total of 15 specimens spanning the bomb 14C chronology were milled for the core region of the otolith, representing the first 1–2 years of growth, and for 13 of these, only the core was extracted. From the two additional individuals (both estimated to have hatched prior to atmospheric nuclear testing in the 1950s and 1960s), multiple samples were extracted per otolith in a radial pattern that began after the core extraction and led into more recent years of formation (Table 1). The goal was to detect the location in the otolith section (year or years of formation) where the time-specific rise of bomb-produced 14C occurred (~1955). This approach can validate age estimates exceeding the minimum maximum age indicated by pre bomb radiocarbon levels in the otolith core. The radial extractions were assigned a mean year of formation by overlaying the annulus structure (an image from the aged lapillus section) on an image of the path extracted by the micromill.

We submitted 28 extracted otolith samples as carbonate to the National Ocean Sciences Accelerator Mass Spectrometry Facility (NOSAMS), Woods Hole Oceanographic Institution in Woods Hole, Massachusetts, for 14C analysis. Radiocarbon measurements were reported by NOSAMS as Fraction Modern (the measured deviation of the 14C/12C ratio from Modern). Modern is defined as 95% of the 14C concentration of the National Bureau of Standards Oxalic Acid I standard (SRM 4990B) normalized to δ13C VPDB (–19‰) in 1950 AD (VPDB = Vienna Pee Dee Belemnite standard)57. Radiocarbon results were corrected for isotopic fractionation using a value measured concurrently during the accelerator mass spectrometry analysis, and these data are reported here as F14C. These values were date corrected based on the estimated year of formation and are reported58 as Δ14C. Stable isotope δ13C measurements were made on a split of CO 2 taken from the CO 2 generated during acid hydrolysis. These values are robust and can be used to infer carbon sources in the formation of the otolith carbonate.

Measured Δ14C values were used to determine the validity of age estimates by comparing the purported year of formation (birth year), calculated from the collection year and estimated age relative to regional Δ14C references (Figs. 4, 8, and 9). Temporal alignment of the measured Δ14C values from otolith material with regional Δ14C reference records from otoliths of other freshwater fishes provided an independent basis for determining fish age, and for evaluating our age reading protocol for Bigmouth Buffalo based on otolith annulus counts. The only thorough 14C reference records available for the freshwater environment of North America were from arctic lakes and mid-continent lakes near the Great Lakes, because very little work has been done in this regard (Fig. 8).

Freshwater radiocarbon references for North America

Overall, bomb radiocarbon dating is considered one of the best methods of age validating long-lived fishes30. The radiocarbon (14C) data used as reference material to validate the age and longevity of Bigmouth Buffalo (Ictiobus cyprinellus) in this study were from a series of rare freshwater sources that used otoliths of two fish species from widely separated regions of North America (Fig. 8). These bomb 14C records were from otoliths of either known-age (juvenile fish) or aged adults (otolith cores) from: salmonids of Arctic lakes (Salvelinus namaycush and S. alpinus)35, and Freshwater Drum of central North America lakes (Aplodinotus grunniens45 and U.S. Fish and Wildlife Service - Northeast Fishery Center, Lamar, Pennsylvania, unpublished data). These data sets were fitted with a Loess curve (spline interpolation smoothing parameter = 0.4, two-parameter polynomial; SigmaPlot v.11.2) to describe the central tendency of each time series (Fig. 4). A caveat of the curve fitting is that one Freshwater Drum specimen from Lake Ontario (2012) was elevated relative to all others from Oneida Lake in 2012–2014 and was considered more likely to be similar to the Arctic references due to hydrography of the Laurentian Basin. This 14C value may have been elevated due to increased atmospheric exposure from greater water mass residence times in the Great Lakes (relative to Oneida Lake) along with other catchment factors associated with the delivery of terrestrial carbon sources that can be 14C-enriched59. The bomb-produced changes in freshwater 14C for North America may begin with what appears to be variable 14C levels in the pre-bomb period (Δ14C ranged from approximately –80‰ to –125‰ before 1955) and become coincident as the sharp bomb-produced 14C rise begins near 1955 (Fig. 4). At mid-rise, near 1960, the regional records (Arctic vs. Central North America lakes) start to diverge and then exhibit differences in peak amplitude and subsequent decline. A separation of the records is maintained through the decline period of the 1970s to the 2010s, but the signal appears to dovetail toward most recent years, provided the elevated specimen from Lake Ontario is an accurate reflection of regional variability. Regardless of the potential for minor variability in 14C levels, the 14C rise due to atmospheric testing provides a valid marker that can be used to determine the validity of age estimates, with further support from the generally consistent pattern of the overall rise and fall of bomb-produced 14C.

Very little work has been done with determining the full bomb-produced 14C signal in freshwater environments — most has been within the marine environment (usually in the mixed layer using various forms of biogenic carbonate). There are differences between the bomb-produced 14C signals in these environments, primarily because of the way 14CO 2 from nuclear testing enters the hydrologic system. While input of the bomb 14C signal to the ocean system relies mostly on air-sea diffusion at the sea surface, the freshwater environment has a more direct advection of bomb 14C from the atmosphere to rivers and lakes via precipitation. Hence, the hydrology of the freshwater environment leads to a more synchronous link to 14C changes in the atmosphere and exhibits a more rapid 14C rise than the marine environment (Fig. 9). The 14C peaks expressed for the Arctic and central North America lakes may be artificially muted because actual peak dates may not have been sampled35. Nonetheless, the marine bomb-produced 14C signal is usually attenuated and phase lagged relative to both freshwater and atmospheric 14C records (Fig. 9). The exceptions are either, close-in fallout that generated a strong regional 14C signal in the marine environment60, or places where there are 14C-depleted sources from either unique hydrogeology (karst topography; AH Andrews, pers. observation) or upwelled waters of the deep-sea61. For the existing freshwater 14C records it is the temporal similarities, despite differences in amplitude, that indicate tracing the bomb-produced 14C signal in other freshwater environments of North America (e.g. river basins of Minnesota for Bigmouth Buffalo). These temporal constraints on otolith 14C measurements can be used to validate age estimates.

In some cases, otolith core material cannot be used as a strong record of support for determining the age of other organisms because of reasoning circularity — a fish of unknown age that was age-validated from a reference 14C record should not be in turn used as a reference to validate the age of other otolith measurements of unknown age. However, this is avoidable when otolith annuli are very well defined and there is little or nothing else to refer to as a regional 14C reference. If the temporal nature of the nearest regional 14C signal is a match with the otolith material’s signal (position in time based on annulus counts from the otolith), then an assumption can be made that adults of the species can provide a bomb-produced 14C timeline where none existed. Hence, this is the case for both the Arctic salmonids and Freshwater Drum used as a reference in the current study on Bigmouth Buffalo. Known age juvenile fish and cored adults with well-defined otolith annuli produced strong evidence of the regional 14C signal of freshwater environments in North America. This data provides a strong basis for validating other freshwater fishes in this region (e.g. Bigmouth Buffalo). These are the most complete records for this environment. The only other records for freshwater environments of North America come from Lake Sturgeon (Acipenser fulvescens)46 and Pallid Sturgeon Scaphirhynchus albus)47, but these 14C records are not as complete.

Mark recapture

Bigmouth Buffalo from Big Pelican and Little Pelican Lakes (Otter Tail County, Minnesota) were captured during 2011–2018 by hook-and-line. Captured individuals were photographed, measured (total length and wet mass, as described previously), sexed (based on visual examination of the urogenital opening, presence of tubercles, or expression of gametes (or combination thereof)), tagged at initial capture using either safety pins or Visible Implant Elastomer tags (Northwest Marine Technology, Inc.), and released in good condition.

Statistics and reproducibility

We used JMP Pro Statistical Discovery™ Software (Version 13.0, SAS Institute, Inc. 2014) for statistical analysis and graphical output. SigmaPlot (Version 11.2) was used to render smoothed curve fits (Loess function, spline interpolation smoothing parameter = 0.4, two-parameter polynomial) to the regional 14C reference data (Figs. 3, 4).

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.