INTRODUCTION

Stable isotope analysis of food webs can be a powerful tool for understanding the effects of large-scale ecological changes, such as the introduction of invasive species and eutrophication (Thorp et al. 1998; Herwig et al. 2007; Delong 2010). Unionid bivalves are cornerstones of aquatic food web condition in stable isotope studies because they integrate the food web base over time, have slow turnover rates, and are not as sensitive to seasonal variability as other measurements of the pelagic food web base (Cabana and Rasmussen 1996; Post 2002). A measure of δ¹⁵N from long-lived primary consumers, such as native mussels, allows calibration of site-specific background conditions that allow calculation of cross-site food chain length and other comparisons. Carbon isotope values from freshwater mussels can be used to identify sources of organic matter and to track the flow of carbon into primary consumers (Rounick et al. 1982; Finlay 2001; Brett et al. 2017), and nitrogen isotopes can be used as an indicator of nutrients entering the watershed (Lefebvre et al. 2009; Atkinson et al. 2014). Stable carbon and nitrogen isotopes can be used to calculate trophic niche space and to explore trophic relationships (Layman et al. 2007). In these and other ways, unionid mussels are a critical component for aquatic community stable isotope analysis to delineate food web dynamics. However, freshwater mussels as a group are sensitive to environmental degradation, with declining populations worldwide. Consequently, scientists increasingly employ nonlethal sampling methods for mussels, but this has resulted in the use of a number of different tissue-sampling protocols for isotope studies.

Stable isotope analysis requires sampling animal tissue or hemolymph and circa 1–2 mg (dry weight) of material is needed for mass spectrometer analysis. Given the conservation status of freshwater mussels, nonlethal and minimally invasive sampling methods for tissue collection are highly desirable. Three main tissue types are currently used for isotope studies of mussels: mantle tissue, foot tissue, and hemolymph from adductor muscles (McKinney et al. 1999; Gustafson et al. 2007; Weber et al. 2017). Mantle tissue biopsies have been shown to have no impact on long-term mussel survival (Berg et al. 1995). Foot biopsies and hemolymph extraction had no adverse effects on long-term survival of larger-bodied mussels (i.e., Amblema plicata, Elliptio complanata, E. crassidens; Naimo et al. 1998; Gustafson et al. 2005; Fritts et al. 2015), but the survival of a smaller-bodied species (Villosa vibex) was adversely affected by both methods (Fritts et al. 2015).

The widespread use of different tissues for isotopic studies of freshwater mussels has raised some concern that different tissues may produce different isotopic signatures and therefore limit the ability to compare isotope values across studies that have used different tissues (Weber et al. 2017). The published literature lacks a comprehensive comparison of variability between tissue sources for both carbon and nitrogen isotopes. Past studies that have conducted isotopic tissue comparisons between a combination of foot, mantle, and hemolymph have quantified the relation only among tissue sources for nitrogen isotopes (McKinney et al. 1999; Gustafson et al. 2007). However, carbon isotopes also provide valuable information about organic matter sources, including whether or not the sources are allochthonous or autochthonous, benthic or pelagic, and littoral or pelagic (Vander Zanden et al. 1999). Evaluating the relation of these isotopes among species can also provide insight into shared resource use, diet, and comparability of signatures among species at a site (Nichols and Garling 2000; Raikow and Hamilton 2001; Christian et al. 2004; Novais et al. 2016; Weber et al. 2017). Most food web models use both carbon and nitrogen isotopes in concert, which establishes the need for a more comprehensive assessment of tissue compartment comparisons for both isotopes.

The first objective of our study was to quantify the relation of carbon and nitrogen isotopes between foot and mantle tissue of two common freshwater mussel species (Amblema plicata and Fusconaia flava) in the Mississippi River Basin and to establish if these two tissue compartments produced comparable isotope results. The second objective of our study was to test for significant differences between these two species within a site for each isotope and tissue source. If the isotopic responses are statistically similar between the two species, this could allow for simplified sampling designs, particularly when a species is not able to be sampled consistently over the spatial gradient of interest.

METHODS

Tissue Sample Collection and Processing

Mantle samples were taken by gently prying open the mussel with a dull, flat-tipped sterile steel diving knife. Mantle tissue was held with a sterile duck billed forceps, and a 1 cm² section was snipped with sterile surgical scissors. Foot tissue was sampled in a similar way or with a biopsy needle (Bard Biopsy, Tempe, AZ). A subset of individuals had a duplicate sample collected from the same tissue source to evaluate variability within a tissue compartment. Tissue samples were immediately put on ice and transported to a –20°C freezer within 6 h. Frozen samples were transported to the Aquatic Resources Ecology Laboratory at Northland College (Ashland, Wisconsin) where they were dried at 60°C for 72 h and homogenized with mortar and pestle. Samples were then weighed, rolled into tin capsules, and shipped to Cornell University Stable Isotope Laboratory, Ithaca, New York. Stable isotope ratios of carbon (¹³C/¹²C) and nitrogen (¹⁵N/¹⁴N) were determined with a Thermo Delta V isotope ratio mass spectrometer interfaced with a NC2500 elemental analyzer. In-house standards (mink animal material and methionine chemical standards) were run every 10 samples. In-house standards were routinely calibrated against international reference materials provided by the International Atomic Energy Association. Isotope data were expressed relative to Vienna PeeDee Belemnite for δ¹³C and atmospheric air for δ¹⁵N. By convention, C and N isotope ratios are expressed as δ, the deviation from standards in parts per thousand (‰), according to the following equation:

where X is ¹³C or ¹⁵N and R is the corresponding ratio ¹³C/¹²C or ¹⁵N/¹⁴N. Instrument precision for calibrating isotope ratios was 0.10–0.33‰ for δ¹³C and 0.12–0.19‰ for δ¹⁵N.

Figure 1.

Sampling locations in the Upper Mississippi River basin, including seven sites in the St. Croix River and three sites in Pool 2 of the Mississippi River.

RESULTS

We sampled both foot and mantle tissue from 73 A. plicata and 87 F. flava (Table 1), with 56 replicates of the same tissue source within an individual to evaluate variability within a tissue compartment. All data are publicly available through ScienceBase (doi.org/10.5066/P9G92506). Among sampling locations, δ¹⁵N values ranged from 5.76 to 12.63‰, and δ¹³C ranged from –29.26 to –33.96‰. Mantle and foot tissue sources were positively correlated with regard to δ¹⁵N and δ¹³C for both species (Figs. 2, 3). Linear regression of δ¹⁵N and δ¹³C data resulted in respective r² values of 0.96 and 0.93 for A. plicata and 0.97 and 0.93 for F. flava (Table 2). Combining the data from both species resulted in r² values of 0.96 and 0.93 for δ¹⁵N and δ¹³C (Table 2).

Paired t-tests indicated that there was not a statistically significant difference in δ¹³C between tissues in A. plicata but δ¹⁵N did differ, while both isotopes were significantly different in F. flava (Table 3). Foot tissue was slightly enriched over mantle tissue for both species for δ¹⁵N (0.10–0.21‰). Mantle tissue was slightly enriched in δ¹³C relative to foot tissue for F. flava, and there was no statistically significant difference between tissue sources for δ¹³C in A. plicata. When analyzing both species combined, δ¹⁵N was enriched in foot relative to mantle, and there was no difference between the tissue sources for δ¹³C. However, the overall difference between the tissues was only 0.04–0.2 l‰ for both isotopes, which is less than the analytical precision of the mass spectrometer (i.e., circa 0.2–0.3‰). There was no statistically significant difference between duplicate samples taken from the same tissue source (Table 4), and the regression relationships were very strong for both species, individually and combined (r² = 0.92–0.99; Table 5).

Table 1.

Amblema plicata and Fusconaia flava sample size (N) and size parameters (mean ± SD) of specimens collected from the Mississippi and St. Croix rivers for this study.

The comparison of isotopes between species across sites indicated that δ¹⁵N was not significantly different between the species across all sites for both foot tissue and mantle tissue (Table 6, Fig. 4A, C). Carbon isotopes from foot tissue were significantly different between the species at two locations, one in the Mississippi River and one in Lake St. Croix, but not at any of the remaining locations, and δ¹³C from mantle tissue was not significantly different between the species at any location (Table 6, Fig. 4B, D). Only one species (F. flava) was able to be collected at M2MC2, therefore species comparisons could not be conducted for this site.

Figure 2.

Simple linear regression of foot and mantle δ¹⁵N per individual, with r² values of 0.955 for Amblema plicata and 0.967 for Fusconaia flava.

Figure 3.

Simple linear regression of foot and mantle δ¹³C per individual, with r² values of 0.925 for Amblema plicata and 0.934 for Fusconaia flava.

DISCUSSION

This is the first study to compare both carbon and nitrogen isotopes between mantle and foot tissue compartments in freshwater mussels. For these two common and widely distributed species in the Upper Mississippi and St. Croix river systems, foot and mantle tissue sources were very similar with regard to δ¹⁵N and δ^l3C. For the isotope/species combinations that had statistically significant per-mil differences in isotope composition between paired tissue compartments, the differences (i.e., 0.10–0.2l‰) were within the range of instrumentation error (i.e., circa 0.2–0.3‰) and suggest that these differences would be unmeaningful in regard to food web analyses. We conclude that the tissue compartments are effectively interchangeable for carbon- and nitrogen-stable isotopes for these two species within this study system.

Table 2.

Simple linear regression results between the stable isotopes (δ¹³C and δ¹⁵N) of foot tissue versus mantle tissue for Amblema plicata, Fusconaia flava, and both species combined.

Table 3.

Stable isotope results of paired t-tests for foot versus mantle tissue for Amblema plicata (N = 73), Fusconaia flava (N = 87), and both species combined (N = 160). Difference (‰) = average difference between tissue types, and SD (dif) = standard deviation of the difference. Statistically significant differences are denoted in bold.

While no studies have compared carbon isotopes between tissue sources, two studies have compared nitrogen isotopes among different tissues. Elliptio sp. foot tissue had slightly less spatial variability for nitrogen isotope signatures across small ponds as compared to mantle and adductor muscle tissue compartments (McKinney et al. 1999). Adductor muscle was enriched by 1.06‰ relative to mantle and foot tissues, and mantle tissue was enriched by 0.13‰ relative to foot tissue. Our findings for A. plicata and F. flava were the opposite, with foot tissue being more enriched in δ¹⁵N relative to mantle tissue (i.e., 0.10–0.21‰). Like our study, the differences between foot and mantle tissues in the McKinney et al. (1999) study were less than the instrumental error and thus unmeaningful for the purposes of the food web analyses typically used.

Gustafson et al. (2007) found a strong positive relation (r² of 0.792) between nitrogen isotopes of foot tissue and hemolymph from Elliptio complanata. Foot tissue was generally enriched relative to hemolymph, but the actual difference in δ¹⁵N between the tissues was not reported (Gustafson et al. 2007). We found even stronger correlations for δ¹⁵N (r² > 0.96) and a difference of only 0.10–0.2 l‰ between mantle and foot tissue for A. plicata and F. flava. The tighter relation in our study compared to Gustafson et al. (2007) suggests that cross-study comparisons where foot or mantle tissues only were used might be more robust than comparisons that included hemolymph and other tissues.

The remarkably low variability in isotopic signatures among freshwater mussels within a location is one of the hallmarks that has made them an ideal taxon for isotopic baseline adjustment in food web studies (Cabana and Rasmussen 1996; Post 2002). Studying the isotopic relationship between/among species within a location can further enhance our understanding of the utility of freshwater mussels in isotope studies. A number of studies have indicated that δ¹⁵N does not differ substantially among unionid species within a location (Nichols and Garling 2000; Raikow and Hamilton 2001; Christian et al. 2004; but see Weber et al. 2017), while the relationship of δ¹³C between/among species has been more mixed (Nichols and Garling 2000; Christian et al. 2004; Weber et al. 2017). Unionid mussels have also been documented to differ in their carbon and nitrogen isotopic ratios relative to the invasive Corbicula fluminea (Atkinson et al. 2010).

Table 4.

Stable isotope results of paired t-tests for duplicate samples taken from the same tissue source from Amblema plicata (N = 22), Fusconaia flava (N = 34), and both species combined (N = 56). Difference (‰) = average difference between duplicate samples, and SD (dif) = standard deviation of the difference.

Our data indicated that there was not a statistically significant difference for either isotope or tissue type between the species at nearly any site in this study. The equivalent isotopic signatures from these two unionid species by site justifies using one, rather than both, species for isotope studies in the Upper Mississippi and St. Croix rivers. These results also lay the foundation for using these two species interchangeably if one is not able to be sampled consistently over a spatial gradient in these Midwestern rivers. In our study system, this opens up more options for comparing food webs in the Mississippi and St. Croix Rivers over larger longitudinal (up and down the river and tributaries) and lateral (on-channel to off-channel) gradients, and we suspect this will also be useful for comparisons over time (Hornbach et al. 2018). The isotopic similarity of A. plicata and F. flava could simplify sampling designs and save time and money on sampling, all without diminishing the usefulness of the data.

Table 5.

Simple linear regression results between the stable isotopes (δ¹³C and δ¹⁵N) of duplicate samples taken from the same tissue within an individual for Amblema plicata, Fusconaia flava, and both species combined.

Table 6.

Two sample t-test comparison of isotopes between species across sites. Statistically significant values after Bonferroni correction are denoted in bold. Sites that begin with M2 are from the Mississippi River Pool 2, and sites that begin with S are locations in the St. Croix River, listed from most downstream to most upstream. N = number of individuals per species per site, SD = standard deviation. Only one species was able to be collected at M2MC2, therefore species comparisons could not be conducted for this site.

This project advances the state of the science for isotopic studies in freshwater mussels by comparing two tissues for two isotopes in two species in two rivers over a large gradient of isotopic values. We argue that the similarity of the isotopic responses of foot and mantle tissue justifies retroactively comparing results between studies that have used these tissue types. However, we urge caution in overextending the implications of these findings outside of this geographic area, these species, or the range of isotopic values encountered in our study. Future research should evaluate the comparability of isotopic signatures between tissues of additional species from different geographic regions and habitats to evaluate the potential for species specific or location-based factors that may result in different patterns than what we observed in our study.

Figure 4.

Box plots of a species comparison of isotopic values between Amblema plicata (white boxes) and Fusconaia flava (grey boxes) among sampling locations. Center horizontal lines are medians, upper and lower margins of the box are 25th and 75th percentiles, and the bars are the 5th and 95th percentile. Outliers are represented as individual points. A = foot tissue δ¹⁵N, B = foot tissue δ¹³C, C = mantle tissue δ¹⁵N, D = mantle tissue δ¹³C. Sites at which the species were significantly different are denoted with an asterisk above the paired boxes.