DOI: 10.5061/dryad.3n5tb2rt5
title: Intraspecific variation in the organismal stoichiometry of the Least Killifish tracks spatial variation in periphyton composition

description: Ecological stoichiometry, the mass-balance relationships in elemental
 composition among species in an ecosystem, is fundamental to understanding
 a wide variety of ecological phenomena, from foraging patterns to nutrient
 cycling (Sterner and Elser 2002). Variation in organismal stoichiometry,
 be it interspecific or intraspecific, can drive substantial variation in
 community and ecosystem dynamics (El Sabaawi et al. 2015, Des Roches et
 al. 2017, Leal et al. 2017a). Here we report a survey of the organismal
 stoichiometry and trophic niche of H. formosa in eight
 populations, four from each of two contrasting habitat types. First, we
 describe variation in water chemistry among a larger set of 17 locations,
 from which we selected our eight populations for study. We also document
 variation among these eight locations in the elemental composition of
 periphyton in the littoral zone, where H. formosa forage. Second, we
 describe the trophic niche of H. formosa in those eight populations*,*
 establishing that the fish occupy a trophic position consistent with their
 being a primary consumer. Third, we examine the organismal stoichiometry
 in those populations. We show that there is less population variation than
 has been reported in other species, but the variation that we detected
 tracks variation in the carbon and nitrogen profiles of the periphyton on
 which these fish feed.
description: We sampled water at 17 locations with populations of <em>H.
 formosa</em>. These locations embraced a variety of habitats,
 including springs, wooded swamps, open canopy lakes, and freshwater
 marshes. We selected eight of these locations for stoichiometric and
 stable isotope analyses: four freshwater springs (McBride’s Slough,
 Newport Bridge, Shepherd’s Spring, Wacissa River) and four lakes (Harper’s
 Eyelet, Little Lake Jackson, Moore Lake, Trout Pond). We chose these eight
 locations for two reasons. First, they spanned a wide range of water
 chemistry parameters. Second, the populations of <em>H.
 formosa</em> in these locations displays a wide range of population
 densities and life histories (Schrader and Travis 2012, Macrae and Travis
 2014).  We collected fish via dip netting in the
 shallow littoral zone, typically at depths less than 0.30 m. We sacrificed
 fish via rapid chilling in an ice slurry, following approved IACUC
 protocols. We obtained periphyton samples by scraping stems and leaves of
 aquatic plants, larger rocks, and fallen limbs wherever we saw
 <em>H. formosa</em> individuals foraging. We placed all
 samples on ice for return to the laboratory.
 <em>Water Chemistry Methods</em>
 We collected water samples by lowering a Nalgene bottle on a long
 pole into clear water, approximately 1.5 meters from the shoreline and
 away from aquatic vegetation. We filtered the water on site by pouring
 through a 10 μl filter and immediately placed the filtered water on ice
 for transport the same day to Akuritlabs, Inc., in Tallahassee (National
 Environmental Laboratory Certification E81350) for analyses. We obtained
 data on pH, concentrations of chlorophyll a, total nitrogen, organic
 nitrogen, nitrite, nitrate, ionized and unionized ammonia, total
 phosphorus, and orthophosphate. For analyses, we used data only on pH,
 chlorophyll a, nitrate, and total nitrogen because only for these
 variables were concentrations above the detection limit at all
 locations. <em>Elemental composition and isotope
 methods</em> We prepared fish tissue and
 periphyton samples for elemental and isotope analyses following Aresco et
 al. (2015). In brief, upon return to the laboratory, we removed and
 discarded all internal organs, including reproductive tissue, gut, and
 liver/viscera, where lipids are stored for future use (McManus and Travis
 1998), and freeze-dried the carcasses. We freeze-dried all periphyton
 samples in the same manner.  We ground all freeze-dried material to a fine
 powder using a WiglBug Model 3110B, rinsing the capsule and grinding
 bearing with ethanol between samples. For analyses of carbon and nitrogen,
 we prepared ground material in small tin capsules with, on average, 1 mg
 fish tissue and 2 mg periphyton. These samples were analyzed using the
 elemental analyzer and continuous flow isotope ratio mass spectrometer at
 the University of California, Davis, Stable Isotope Facility.
 We analyzed total phosphorus concentration in fish tissue as
 concentration of <sup>31</sup>P with the Thermo 2 inductively
 coupled plasma mass spectrometry system (ICP-MS) in the Geochemistry Lab
 at the National High Magnetic Field Laboratory in Tallahassee, Florida.
 The ICP-MS method is especially accurate for small amounts of sample
 material or samples with low concentrations of total phosphorus (Cooper et
 al. 2005; Wilschefski and Baxter 2019). We prepared samples by dissolving
 them in nitric acid, following protocols described in Wilschefski and
 Baxter (2019), using 1 ppm phosphorus and 2 ppb indium as internal
 standards. <em>Calculation of baseline δ15N
 values and H. formosa trophic position</em> We
 estimated the trophic position of individual <em>H.
 formosa</em> by correcting for the variation in δ15N values of the
 identifiable primary producers (trophic level 1; Vander Zanden et al.,
 1999). For baseline δ15N values, we used those estimated from the
 periphyton samples at each site. We used the equation
 Trophic Position =
 [(δ^15^N-δ^15^N<sub>baseline</sub>)/3.4]+1,
 where 3.4% is the mean enrichment of δ15N between trophic levels
 as determined in previous studies of fish that have the same range of
 longevity and tissue turnover rates as fish species in the sites we
 sampled (Vander Zanden et al., 1999; Aresco et al., 2015).
description: # Intraspecific variation in the organismal stoichiometry of the Least
 Killifish tracks spatial variation in periphyton composition
 [https://doi.org/10.5061/dryad.3n5tb2rt5](https://doi.org/10.5061/dryad.3n5tb2rt5) ## Description of the data and file structure We sampled water at 17 locations with populations of *H. formosa*. We selected eight of these locations for stoichiometric and stable isotope analyses: four freshwater springs (McBride’s Slough, Newport Bridge, Shepherd’s Spring, Wacissa River) and four lakes (Harper’s Eyelet, Little Lake Jackson, Moore Lake, Trout Pond). We collected fish via dip netting and sacrificed fish via rapid chilling in an ice slurry, following approved IACUC protocols. We obtained periphyton samples by scraping stems and leaves of aquatic plants, larger rocks, and fallen limbs wherever we saw *H. formosa* individuals foraging.  ### Files and variables #### File: 2010-2013_Water_Data.xlsx **Description:** Water data collected from sites in the years 2010, 2011, 2012, & 2013 ##### Variables * Site- Location of water collection: McBride’s Slough, Newport Bridge, Shepherd’s Spring, Wacissa River, Harper’s Eyelet, Little Lake Jackson, Moore Lake, Trout Pond * Chlorophyll_a- Chlorophyll_a Corrected, EPA 445.0 (mg/m^3) - concentration of chlorophyll-a, a pigment found in algae, used to estimate algal biomass in water bodies * pH- Laboratory pH, SM 4550-H+ -B (mg/L)- acidity or alkalinity of the water. It is important for aquatic life, as most organisms thrive in a narrow pH range. * Nitrate- Nitrate-N, SM 4500-NO3 E (mg/L)- Nitrate concentration (measured as nitrate-nitrogen) represents the levels of nitrogen in the form of nitrate. Elevated nitrate levels can be a sign of pollution from fertilizers, sewage, or industrial waste, which can harm aquatic ecosystems. * Total_N (mg/L)- Total Nitrogen, Calculated- Total nitrogen represents the sum of all nitrogen compounds in the water, including nitrate, nitrite, ammonia, and organic nitrogen. It is an indicator of nutrient load and can contribute to eutrophication. * OrthoPhosphate (mg/L)- Orthophosphate, SM 4500-P E - Orthophosphate is the form of phosphate that is immediately available for biological uptake. Phosphates are essential nutrients for aquatic plants and algae, but excessive amounts can cause water quality issues like algal blooms. * Total Phosphorus (mg/L)- Total Phosphorus, SM 4500-P E - Total phosphorus represents both organic and inorganic forms of phosphorus in water, including orthophosphate. Like nitrogen, it is a key nutrient that can drive eutrophication if present in excess. #### File: 2012_2013_Fish_Data.xlsx **Description:** Fish data collected from sites in the years 2012 & 2013 ##### Variables * Indiv. #- Which individual the data was collected from * Population- Location of water collection: McBride’s Slough, Newport Bridge, Shepherd’s Spring, Wacissa River, Harper’s Eyelet, Little Lake Jackson, Moore Lake, Trout Pond * SL (Standard Length) (mm)- The measurement of a fish from the tip of the snout to the base of the tail (caudal peduncle), excluding the tail fin. It is a standard metric used to assess fish size and growth. * % Nitrogen- The percentage of nitrogen by dry weight in fish tissue. Nitrogen is a key component of proteins and provides insights into the nutritional and metabolic status of the fish. * % Carbon- The percentage of carbon by dry weight in fish tissue. Carbon is the primary element in organic matter and indicates energy content in the fish’s body. * % Phosphorus- The percentage of phosphorus by dry weight in fish tissue. Phosphorus is essential for bone formation, cellular energy (ATP), and nucleic acids, and is a limiting nutrient in ecosystems. * C:N- C:N (Carbon to Nitrogen Ratio): A stoichiometric ratio indicating the balance between carbon and nitrogen in fish tissue. It helps infer protein content and overall nutrient quality. * C:P- C:P (Carbon to Phosphorus Ratio): This ratio reflects the balance of carbon to phosphorus in fish tissue, providing insight into nutrient demands and growth limitations, especially in relation to skeletal development and metabolic processes. * N:P- N:P (Nitrogen to Phosphorus Ratio): The ratio of nitrogen to phosphorus in fish tissue. It is used to assess nutrient allocation strategies and can reflect the nutritional ecology and physiological condition #### File: 2013_Algae_Data.xlsx **Description:** Algae/Periphyton data collected from 8 sites in the year 2013 ##### Variables * Sample ID- ID of the Periphyton sample * d13C (δ13C)- The ratio of the stable isotopes 13C to 12C in periphyton, expressed in ‰ (per mil) relative to a standard. It provides insights into the carbon source used by the algae (e.g., atmospheric CO₂ vs. bicarbonate) and can help trace food web carbon flow. * d15N (δ15N)- The ratio of 15N to 14N, also expressed in ‰ relative to a standard. δ15N is commonly used to infer nitrogen sources (e.g., agricultural runoff, sewage) and trophic position within food webs. * C Amount (µg)- The absolute amount of carbon in micrograms measured in the periphyton sample. It reflects the total carbon content in the analyzed mass. * N Amount (µg)- The absolute amount of nitrogen in micrograms in the sample. It indicates the total nitrogen content present in the sample. * Amount (mg)- The total dry mass of the periphyton sample used for analysis, measured in milligrams. This contextualizes the elemental concentrations. * %P- The proportion of phosphorus by weight in the periphyton sample. Phosphorus is a key nutrient that often limits algal growth, making %P important in assessing nutrient status. * %C- The proportion of carbon by dry weight in the periphyton. It is a fundamental measure of organic matter content and energy potential. * %N- The proportion of nitrogen by dry weight in the periphyton. It reflects protein and nutrient content, important for ecological and stoichiometric studies. * C:N- The ratio of total carbon to nitrogen in the sample. A higher ratio may indicate carbon-rich, nutrient-poor material, while a lower ratio can suggest higher nutrient quality or nitrogen enrichment. ## Code/software N/A