Periphyton collection and processing:
The rate of periphytic production of minerals along the BBCW gradient was measured on artificial substrates called periphytometers. Periphytometers consist of 20 glass microscope slides placed inside a plastic box that is open on all sides and attached to Styrofoam floatation devices (Gaiser et al., 2005). Three periphytometers were deployed on July 19, 2022, at each site along the three transects and left to accumulate periphyton for two months for retrieval on September 13, 2022. Deployment was repeated on September 13, 2022, with retrieval on November 18, 2022. Surface water salinity, pH, and conductivity were measured using a multiparameter probe (OAKTON WD-35607-32 CON), and water and soil depth were measured using a meter stick and soil probe. The retrieved slides were frozen on the collection day in the laboratory and later thawed in a refrigerator for approximately 4 hours. Once thawed, slides were scraped with a metal razor into a sanitized pan and rinsed with deionized water into a beaker. The material was then homogenized with a hand-held blender and diluted with deionized water to a measured volume. Measured sub-samples were removed from each of the diluted and measured samples for dry weight and ash-free dry weight (AFDW) (40 ml), nutrients (120 ml), and diatoms (10 ml). The dry weight and nutrient subsamples were dried to constant weight at 80 ºC (~2 days) and re-weighed. The dry weight subsample was combusted in a muffle furnace for one hour at 500 ºC to estimate the entire sample's mineral and ash-free dry mass (g/m2). The nutrient subsample was subsequently analyzed for periphyton TP, total carbon (TC), and total nitrogen (TN) using a CARLO-ERBA ® NA1500 analyzer (Solaranzo and Sharp 1980, EPA 365.1). The rate of periphyton production was calculated as the particulate organic (biomass production, ash-free dry) and inorganic (mineral production, ash -12% carbon) accretion rate on the periphytometer slides in g/m2/d. The mineral/ash weight calculated periphyton mineral mass in the total sample (g/m2). The AFDW calculated periphyton biomass in the total sample (g/m2).
Diatom composition:
To determine whether diatom assemblages reflect mineral production/accretion gradients, periphytic diatoms were identified and counted in samples from two of the three periphytometer samples from each site and date. In the laboratory, frozen diatom subsamples (10 ml) were thawed for approximately 2 hours in a refrigerator. A chemical oxidizing process was then used to remove calcite and organic matter from within and around diatoms by adding ~25 ml 70% sulfuric acid, followed by successive additions of a saturated solution of potassium permanganate until the solution turned dark violet/ brown and unreactive. Oxalic acid was then added to the sample to clear the solution. It was then diluted with deionized water (Hasle & Fryxell, 1977). Samples were left to settle for at least 6-hour intervals for the material to settle to the bottom and then partially decanted. They were then refilled with deionized water and decanted until neutralized. This process was replicated several times until the sample reached a neutral pH using a litmus test strip. Samples were then decanted to the 50 ml mark and transferred into sanitized glass test tubes that were left to settle for at least 6 hours.
The samples were then decanted to 5 ml using a vacuum suction to remove only surface fluids. The now cleaned diatom samples were transferred into pre-weighed micro-vials that were then re-weighed with the sample inside to estimate its volume. A measured ~0.25 ml subsample was removed with a calibrated volumetric micropipette and placed on a #1 glass coverslip. The coverslip was then filled with deionized water (~0.75 ml) and placed on a warmer to dry slowly. Once dried, the coverslip was inverted and permanently fixed to a glass slide using Naphrax® mounting medium on a hot plate. At least 500 diatom valves were counted and identified using random counting transects measured on a compound light microscope at 1000X/1—40 oil emersion lens. Standards and regional references to count and identify diatoms were used (Schmidt et. al., 1874-1959; Peragallo and Peragallo, 1897-1908; Hustedt, 1930a,b, 1955; Patrick and Reimer, 1966, 1975; Sullivan & Reimer, 1975; Montgomery & Miller, 1978; Sullivan, 1979, 1990; Krammer, 1980, 1997a,b; Lange-Bertalot, 1980, 1983; Germain, 1981; Navarro, 1982, 1983; Foged, 1984; Podzorski, 1984, 1985; Krammer and Lange-Bertalot 1986, 1988, 1991a,b; Podzorski & Håkansson, 1987; Schoeman & Archibald, 1987; Archibald & Schoeman, 1987; Navarro et al., 1989; Round et al., 1990; John, 1990; Lange- (Bertalot & Moser, 1994; Metzeltin & Lange-Bertalot, 1998; Reichardt, 1999; Lange-Bertalot & Genkal, 1999; Witkowski et al., 2000; Danielidis & Mann, 2003; Wachnicka & Gaiser, 2007 ; Loban & Frankovich, 2023; Frankovich & Wachnicka, 2015; Tobias & Gaiser, 2007; Talgatti et al., 2014; Li et al., 2022). Morphologically distinct taxonomic units (MOTUs) that remained unidentifiable after a thorough literature review were given a unique and consistent numeric designation.
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