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Sampling methods are described by Szydlowski et al. "Macrophyte and snail community responses to 30 years of population declines of invasive rusty crayfish (Faxonius rusticus)," but are provided here for convenience. Instrumentation is further documented in the supplementary information of the paper.

Macrophytes were sampled during July and August at a subset of crayfish sampling sites within our ten study lakes (n = 6–14 sites per lake) that were selected in 1987 to capture a variety of substrates and both east and west sun exposure. Sampling depths were randomly assigned to sites during initial sampling in 1987 as either 0.75 m, ½ of Secchi depth, or ¾ of Secchi depth, with 1987 Secchi depths used for all subsequent sampling years for macrophyte surveys. We followed the line-intercept method to sample macrophytes, using snorkeling and SCUBA to visually identify and determine the presence or absence of macrophyte species along a 25 m transect set parallel to shore at the pre-determined depth for each sampling site. Transects were marked at 1 m intervals, with the first 10 cm of each interval marked by a band of tape. Divers moved along the transect recording the presence or absence of each macrophyte species crossing the vertical plane of each 10 cm band. The line-intercept method allowed us to obtain a measure of both macrophyte species richness and abundance. Because just presence or absence of macrophyte species was recorded, and only at each 10 cm band, our measurements provide an index for abundance and a minimum estimate for species richness.

Freshwater snails were sampled at locations historically sampled for crayfish (n = 24 or 36 sites per lake) between late June and early August. As with macrophytes, snails were sampled at randomly assigned depths of either 0.75 m, ½ of Secchi depth, or ¾ of Secchi depth. While the same absolute depths were used in 1987 and 2002 based on 1987 Secchi values, depths in 2011 and 2020 were determined using year-specific Secchi values. Most sampling depths in 2011 and 2020 varied only slightly from the 1987 and 2002 values, but in two lakes the change in sampling depth was greater than one meter due to larger shifts in water clarity. The greatest changes in sampling depth (2.7 m in Papoose Lake and 1.5 m in Little John Lake) occurred at the ¾ Secchi depth sites, whereas the ½ Secchi depth sites were less affected by the change in water clarity in these two lakes.

We sampled snails using methods and equipment designed for each habitat type present in our study lakes (soft substrates, macrophytes, and cobble). For soft substrates such as sand and muck (flocculent sediment or sediment rich in organic material), we used a cylindrical polyvinyl chloride (PVC) sediment corer (0.018 m2), which we used to take a 5 cm sediment core. For sites with soft substrates where macrophytes were present, we used a modified PVC sampler of the same size but with two hinged PVC halves, and a net made of 1-mm mesh attached to the top. We carefully closed the two halves of the PVC sampler around macrophytes growing at the surface and zippered the mesh net around taller macrophytes before pushing the corer into the sediment to collect a 5 cm core. Collecting the macrophyte material along with the sediment allowed us to sample any snails on the macrophytes along with those in the sediment. At the water’s surface we sieved (with 1 mm mesh) all cores from soft substrates to remove fine sediments and large particles and picked through macrophyte material for snails. Finally, for cobble habitats, we placed a ring (0.1 or 0.5 m2) on the substrate at each site to define a sampling area. In 1987 and 2002, the 0.1 m2 ring was used for sites with a high density of snails, and the 0.5 m2 ring was used for sites with a low density of snails. In 2011 and 2020, we used the 0.5 m2 ring at all sites. We gently collected the surface layer of rocks within the sampling ring and briefly brought the rocks to the surface, where we scraped attached material into a collection pan and funneled it through a 1 mm mesh sieve to gather snails. We stored snails collected using all sampling methods in 70% ethanol for later identification.

In the lab, we picked snails from all samples and identified them to species or genus (for Physella sp.) according to Burch (1989) and Johnson et al. (2013), with revisions for Lymnaeidae (Hubendick 1951) and Planorbidae (Hubendick and Rees 1955). We calculated snail abundance as density to account for differences between the sediment corers and the rings in area sampled. Snail samples from 1987 were lost in a laboratory flood, but specimens from 2002 and 2011 are vouchered at the Notre Dame Museum of Biodiversity in Notre Dame, Indiana, USA. Specimens from 2020 are vouchered at the Illinois Natural History Survey Mollusk Collection at the University of Illinois in Champaign, Illinois, USA.

In 2020, we were not able to sample macrophytes and snails using SCUBA due to limitations from the COVID-19 pandemic. Therefore, we excluded a small portion of deeper sites (approximately 2% of total macrophyte sites and 13% of total snail sites) that could not be sampled accurately and safely while snorkeling. In addition, because of a few lost samples, data from previous sampling years were not always available for each site. Consequently, in our datasets of macrophytes and snails, we only include sites for which we had data in all four sampling years (n = 100 sites/year for macrophytes, n = 208 sites/year for snails). In our snail data, we only included snails which were alive at the time of sampling (i.e., we did not include empty shells).

Between 1975 and 2020, each of our ten study lakes was sampled for crayfishes between nine and 17 times using standard methods for our study system (Table 1; Capelli and Magnuson 1983, Kreps et al. 2012, Baldridge and Lodge 2014, Larson et al. 2019). Wire-mesh Gee minnow traps with two 3.5 cm diameter openings were baited with approximately 120 g of beef liver and set overnight at depths of 1–3 m. Each lake was sampled at either 24 or 36 sites, located at least 100 m apart, to allow for 12 traps per major habitat type present in the lake (cobble, open sand, or macrophyte; Kershner and Lodge 1995, Lodge et al. 1998). Sites were located with marked bathymetric maps in early years of sampling and later with a handheld global positioning system (GPS). Trap numbers were generally consistent between years within lakes, with only minor inter-annual variation due to trap theft or damage. Crayfish species, size, and sex (including reproductive form for males) were recorded for each trap. Because trapping is biased towards adult males, we calculated catch-per-unit effort (CPUE) as male F. rusticus per trap, which compares well to other methods of measuring crayfish in our study system such as SCUBA surveys or biomass (Capelli and Magnuson 1983, Olsen et al. 1991). Consequently, all subsequent references to F. rusticus relative abundance are male crayfish per trap. Finally, trapping almost always occurred between mid-July and late-August, after F. rusticus had molted from its reproductively inactive Form II to its reproductively active or mature Form I (Larson et al. 2019).