DOI: 10.5061/dryad.6t1g1jx71
title: Distribution and food chain assimilation of New Zealand mud snails in Spirit Lake, Washington, USA

description: Mount St. Helens National Volcanic Monument was designated by the U.S.
 Congress in 1982 to conserve the landscape for natural regeneration,
 scientific research, education, and cultural resource preservation.
 However, this designation has not eliminated threats from the introduction
 of non-native species. The non-native New Zealand mud snail (NZMS),
 Potamopyrgus antipodarum, was first observed in 2016 along the SW shore of
 Spirit Lake at the foot of Mount St. Helens, despite the lake’s closure to
 public recreation and isolation from other known sites harboring NZMS. Our
 study mapped native and non-native snails on aquatic macrophytes in Spirit
 Lake, analyzed NZMS eDNA in Spirit Lake and surrounding waters, measured
 stable isotopes in snails and their food sources, and analyzed rainbow
 trout (Oncorhynchus mykiss) gut contents from a twenty-year survey to
 examine the patterns of spatial distribution, habitat occurrence, and
 resource use. Our results show that NZMS colonies were likely first
 established along the SW shore of Spirit Lake in 2015, and presently
 remain largely confined to the vegetated littoral zone along this same
 shoreline. The native snail species Gyraulus deflectus and NZMS co-occur
 on multiple macrophyte species, and δ¹⁵N and δ¹³C isotope data reveal they
 are consuming the same food sources, but no evidence was seen for
 competitive exclusion. The abundance and frequency of NZMS found in
 rainbow trout gut contents have increased since 2015 with a significant
 portion undigested. In addition, stable isotope analysis shows a
 negligible trophic tie between snails (both NZMS and G. deflectus) and
 rainbow trout, which may signal longer-term impacts on fish populations.
 Characterizing this invasion spatially and temporally elucidates the
 factors facilitating and hindering the spread of NZMS in a relatively
 young and dynamic subalpine lake ecosystem closed to public recreation,
 and may inform current and future management decisions.
description: <em>eDNA Sampling</em> On two
 dates in October 2019 we gathered water samples from 18 sites for eDNA
 testing. Sites included nearshore waters along the SW shore of Spirit Lake
 (where NZMS had been observed annually since 2016), tributary streams
 flowing into Spirit Lake along this shoreline, nearby isolated ponds, and
 downstream in Coldwater Creek and Coldwater Lake. Flow rates in the
 tributary streams entering Spirit Lake were generally low in October
 (0.5-90 L s<sup>-1</sup> measured in 2008; Gawel et al. 2018).
 Flow rates exiting the tunnel outlet and into Coldwater Creek were
 approximately 2800 L s<sup>-1</sup> at the time of sampling
 (Gawel et al. 2018; US Geological Survey 2024).  At each site two
 replicate 1000 mL samples were collected in sterile one-liter Nalgene
 bottles following published protocol (Goldberg and Strickler 2017).
 Samples were placed on ice and filtered within 12 hours of collection.
 Both replicates were analyzed for 13 of the sampling sites with single
 samples run for the remaining 5 sites, in addition to 2 field blanks.
 Filtered samples and field blanks were delivered to Dr. Caren Goldberg at
 Washington State University for analysis using published methods (Goldberg
 et al. 2013). <em>Aquatic Macrophyte and Snail
 Sampling</em> Sampling for aquatic macrophytes
 with attached snails occurred on multiple dates during the summers of 2021
 and 2022. Samples of common and abundant macrophyte taxa
 (<em>Myriophyllum spicatum</em>, Charales (Class,
 Charophyceae), <em>Ceratophyllum demersum</em>,
 <em>Potamogeton amplifolius</em>, and filamentous algae) were
 gathered using a plant rake at water depths ranging from 1 to 5 m.
 Macrophyte samples were floated in water in a clear plastic tub and
 agitated by hand for 30 s to release any attached snails from the
 vegetation. Snails were allowed to sink to the bottom and separated from
 the vegetation. Subsequent periodic examination of the vegetation after
 agitation showed this method to be very effective at separating snails
 from vegetation, with &lt; 1% of total snail abundance remaining on
 any vegetation sample. Any additional snails found in rechecked samples
 were not included in sample totals for method consistency. Macrophytes
 were then removed by hand, identified, and vegetation volume was estimated
 using a graduated beaker. The area of vegetation sampled was estimated
 using the diameter of the rake head, which was rotated on the pole axis to
 collect a circular sample of macrophytes. The remaining solids and
 detached snails were then transferred to plastic containers on ice for
 transport to University of Washington Tacoma for analysis. Snails were
 separated from any remaining solids, identified, and manually counted
 under a dissecting microscope. A subset of macrophyte and snail samples
 collected in 2021 were kept for stable isotope analysis.
 <em>Fish Sample Collection and Gut Content
 Analysis</em> Annual sampling of rainbow trout
 began at Spirit Lake in 2000 using hook and line and gillnet methods
 (Blackman et al. 2018). The majority of these surveys took place in Duck
 Bay in the SW quadrant of the lake. In 2021 additional hook and line
 sampling was conducted in locations paired with macrophyte surveys to
 provide more insight into snail distribution throughout Spirit Lake.
 Rainbow trout caudal fins from fish collected in 2021 (n= 42) were
 removed, transported on ice, and dried for stable isotope analysis.
 Gastrointestinal tracts (fish guts) were collected from destructive hook
 and line sampling, gill netting, and opportunistically from
 catch-and-release sampling when fish perished unintentionally. Rainbow
 trout gut samples were transported to our lab in 90% ethanol where they
 were dissected, with contents removed from the entirety of the
 gastrointestinal tract and stored again in 90% ethanol. Snails were then
 separated from the gut contents, identified, and counted under a
 dissecting microscope. <em>P. antipodarum</em> and
 <em>G. deflectus</em> individuals from a subset of rainbow
 trout guts sampled in 2021 containing both species (n = 10 fish) were
 stored after counting in 90% ethanol for stable isotope analysis. Midge
 larvae (Chironomidae) were also present in the gut contents of 9 of these
 10 trout, and they were also stored in 90% ethanol for stable isotope
 analysis. <em>Arthropod and Amphibian Sample
 Collection</em> Aquatic insect larvae samples
 (Ephemeroptera, Plecoptera, Trichoptera, Odonata), aquatic mite samples
 (<em>Hydrachna </em>species), and amphibian tadpole samples
 (<em>Anaxyrus boreas</em>) were collected from benthic (log
 mat and littoral) substrates in the summers of 2018 and 2021. Arthropod
 specimens were scraped off submerged substrates with a modified brush and
 picked off substrates with forceps, and amphibians were captured with
 handheld dip nets. Emergent Ephemeroptera were collected from emergence
 traps (amphibious emergence trap - black and white, BugDorm, MegaView
 Science Co.) over logs and the lake surface. All samples were transported
 on ice, and stored frozen until they were prepared for stable isotope
 analysis. <em>Stable Isotope Sample Preparation
 </em> Macrophyte samples were dried at 40 °C to a
 constant weight, and then pulverized in a mortar and pestle. Aquatic
 arthropod samples that were large enough were dissected, and head, thorax
 and legs were preferentially subsampled for analysis. Smaller arthropods,
 including mites and midge larva, were kept as intact individuals, and
 whole snail bodies were carefully extracted from their shells. Tadpole
 tails and caudal fins were subsampled and sonicated in a 2:1 chloroform
 and methanol solution twice for 30 minutes to remove lipids. Caudal
 rainbow trout fins were wiped with methanol to clean surface contaminants.
 The distal 1-2 mm tip of the fin was subsampled for analysis. All animal
 samples were dried at 40 °C to a constant weight. Macrophyte and animal
 samples were weighed into tin capsules to a target mass of 1-3 mg, and
 multiple individuals (n = 3-5) were combined into composited samples for
 the snails and the smaller arthropods. 
 <em>Stable Isotope Sample Analysis</em>
 All samples were analyzed for carbon and nitrogen stable isotope
 ratios (reported as δ¹³C and δ¹⁵N values) at the University of Colorado
 Earth Systems Stable Isotope Laboratory using an elemental analyzer
 coupled to a Thermo Delta V Isotope Ratio Mass Spectrometer. Sample δ¹³C
 and δ¹⁵N values are reported relative to Vienna Pee Dee Belemnite and air
 N<sub>2</sub> standards, respectively.
 <em>Stable Isotope Data Analysis</em>
 We assumed that the isotopic values of all samples were
 seasonally comparable and reflected the spring and summer growth season
 (May-September) in Spirit Lake. We used elemental C and N content to
 verify that invertebrate samples (comprised of chitin, proteinaceous
 materials, and soft tissues) were comparable among individuals; weight
 percent C/N ratios were within the range of 3.2 – 5.5 (except for 6
 samples with higher ratios that were not isotopically anomalous and thus
 retained in the dataset). The tadpole tail and fin samples were fleshy and
 assumed to be primarily composed of skin, muscle, and connective tissues,
 and all C/N ratios fell within a range of 4.2 - 4.3. The rainbow trout fin
 tip sample C/N ratios were relatively invariant (range = 2.9 - 3.4), which
 suggested there were not substantial differences in tissue composition. We
 confirmed that the larval and emergent Ephemeroptera δ¹³C and δ¹⁵N values
 were not significantly different (t-test, p &gt; 0.05) and combined
 these into a single dataset for that taxon. We used data collected from
 the few 2021 macrophyte samples to supplement a published Spirit Lake 2018
 macrophyte dataset (Shinneman et al. 2024) and confirmed that the isotopic
 values were comparable, with only one δ¹⁵N value slightly outside the
 range of the published values. We also used isotopic values for Spirit
 Lake periphyton reported in Shinneman et al. (2024). We used average
 isotopic trophic discrimination factors (δ¹³C = 1.5‰, δ¹⁵N = 1.3‰)
 calculated from a snail feeding study (Li et al. 2018) to directly compare
 snail isotopic values to those of their most abundant diet sources
 (periphyton and macrophytes). Since this was not a comprehensive dietary
 analysis for either snail species, we did not attempt to quantitatively
 estimate the contributions of diet sources with an isotopic mixing model.
 We used isotopic trophic discrimination factors determined experimentally
 from trout (δ¹³C = 1.0‰, Jensen et al. 2012 and δ¹⁵N = 1.7‰, Heady and
 Moore 2012) to directly compare trout and snail values and demonstrate the
 improbability of snails as a substantial assimilated diet source. Our
 study was not a dietary analysis for the rainbow trout and future research
 may focus on determining the relative contributions of diet sources in
 Spirit Lake.
description: # Distribution and food chain assimilation of New Zealand mud snails in
 Spirit Lake, Washington, USA
 [https://doi.org/10.5061/dryad.6t1g1jx71](https://doi.org/10.5061/dryad.6t1g1jx71) This data set contains measurements of snail (*Potamopyrgus antipodarum*, *Gyraulus deflectus*, and *Lymnaea* sp.) density per vegetation volume distributed spatially and per *Oncorhynchus mykiss* digestion track over time and distributed spatially in Spirit Lake, Mount St. Helens National Volcanic Monument, Washington, USA. *P. antipodarum* relative density is also measured using eDNA samples collected in Spirit Lake and surrounding water bodies. Stable isotope analyses (d13C and d15N) and %C and %N of the various food web members (periphyton, macrophytes, macroinvertebrates, amphibians, and fish) are also included.   ## Description of the data and file structure All data are in one .XLSX file with 5 tabs as described below. #### eDNA Data (New Zealand mud snail eDNA relative abundance in water samples from Spirit Lake and nearby waters) * *Date Filtered* - date sample collected and filtered in the field * *Site Name* - location common name * *WGS84 UTM East* - longitude UTM coordinates using the WGS84 datum * *WGS84 UTM North* - latitude UTM coordinates using the WGS84 datum * *Replicate* - denotes sample replicate analyzed (note that only one replicate was analyzed for locations H55, H48, COLDWATER LAKE OUTLET, COLDWATER LAKE 1, COLDWATER LAKE 2, and the two FIELD BLANK samples) * *Sample Collected (mL)* - volume of sample collected * *Volume Filtered (mL)* - volume of sample filtered (filter clogged after less than 1000 mL for some samples) * *NZMS copies in reaction* - number of copies of New Zealand mud snail DNA detected in the analytical reaction * *NZMS copies in sample *- number of copies of New Zealand mud snail DNA in entire sample filtered * *NZMS copies per mL *- number of copies of New Zealand mud snail DNA detected per mL of sample filtered * *Sample Collection Notes* - notes regarding deviations in sample filtering * *Analysis Notes* - notes from analyst regarding observations during analysis #### Snail-Vegetation Data** (snail spatial distribution counted on aquatic macrophyte samples in Spirit Lake)** * *Sample ID* - sample identification used by authors * *Longitude* - longitude in decimal degrees using the WGS84 datum (GPS location lost for one sample, but quadrant known) * *Latitude* - latitude in decimal degrees using the WGS84 datum (GPS location lost for one sample, but quadrant known) * *Quadrant* - lake quadrant as defined in the related manuscript (Myers et al. 2024; Figure 4) * *Date* - sample collection date (note for some only year was recorded) * *Myriophyllum spicatum* - presence (or null for absence) of vegetation type in sample * *Charales (Charophyceae)* - presence (or null for absence) of vegetation type in sample * *Ceratophyllum demersum* - presence (or null for absence) of vegetation type in sample * *Potamogeton amplifolius* - presence (or null for absence) of vegetation type in sample * *Filamentous Algae* - presence (or null for absence) of vegetation type in sample * *Other* - presence (or null for absence) of other miscellaneous vegetation types (as indicated) in sample * *P.antipodarum* - *Potamopyrgus antipodarum* individuals counted in vegetation sample collected * *G. deflectus* - *Gyraulus deflectus* individuals counted in vegetation sample collected * *Lymnaea sp.* - sum of *Lymnaea humilis* and *Lymnaea auricularia* individuals counted in vegetation sample collected * *Plant volume (mL)* - volume of aquatic plant material collected #### Snail-Onmy Data - Duck Bay only **(snail temporal abundance counted in gut contents of rainbow trout collected in Duck Bay, Spirit Lake)** * *sample_id* - sample identification used by authors * *sample_year* - year rainbow trout were sampled * *sample_date* - date rainbow trout were sampled * *P.antipodarum* - *Potamopyrgus antipodarum* individuals counted in each rainbow trout individual collected * *G. deflectus* - *Gyraulus deflectus* individuals counted in each rainbow trout individual collected * *Lymnaea sp.* - sum of *Lymnaea humilis* and *Lymnaea auricularia* individuals counted in each rainbow trout individual collected * *Location* - all samples included in this data were collected in Duck Bay, Spirit Lake #### Snail-Onmy Data - 4 locations **(snail spatial abundance counted in gut contents of rainbow trout collected from four locations in Spirit Lake)** * *sample_id* - sample identification used by authors * *sample_year* - year rainbow trout were sampled * *sample_date* - date rainbow trout were sampled * *P.antipodarum* - *Potamopyrgus antipodarum* individuals counted in each rainbow trout individual collected * *G. deflectus* - *Gyraulus deflectus* individuals counted in each rainbow trout individual collected * *Lymnaea sp.* - sum of *Lymnaea humilis* and *Lymnaea auricularia* individuals counted in each rainbow trout individual collected * *Location* - location of rainbow trout sample collection as defined in the related manuscript (Myers et al. 2024; Figure 5) * *GPS Coordinate - *longitude and latitude UTM coordinates using the WGS84 datum #### Stable Isotope Data * *Taxon* - taxa sampled for stable isotope analysis * *Year Collected* - year taxa samples were collected * δ¹³C - carbon stable isotope ratios (reported as per mil (‰) δ¹³C values) analyzed at the University of Colorado Earth Systems Stable Isotope Laboratory using an elemental analyzer coupled to a Thermo Delta V Isotope Ratio Mass Spectrometer; sample δ¹³C values are reported relative to Vienna Pee Dee Belemnite standards * δ¹⁵N - nitrogen stable isotope ratios (reported as per mil (‰) δ¹⁵N values) analyzed at the University of Colorado Earth Systems Stable Isotope Laboratory using an elemental analyzer coupled to a Thermo Delta V Isotope Ratio Mass Spectrometer; sample δ¹⁵N values are reported relative to air N2 standards * *Weight %C* - weight fraction of carbon normalized to total dry mass of organism  * *Weight %N *- weight fraction of nitrogen normalized to total dry mass of organism * *Source* - source of sample data either from Myers et al. 2024 or Shinneman et al. 2024 (included with permission of coauthor KFD)