# Greater than the sum of your parts: Nonlethal stable isotope sampling methods in spiders

Jeffery Marker, Denis Lafage, Eva Bergman, Rachel E. Bowes

► To cite this version:

Jeffery Marker, Denis Lafage, Eva Bergman, Rachel E. Bowes. Greater than the sum of your parts: Nonlethal stable isotope sampling methods in spiders. Ecosphere, 2022, 13 (1), pp.e3903. 10.1002/ecs2.3903. hal-03592519

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# Greater than the sum of your parts: Nonlethal stable isotope sampling methods in spiders

Jeffery Marker<fnref n="1" /> ID

Denis Lafage<fnref n="1" />,<fnref n="2" /> ID

Eva Bergman<fnref n="1" /> ID

Rachel E. Bowes<fnref n="1" />,<fnref n="3" /> ID

**Correspondence**

Jeffery Marker

Email: jeffery.markers@kau.se

**Funding information**

Karlstad University; Svenska

Forskningsrådet Formas, Grant/Award

Number: 2018-00412

**Handling Editor:** Debra P. C. Peters.

**Abstract**

As top consumers and generalist predators, spiders are ideal organisms to study food webs and complex ecological functions using stable isotopes. Most researchers use whole-body samples to analyze stable isotope ratios in spiders. Spiders can regrow lost legs and produce multiple molts during a life cycle, and nonlethal sampling utilizing legs and molts may provide a useful alternative to whole-body sampling especially in larger bodied or threatened species. Furthermore, removing spider abdomens and thus leftover prey in the gut contents may provide a more accurate isotopic value. We tested the hypothesis that the δ¹⁵N, δ¹³C, or δ²H isotopic values in spider legs are reliable proxies for spider prosomas, abdomens, or whole bodies. We used laboratory-reared large-bodied spiders (*Pterinochilus murinus*) and field-collected Lycosidae to compare lethal and nonlethal tissue isotopic values. We found that nonlethal samples of spider legs and molts are acceptable alternatives to lethal whole-body samples to determine δ¹³C and δ¹⁵N stable isotope signatures. Nonlethal samples are not suitable proxies for whole-body samples to determine δ²H isotopic values. Using nonlethal leg or molts samples in stable isotope investigations of spiders will allow researchers to promote conservation efforts and study threatened species while ensuring accurate and repeatable results.

**KEYWORDS**

discrimination values, hydrogen, Lycosidae, nonlethal sampling, Theraphosidae, trophic shift

## INTRODUCTION

Understanding food web structure within an ecological community is essential to track the effects of natural or anthropogenic disturbances on an ecosystem. Ecologists have readily accepted the use of stable isotopes to investigate the food webs (i.e., trophic position and diet composition) of different organisms in their natural environments and in the laboratory (Boecklen et al., 2011; Wolf

et al., 2009). In many cases, sampling for stable isotope analysis (SIA) in smaller animals such as juvenile fishes, spiders, or other arthropods requires sacrificing the organism to obtain sufficient sample tissue. This sampling technique limits the inclusion of threatened or endangered species in isotopic studies, thus hindering critical research. Furthermore, whole-body sampling in arthropods can be problematic as prey remains in guts and tissue turnover rates between different body parts may skew or

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

© 2022 The Author(s). *Ecosphere* published by Wiley Periodicals LLC on behalf of The Ecological Society of America.

<footnote n="1">¹Department of Environmental and Life Sciences, Karlstad University, Karlstad, Sweden</footnote>

<footnote n="2">²UMR Ecobio, Université de Rennes, Rennes Cedex, France</footnote>

<footnote n="3">³Department of Biological Sciences, Emporia State University, Emporia, Kansas, USA</footnote>
misrepresent the true isotopic values of the target organism (Belivanov & Hambäck, 2015; Gratton & Forbes, 2005; Hill & McQuaid, 2011). The problems associated with lethal sampling have led many researchers to pursue non-lethal alternatives. Nonlethal sampling in SIA is very common in mammals (Crawford et al., 2008) and fish (Cano-Rocabayera et al., 2014; Hayden et al., 2017; Valladares & Planas, 2012; Willis et al., 2013) but has also been applied in salamanders (Gillespie, 2013) and amphipods (Wilhelm & Nelson, 2012), and such a method would be especially valuable to study organisms where, in current research, the whole animal is typically sacrificed.

The use of stable isotopes is now more commonplace, but the complexity and variables that affect discrimination values (differences in δ values between an animal and its prey) through a food web may create flawed SIA models and lead to misleading ecological interpretations (Phillips et al., 2014; Vander Zanden et al., 2016). For example, a spider's isotopic value varies depending on the base diet of their prey (i.e., aquatic algae vs. terrestrial leaf litter) and, typically, the isotopic values in spiders will reflect a mixture of prey basal food sources (Collier et al., 2002; Herwig et al., 2007; Kato et al., 2004). Additionally, discrimination values will vary depending on diet quality, water source, and type of organism (Caut et al., 2009; Vanderklift & Ponsard, 2003; Webb et al., 1998). Furthermore, many of the discrimination values used in SIA models are derived from meta-analyses combining isotopic values from different animal groups often tested under significantly different conditions (Caut et al., 2009; Oelbermann & Scheu, 2002; Post, 2002) and thus may not be suitable to accurately model a specific animal's diet. SIA is a powerful tool, but accurate calculation of trophic positons, food source, and the analysis of isotopic values may be reduced or misinterpreted in investigations where the discrimination values of the research organism are understudied.

In most environments, arthropods are abundant, widespread, easy to sample, and represent an extensive range of resource consumption and predation strategies. For these reasons, insects and spiders are ideal organisms to study diet assimilation, dispersal methods, population, and feeding behavior using SIA (Hood-Nowotny & Knols, 2007). As ubiquitous and generalist predators, spiders are used in SIA investigations to track energy flow and subsidy sources in a range of environments such as riparian zones, agroecosystems, and urban areas (Akamatsu et al., 2004; Krell et al., 2015; Sullivan et al., 2019). Spiders are easy to sample and certain families can be targeted depending on the environment and collection method. Like most arthropods, the small size of spiders usually leads a researcher to utilize whole bodies or pool several individuals in one sample for SIA. Fortunately,

spiders have two biological strategies making them ideal for nonlethal sampling. First, spiders can lose multiple legs (autotomy) and regrow them between molts, while still experiencing full lives with little loss in overall fitness (Brueseke et al., 2001). Second, spiders go through five or more molts as they mature from juveniles to adulthood (Foelix, 2010) and the molted remains may be especially suitable for SIA (Belivanov & Hambäck, 2015). Using spider molts in a laboratory or field setting may provide researchers isotopic values over a longer period, assist in calculating the timing of a diet shift, or indicate an ecosystem disturbance.

Although arthropods are used in SIA, when compared to their enormous group size and range of environments they inhabit, they remain an underutilized resource for isotopic investigations (Quinby et al., 2020). Spiders can signal trophic shifts from ecosystem disturbances (Krell et al., 2015; Stenroth et al., 2015; Sullivan et al., 2019) and indicate the effects of ecosystem restoration (Kupilas et al., 2020). Conversely, ecosystem disturbances and changes in ecosystem structure have a direct effect on spider diversity, community composition, and dispersal ability (Lafage et al., 2019; Öberg et al., 2007; Prieto-Benítez & Méndez, 2011). As top terrestrial predators and critical links in complex ecosystems, the more frequent use of spiders in SIA and food web studies is necessary to understand the effects of natural and anthropogenic disturbances on both ecosystems and spiders themselves. As such, researchers should make an effort toward ecological stewardship and conservation, the first steps of which can be accomplished with increased non-lethal sampling in SIA studies.

Our study experimentally tested whether nonlethal spider tissues accurately reflect the isotopic values of other spider body tissues. We assumed spider legs and molts represented nonlethal samples compared to the lethal samples of other tissues. We performed a controlled laboratory study by feeding spiders with prey representing two distinct basal food sources. Using these spiders, we compared the isotopic relationship between spider legs or molts and spider prosomas, abdomens, and whole bodies as well as the basal food source. In addition, we examined the isotopic relationship between legs and abdomens of field-collected spiders with an unknown prey source. We hypothesized that the isotopic values of laboratory-reared spider legs were comparable to isotopic values in the same spider abdomens, prosomas, and whole spiders. We hypothesized that the isotopic values of the field-collected spider legs were comparable to the isotopic values in their respective abdomens. Finally, we investigated the discrimination values of spider body tissues to examine how they may effect stable isotope investigations.
# METHODS

## Study species and rearing conditions

To trace food source stable isotope signatures, we chose the spider *Pterinochilus murinus* (Araneae: Theraphosidae) as it is easily kept in captivity, rarely rejects prey, and is fast growing providing substantial material for analysis (Charpentier, 1993). All spiders came from the same egg sac ensuring uniform initial stable isotope signatures and developmental stage. Spiders were individually kept in separate 12 cm high, 520-ml plastic containers containing a 3:1 mixture of coconut fiber and vermiculite substrate filled to 6 cm and sprayed with water once a week. Spiders were acclimated to containers and environment without disturbance for 1 week. Growth data and spider size were not recorded to minimize disturbance and as a safety precaution for laboratory technicians.

For prey, we used a single colony of Turkestan cockroaches (*Shelfordella lateralis*) as they are easily kept in captivity and will accept a wide range of food. The roach colony was separated into two 40-L plastic containers (40 × 60 × 25 cm) with polyethylene tubing substrate. We chose the roach prey's basal food sources to represent two extremes (terrestrial vs. aquatic) of potential carbon resources that spiders may feed on in a field setting. One group was fed organic kale (*Brassica oleracea*) ad libitum (hereafter, terrestrial prey) and the other group was fed 100% organic flaked *Chlorella* spp. (Superfruit Scandinavia AB) ad libitum (hereafter, aquatic prey). Dried *Chlorella* powder was formed into flakes by adding equal parts by volume water and *Chlorella*, spreading the mixture out on a flat surface, and allowing it to dry out completely. Both cockroach groups were provided water using superabsorbent water gel to prevent drowning. Roaches were acclimated to the environment and were allowed to feed on each food source for 2 weeks prior to the start of the feeding trials. Spiders and roaches were kept in a controlled environment at a constant 26°C with a light regime of 16:8 light : dark. Stable isotope values differed among feeding groups in the cockroach prey. The cockroaches showed a large difference for both $\delta^{13}$C (aquatic vs. terrestrial group: $\bar{x}$ = -19.7 ± 0.31 vs. -27.3 ± 0.37; t(8) = -35.5, p < 0.0001) and $\delta^{15}$N (aquatic vs. terrestrial group: $\bar{x}$ = 1.5 ± 0.61 vs. 4.8 ± 0.64; t(8) = 8.3, p < 0.0001). There was no difference in $\delta^{2}$H for cockroaches (aquatic vs. terrestrial group: -94.0 ± 10.3 vs. -106.4 ± 27.1; t(8) = -1.0, p = 0.37).

## Feeding trials

Spiders ($n$ = 28) were randomly separated into two equal groups: (1) terrestrial and (2) aquatic. Each group of

spiders was fed from their respective prey group as often as they would accept prey. We watched each spider to ensure that it captured the offered prey. If a spider captured and pulled a roach into a burrow, we assumed it was eaten. We removed all accessible prey remains after feeding and saved all accessible spider molts. We recorded the assumed molt sequence number and estimated date of molt.

The experiment was ended after maintaining and feeding spiders for 220 days. All spiders were sacrificed 7 days after the final offer of prey to minimize the effects of any prey remains in the gut. Three spiders died during the feeding trial leaving a total of 25 for SIA. In addition, we sampled terrestrial and aquatic prey for SIA. Substrate in each spider container was searched for any overlooked molts, and if molts were found, we estimated the molting sequence based on size comparison with those previously removed. Each spider in the aquatic feeding group completed a maximum of two molting events resulting in 18 total molts and the terrestrial feeding group completed a maximum of four molting events resulting in 27 total molts.

## Field-collected spiders

Lycosidae spiders were sampled using a suction sampler and identified to species at three riparian sites along the Klarälven River (Sweden) during spring 2018 (Lafage et al., 2020). We collected 378 spiders representing 16 species. Species with less than 10 individuals were removed leaving five species and 339 individuals for analysis. Prior to SIA, field-collected spiders were dissected into abdomens and legs. For comparisons in this study, abdomens and legs were assumed to represent lethal and nonlethal tissue, respectively.

## Stable isotope analysis

All spiders and prey were placed in 70% ethanol in individual vials until SIA preparation. Laboratory spiders were randomly selected for whole-body preparation or dissection into parts. Dissected laboratory spiders were separated into legs, prosoma, and abdomen. Whole spiders, dissected spider parts, and prey were individually placed in sterile glass vials and dried at 60°C for 48 h. Each dried sample was completely pulverized to a homogeneous powder, sealed in clean glass vials, and held in a desiccator until sent for analysis. SIA was performed by Iso-Analytical (Crewe, UK) using elemental analysis isotope ratio mass spectrometry. Analysis for each element (carbon, nitrogen, and deuterium) included the $\delta$ isotope ratios expressed per mil (‰).
## Statistical analysis

To determine whether the δ¹³C, δ¹⁵N, or δ²H isotopic values of whole bodies, prosomas, or abdomens of laboratory-fed spiders could be predicted as a function of the δ¹³C, δ¹⁵N, or δ²H values of spider legs, we used a linear mixed model. For this analysis, we created a pseudo-whole-body spider for each isotope by averaging and combining the δ¹³C, δ¹⁵N, and δ²H isotopic values of each tissue (legs, abdomen, and prosoma). We determined that the pseudo-whole body (n = 12, for each isotope) was a valid representation for modeling by comparing it against our whole-body samples (n = 13, for each isotope) (δ¹³C: t(23) = 0.15, p = 0.88; δ¹⁵N: t(23) = -0.27, p = 0.79; δ²H: t(23) = 2.02, p = 0.06). Pseudo-whole body, abdomen, and prosoma isotope values were the response variables; leg tissue isotope values were the predictor variables, and feeding group (aquatic or terrestrial) was a fixed factor. Abdomens and prosomas were included in the analysis to examine the isotopic relationship between lethal and nonlethal tissues. This relationship is especially important when a whole body of a single specimen is divided up for isotopic or genetic analysis. We combined isotopic values from the aquatic and terrestrial feeding groups to represent an average range of isotopic values for tissue comparison analysis. To further explore the laboratory spider data, we tested each tissue–leg combination against a hypothetical 1:1 fit line (x = y) using one-way ANOVA.

To compare δ¹³C or δ¹⁵N isotopic values of molts with prey and spider tissue, we used a linear mixed model where either δ¹³C or δ¹⁵N isotopic value was the response variable, tissue type was the predictor variable, and individual spiders was a random factor. We separated molts, prey, and spider tissues into feeding groups for the analysis. To compare molt, prey, and spider tissue values, we performed post hoc analysis using pairwise comparisons of estimated marginal means for each molt sequence with prey and other tissues adjusting for multiple comparisons.

To determine whether δ¹³C or δ¹⁵N leg isotopic values are suitable predictors of δ¹³C or δ¹⁵N abdomen isotopic values in field-collected spiders, we used a least squares linear model with abdomen isotope values as the response variable and leg isotope values as the predictor variable. To further explore the field-collected spider data, we tested each regression model against a hypothetical 1:1 fit line (x = y) using one-way ANOVA.

Finally, to calculate discrimination factors, we separated laboratory spider tissues and prey into feeding groups for isotopic discrimination analysis. Discrimination factors were calculated for each tissue–prey combination by averaging the isotopic values of the prey and

subtracting that value from all isotopic values of each tissue and averaging the result. We then compared δ¹³C, δ¹⁵N, and δ²H isotope values for prey and all spider tissues using separate one-way ANOVAs for each isotope. To confirm significant discrimination values, we performed post hoc analysis using pairwise comparisons of estimated marginal means between each tissue and the prey in the respective feeding groups adjusting for multiple comparisons.

All analyses were performed in R Studio v.1.2.5033 (RStudio Team, 2021) with R v.3.6.3 (R Core Team, 2020) using the libraries “nlme” (Pinheiro et al., 2020) and “emmeans” (Lenth, 2020) and assuming the alternative hypothesis at *p* < 0.05.

## RESULTS

### Laboratory experiment

Our results (Tables 1 and 2) showed a significant relationship for the δ¹³C and δ¹⁵N isotopic values of prosomas, abdomens, and pseudo-whole bodies as a function of the leg tissue (Figure 1 and Table 3). There were no significant relationships for δ²H between all tissues and leg tissue (Figure 1 and Table 3). δ¹³C and δ¹⁵N regression analysis showed very strong relationships (all models: conditional R² > 0.79). In all tissues, δ¹³C and δ¹⁵N model regression slopes were not significantly different from the slope of the hypothetical 1:1 fit line (Table 3).

δ¹⁵N values of molts were significantly different compared to prey and other tissues in the terrestrial group, while no δ¹³C values were different from any other tissues tested in the same group (model root mean squared error [RMSE]; N molts = 0.59; C molts = 0.92). Both δ¹⁵N and δ¹³C values of molts were significantly different within the aquatic group (model RMSE; N molts = 0.23; C molts = 1.85). Post hoc analysis of δ¹⁵N in both feeding groups showed a distinct trend in increasing isotopic value from prey through each subsequent molt to final spider tissues (Figures 2 and 3). One molt with a δ¹⁵N outlier value, likely because of analysis error, was removed from the analysis.

Discrimination values of δ¹³C, δ¹⁵N, and δ²H showed significant differences between tissues and prey within both feeding groups (terrestrial, C: F₅,₂₉ = 19.9, *p* < 0.001; N: F₅,₂₉ = 34.6, *p* < 0.001; H: F₅,₂₉ = 26.94, *p* < 0.001; aquatic, C: F₅,₃₀ = 8.04, *p* < 0.001; N: F₅,₃₀ = 114.2, *p* < 0.001; H: F₅,₃₀ = 73.9, *p* < 0.001). Post hoc testing showed a significant increase in δ¹⁵N values in all tissues in both feeding groups (Table 4). δ¹³C values
increased in prosomas, legs, and pseudo-whole tissues, and prosomas and legs in the aquatic and terrestrial groups, respectively (Table 4). δ²H values showed a significant decrease in abdomens in both feeding groups, an increase

in leg tissue values in both feeding groups, and an increase in prosoma values in the aquatic feeding group (Table 5).

<table><caption>TABLE 1 Summary statistics for ¹³C, ¹⁵N, and ²H for whole spider, pseudo-whole spiders, each tissue analyzed, and prey</caption><thead><tr><th>Isotope</th><th>Tissue</th><th>n</th><th>Mean ± SD</th><th>Min</th><th>Max</th></tr></thead><tbody><tr><td rowspan="6">¹³C</td><td>Whole</td><td>13</td><td>-22.90 ± 4.32</td><td>-27.76</td><td>-18.42</td></tr><tr><td>Pseudo-whole</td><td>12</td><td>-22.64 ± 4.10</td><td>-27.19</td><td>-18.28</td></tr><tr><td>Leg</td><td>12</td><td>-21.96 ± 3.92</td><td>-26.28</td><td>-17.87</td></tr><tr><td>Prosoma</td><td>12</td><td>-22.28 ± 3.86</td><td>-26.83</td><td>-18.21</td></tr><tr><td>Opisthosoma</td><td>12</td><td>-23.70 ± 4.53</td><td>-28.62</td><td>-18.73</td></tr><tr><td>Prey</td><td>10</td><td>-23.48 ± 4.05</td><td>-27.80</td><td>-19.31</td></tr><tr><td rowspan="6">¹⁵N</td><td>Whole</td><td>13</td><td>8.94 ± 1.11</td><td>6.63</td><td>10.25</td></tr><tr><td>Pseudo-whole</td><td>12</td><td>8.84 ± 0.78</td><td>7.75</td><td>10.10</td></tr><tr><td>Leg</td><td>12</td><td>9.05 ± 0.75</td><td>8.11</td><td>10.26</td></tr><tr><td>Prosoma</td><td>12</td><td>9.15 ± 0.65</td><td>8.24</td><td>10.33</td></tr><tr><td>Opisthosoma</td><td>12</td><td>8.32 ± 1.09</td><td>6.33</td><td>9.98</td></tr><tr><td>Prey</td><td>10</td><td>3.13 ± 1.84</td><td>0.77</td><td>5.60</td></tr><tr><td rowspan="6">²H</td><td>Whole</td><td>13</td><td>-99.91 ± 11.15</td><td>-123.68</td><td>-84.86</td></tr><tr><td>Pseudo-whole</td><td>12</td><td>-91.40 ± 9.93</td><td>-111.31</td><td>-77.34</td></tr><tr><td>Leg</td><td>12</td><td>-55.32 ± 7.38</td><td>-66.70</td><td>-42.84</td></tr><tr><td>Prosoma</td><td>12</td><td>-75.31 ± 10.19</td><td>-99.57</td><td>-63.32</td></tr><tr><td>Opisthosoma</td><td>12</td><td>-143.58 ± 20.78</td><td>-190.52</td><td>-122.99</td></tr><tr><td>Prey</td><td>10</td><td>-100.2 ± 20.41</td><td>-140.76</td><td>-83.51</td></tr></tbody></table>

*Note*: Spiders from both feeding groups (aquatic and terrestrial) were combined to calculate summary statistics. Min and Max for pseudo-whole spiders represent an average of the combined minimum and maximum values from individual tissues.

## Field spiders

Field-collected spiders (Table 6) regression analysis showed significant relationships between the δ¹³C and δ¹⁵N isotope signatures of abdomens as a function of the leg tissue for all field-collected spiders combined and each species except δ¹³C in *Piratula hygrophila* (p = 0.29) (Table 7 and Figure 4). Regression models reflected weak-to-moderate relationships between legs and abdomens in all significant models (all models: R² 0.06–0.59, RMSE 0.62–0.96). δ¹³C model regression slopes were not significantly different from the slope of the hypothetical 1:1 fit line for all tested species except *Pardosa* spp. and *P. hygrophila* (Table 5). δ¹⁵N model regression slopes were not significantly different from the slope of the hypothetical 1:1 fit line for all species except *Pardosa lugubris* and *Pardosa* spp. (Table 7).

## DISCUSSION

### Nonlethal sampling as a viable method

Our study highlights the use of nonlethal sampling methods with large-bodied spiders for future stable isotope studies. In general, our results indicate that nonlethal samples of spider legs or molts are acceptable alternatives to lethal whole-body sampling to determine

<table><caption>TABLE 2 Summary statistics of C, N, and H% content and C:N ratio for food source, prey, whole spiders, and each tissue analyzed separated by feeding group</caption><thead><tr><th>Group</th><th>Food source/tissue</th><th>n</th><th>%C ± SD</th><th>%N ± SD</th><th>C:N</th><th>%H ± SD</th></tr></thead><tbody><tr><td rowspan="6">Terrestrial</td><td>Kale</td><td>4</td><td>42.15 ± 0.64</td><td>3.39 ± 0.71</td><td>12.43</td><td>-</td></tr><tr><td>Whole</td><td>6</td><td>51.32 ± 1.68</td><td>11.55 ± 0.51</td><td>4.44</td><td>7.70 ± 0.28</td></tr><tr><td>Prosoma</td><td>6</td><td>46.98 ± 1.26</td><td>12.22 ± 0.83</td><td>3.84</td><td>7.18 ± 0.36</td></tr><tr><td>Abdomen</td><td>6</td><td>56.08 ± 3.75</td><td>10.55 ± 10.59</td><td>5.32</td><td>9.04 ± 0.55</td></tr><tr><td>Leg</td><td>6</td><td>47.17 ± 1.12</td><td>13.05 ± 8.61</td><td>3.61</td><td>6.74 ± 0.23</td></tr><tr><td>Prey</td><td>4</td><td>48.02 ± 5.29</td><td>15.13 ± 1.24</td><td>3.17</td><td>6.29 ± 1.41</td></tr><tr><td rowspan="6">Aquatic</td><td>Algae</td><td>5</td><td>50.10 ± 1.26</td><td>10.28 ± 1.28</td><td>4.87</td><td>-</td></tr><tr><td>Whole</td><td>7</td><td>51.41 ± 2.42</td><td>13.31 ± 3.18</td><td>3.86</td><td>7.50 ± 0.54</td></tr><tr><td>Prosoma</td><td>6</td><td>49.26 ± 1.88</td><td>12.64 ± 3.56</td><td>3.90</td><td>6.89 ± 0.30</td></tr><tr><td>Abdomen</td><td>6</td><td>57.67 ± 2.05</td><td>9.26 ± 5.07</td><td>6.23</td><td>9.18 ± 0.40</td></tr><tr><td>Leg</td><td>6</td><td>47.43 ± 2.43</td><td>12.92 ± 4.48</td><td>3.67</td><td>6.80 ± 0.24</td></tr><tr><td>Prey</td><td>5</td><td>40.96 ± 1.31</td><td>22.65 ± 3.73</td><td>1.81</td><td>3.91 ± 0.33</td></tr></tbody></table>

*Note*: H% content was not analyzed for food sources.
<figure><img src="image_2.png" /><figcaption>**FIGURE 1** δ¹³C, δ¹⁵N, and δ²H isotopic values of abdomen, prosoma, and pseudo-whole tissues as a function of leg tissue in laboratory-reared *Pterinochilus murinus* spiders. Isotopic values from both feeding groups were combined for regression analysis. Each plot shows a 1:1 line for comparison (dashed line). Note that the fitted line is shown only in significant relationships (see Table 3). Each tissue tested is represented by red-highlighted body parts</figcaption></figure>
<table><caption>TABLE 3 Linear mixed model regression equations to estimate laboratory-reared spider abdomen, prosoma, or pseudo-whole-body δ¹³C, δ¹⁵N, or δ²H isotope values based on the respective isotope values in the legs</caption><thead><tr><th>Isotope</th><th>Model</th><th>Regression equation</th><th>H₀: a = 0, p<sup>a</sup></th><th>H₀: a = 1, p<sup>a</sup></th><th>H₀: b = 0, p<sup>a</sup></th><th>R<sup>2b</sup></th></tr></thead><tbody><tr><td rowspan="3">¹³C</td><td>Abd ~ Leg</td><td>Abd = 1.12(Leg) + 1.00</td><td><strong>0.021</strong></td><td>0.774</td><td>0.895</td><td>0.99</td></tr><tr><td>Pro ~ Leg</td><td>Pro = 1.17(Leg) + 2.68</td><td><strong>&lt;0.001</strong></td><td>0.291</td><td>0.353</td><td>0.99</td></tr><tr><td>PW ~ Leg</td><td>PW = 1.10(Leg) + 1.26</td><td><strong>&lt;0.001</strong></td><td>0.571</td><td>0.687</td><td>0.99</td></tr><tr><td rowspan="3">¹⁵N</td><td>Abd ~ Leg</td><td>Abd = 1.72(Leg) - 6.71</td><td><strong>0.003</strong></td><td>0.133</td><td>0.104</td><td>0.79</td></tr><tr><td>Pro ~ Leg</td><td>Pro = 0.72(Leg) + 2.53</td><td><strong>0.002</strong></td><td>0.143</td><td>0.119</td><td>0.85</td></tr><tr><td>PW ~ Leg</td><td>PW = 1.15(Leg) - 1.41</td><td><strong>&lt;0.001</strong></td><td>0.425</td><td>0.379</td><td>0.89</td></tr><tr><td rowspan="3">²H</td><td>Abd ~ Leg</td><td>Abd = -0.65(Leg) - 161.86</td><td>0.519</td><td>-</td><td>0.009</td><td>NA</td></tr><tr><td>Pro ~ Leg</td><td>Pro = 1.08(Leg) - 18.21</td><td>0.079</td><td>-</td><td>0.524</td><td>NA</td></tr><tr><td>PW ~ Leg</td><td>PW = 0.48(Leg) - 60.02</td><td>0.243</td><td>-</td><td>0.013</td><td>NA</td></tr></tbody></table>

Note: Bold indicates a significance at *p* < 0.05.

Abbreviations: Abd, abdomen; Pro, prosoma; PW, pseudo-whole.

<sup>a</sup>Models were tested for significance against a null hypothesis (H<sub>0</sub>) of slope (a) = 0. If model was significant, we tested it against a 1:1 fit line of slope = 1. Intercept (b) coefficient tested against the null hypothesis of intercept = 0.

<sup>b</sup>R<sup>2</sup> is calculated as the conditional R<sup>2</sup>, which describes the proportion of variance explained by both the fixed and random factors (see Nakagawa & Schielzeth, 2013). Conditional R<sup>2</sup> is not relevant for nonsignificant models.

<figure><img src="image_3.png" /><figcaption>FIGURE 2 Boxplot of δ¹⁵N isotopic values of prey, molts, tissue, and whole spiders in the laboratory terrestrial feeding group. Average days between molting events are listed above subsequent molt boxplots. Note that spiders in the terrestrial group underwent a maximum of four molting events. Letters under each boxplot indicate significant differences between tissues based on calculation of estimated marginal means post hoc testing with Tukey adjustment for multiple comparisons (*p* < 0.05). Each tissue tested is represented by red-highlighted body parts and molts are uncolored</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>FIGURE 3 Boxplot of δ¹⁵N isotopic values of prey, molts, tissue, and whole spiders in the laboratory aquatic feeding group. Average days between both molting events are listed above subsequent molt boxplots. Note that spiders in the aquatic group underwent a maximum two molting events. Letters under each boxplot indicate significant differences between tissues based on estimated marginal means post hoc testing with Tukey adjustment for multiple comparisons (*p* < 0.05). Each tissue tested is represented by red-highlighted body parts and molts are uncolored</figcaption></figure>

δ¹³C and δ¹⁵N stable isotope signatures in laboratory and field experiments. In our laboratory study, spider legs and prosomas had similar isotopic values in both δ¹³C and δ¹⁵N, which agree with past studies measuring δ¹³C and δ¹⁵N values in field-collected spiders (Beaubien et al., 2019; Collier et al., 2002). In some situations, it may be unrealistic to remove only spider legs for SIA due to the size of the target family or species. This has often motivated other researchers to use the entire spider body to calculate stable isotope values (Akamatsu et al., 2004; González Macé et al., 2019; Kennedy et al., 2018). However, this approach can be problematic as prey remains may be present in specimen guts during preparation for SIA and can skew isotopic values (Hill & McQuaid, 2011). Our results show that legs alone, for larger spider species, or both spider legs and prosomas, for small spider species, can be combined to determine δ¹³C and δ¹⁵N isotopic values.

Despite the fact that we tested four different species, collected from three different sites, >35 days (Lafage

et al., 2020), in a variety of conditions, and with all species presumed to be feeding on different food sources, we found strong correlations between legs and abdomens in our field-collected spiders. We found strong correlations for all species for δ¹⁵N and for all but *P. hygrophila* for δ¹³C. We believe that the absence of correlation in *P. hygrophila* is connected to its small sample size (*n* = 14). In fact, other species that were removed from this analysis due to small sample sizes failed to achieve significance in δ¹³C (Table S1). We believe testing with larger sample sizes would reveal a similar pattern of isotopic predictive capacity of abdomens as a function of legs. Deviations of fitted regression lines from the 1:1 hypothetical fit line for some field species is likely associated with diets and the different tissue turnover rates between the tested tissues (Willis et al., 2013). Spider leg tissue has an average turnover time 2–3 times longer than abdomens (Belivanov & Hambäck, 2015). Leg and abdomen tissues in the field-collected spiders were likely reflecting an isotopic value corresponding to different
<table><caption>**TABLE 4** Average (±SD) δ¹³C and δ¹⁵N isotopic values and C and N discrimination values for each tissue and prey separated by feeding group</caption><thead><tr><th>Group</th><th>Tissue</th><th>n</th><th>δ<sup>13</sup>C ± SD</th><th>Δ<sup>13</sup>C<sup>a</sup></th><th>δ<sup>15</sup>N ± SD</th><th>Δ<sup>15</sup>N<sup>a</sup></th></tr></thead><tbody><tr><td rowspan="6">Terrestrial</td><td>Whole</td><td>6</td><td>-27.37 ± 0.29</td><td>-0.05 ± 0.29</td><td>9.86 ± 0.31</td><td><strong>5.07</strong> ± 0.31</td></tr><tr><td>Pseudo-whole</td><td>6</td><td>-26.55 ± 0.52</td><td>0.77 ± 0.52</td><td>9.34 ± 0.75</td><td><strong>4.56</strong> ± 0.75</td></tr><tr><td>Prosoma</td><td>6</td><td>-25.95 ± 0.58</td><td><strong>1.36</strong> ± 0.58</td><td>9.64 ± 0.50</td><td><strong>4.86</strong> ± 0.5</td></tr><tr><td>Abdomen</td><td>6</td><td>-28.00 ± 0.65</td><td>-0.68 ± 0.65</td><td>8.76 ± 1.28</td><td><strong>3.98</strong> ± 1.28</td></tr><tr><td>Leg</td><td>6</td><td>-25.69 ± 0.43</td><td><strong>1.62</strong> ± 0.43</td><td>9.61 ± 0.63</td><td><strong>4.83</strong> ± 0.63</td></tr><tr><td>Prey</td><td>4</td><td>-27.31 ± 0.37</td><td>-</td><td>4.78 ± 0.64</td><td>-</td></tr><tr><td rowspan="6">Aquatic</td><td>Whole</td><td>7</td><td>-19.07 ± 0.50</td><td>0.59 ± 0.50</td><td>8.16 ± 0.91</td><td><strong>6.67</strong> ± 0.91</td></tr><tr><td>Pseudo-whole</td><td>6</td><td>-18.74 ± 0.41</td><td><strong>0.91</strong> ± 0.41</td><td>8.34 ± 0.41</td><td><strong>6.86</strong> ± 0.41</td></tr><tr><td>Prosoma</td><td>6</td><td>-18.61 ± 0.36</td><td><strong>1.04</strong> ± 0.36</td><td>8.67 ± 0.32</td><td><strong>7.18</strong> ± 0.32</td></tr><tr><td>Abdomen</td><td>6</td><td>-19.40 ± 0.62</td><td>0.26 ± 0.62</td><td>7.88 ± 0.71</td><td><strong>6.40</strong> ± 0.71</td></tr><tr><td>Leg</td><td>6</td><td>-18.22 ± 0.33</td><td><strong>1.43</strong> ± 0.33</td><td>8.48 ± 0.25</td><td><strong>7.00</strong> ± 0.25</td></tr><tr><td>Prey</td><td>5</td><td>-19.65 ± 0.31</td><td>-</td><td>1.49 ± 0.61</td><td>-</td></tr></tbody></table>

Note: Bold values indicate a significant difference between prey δ values and tissue δ values at *p* < 0.05.

ᵃΔ¹³C and Δ¹⁵N are discrimination values calculated by subtracting the average δ value of prey from each individual tissue sample δ value and averaging the result.

<table><caption>**TABLE 5** Average (±SD) δ²H isotopic values and H discrimination values for each tissue and prey separated by feeding group</caption><thead><tr><th>Group</th><th>Tissue</th><th>n</th><th>δ<sup>2</sup>H ± SD</th><th>Δ<sup>2</sup>H<sup>a</sup></th></tr></thead><tbody><tr><td rowspan="6">Terrestrial</td><td>Whole</td><td>6</td><td>-104.46 ± 11.57</td><td>1.96 ± 11.57</td></tr><tr><td>Pseudo-whole</td><td>6</td><td>-98.87 ± 7.96</td><td>7.54 ± 7.96</td></tr><tr><td>Prosoma</td><td>6</td><td>-78.37 ± 12.34</td><td>28.04 ± 12.34</td></tr><tr><td>Abdomen</td><td>6</td><td>-157.69 ± 21.10</td><td><strong>-51.27</strong> ± 21.10</td></tr><tr><td>Leg</td><td>6</td><td>-60.56 ± 3.60</td><td><strong>45.86</strong> ± 3.60</td></tr><tr><td>Prey</td><td>4</td><td>-106.42 ± 27.10</td><td>-</td></tr><tr><td rowspan="6">Aquatic</td><td>Whole</td><td>7</td><td>-96.02 ± 9.94</td><td>-2.04 ± 9.94</td></tr><tr><td>Pseudo-whole</td><td>6</td><td>-83.93 ± 4.42</td><td>10.05 ± 4.42</td></tr><tr><td>Prosoma</td><td>6</td><td>-72.26 ± 7.33</td><td><strong>21.73</strong> ± 7.33</td></tr><tr><td>Abdomen</td><td>6</td><td>-129.47 ± 5.15</td><td><strong>-35.49</strong> ± 5.15</td></tr><tr><td>Leg</td><td>6</td><td>-50.08 ± 6.39</td><td><strong>43.91</strong> ± 6.39</td></tr><tr><td>Prey</td><td>5</td><td>-93.98 ± 10.30</td><td>-</td></tr></tbody></table>

Note: Bold values indicate a significant difference between prey δ values and tissue δ values at *p* < 0.05.

ᵃΔ²H are discrimination values calculated by subtracting the average δ value of prey from each individual tissue sample δ value and averaging the result.

times and thus different predation events where unique prey were consumed. However, our results from field-collected spiders provide strong evidence, in addition to the laboratory results, that legs can be used as nonlethal alternatives to determine δ¹³C and δ¹⁵N isotopic values or can be used in cases where abdomens are required for other analyses.

Distinct differences over time were much more evident in δ¹⁵N isotopic values in molts compared to δ¹³C. The single prey source in our study revealed a lack of a shift in δ¹³C values in molt remains suggesting that molts may be used to track or expose diet shifts when sampled over time in the field where a spider diet is expected to fluctuate. These results highlight a need for further exploration of δ¹³C shifts in spider molts. Belivanov and Hambäck (2015) also used spider molts to track isotopic shifts over time focusing on a diet switch and using spiders similar to ours (Lasiodora parahybana). They found a significant and traceable shift in δ¹³C values, but their δ¹⁵N values were too small to detect any meaningful change. In our study, δ¹⁵N isotopic values show a significant trend of shifting tropic position through each molt stage to the final tissue samples suggesting that spider molts can be used to track distinct and significant shifts in δ¹⁵N isotopic values. However, further investigation is needed to understand the exact mechanism of the shifting δ¹⁵N values of spider molts. Previous studies of trophic position point out that isotopic enrichment in tissues is related to a combination of tissue assimilation rate, excretion, and prey source (Olive et al., 2003), each of which may have affected the δ¹⁵N values in our results. Furthermore, the isotopic signature of molts should be interpreted as a past diet, not the current spider diet, and multiple molts are necessary to establish diet trends or trophic shifts. Regardless, spider molts have strong potential to investigate δ¹⁵N values and trophic position in field and laboratory studies. However, the authors recognize the difficulty in molt sampling in
<table><caption>TABLE 6 Isotopic values (mean ± SD) for ¹³C and ¹⁵N for individual species and all species combined of field-collected spiders</caption><thead><tr><th rowspan="2">Species</th><th rowspan="2">n</th><th rowspan="2">Tissue</th><th colspan="3">δ<sup>13</sup>C</th><th colspan="3">δ<sup>15</sup>N</th></tr><tr><th>Mean ± SD</th><th>Min</th><th>Max</th><th>Mean ± SD</th><th>Min</th><th>Max</th></tr></thead><tbody><tr><td rowspan="2">Pardosa amentata</td><td rowspan="2">124</td><td>Abd</td><td>-27.23 ± 1.02</td><td>-30.57</td><td>-25.33</td><td>6.00 ± 1.33</td><td>2.90</td><td>9.64</td></tr><tr><td>Leg</td><td>-26.87 ± 0.50</td><td>-28.17</td><td>-25.88</td><td>5.54 ± 0.79</td><td>3.74</td><td>7.45</td></tr><tr><td rowspan="2">Pardosa lugubris</td><td rowspan="2">105</td><td>Abd</td><td>-26.96 ± 0.92</td><td>-28.63</td><td>-23.32</td><td>4.25 ± 1.13</td><td>1.75</td><td>7.50</td></tr><tr><td>Leg</td><td>-26.34 ± 0.39</td><td>-27.41</td><td>-25.39</td><td>3.82 ± 1.19</td><td>1.33</td><td>6.71</td></tr><tr><td rowspan="2">Pardosa prativaga</td><td rowspan="2">35</td><td>Abd</td><td>-27.42 ± 0.96</td><td>-30.40</td><td>-25.91</td><td>5.15 ± 1.40</td><td>2.03</td><td>7.73</td></tr><tr><td>Leg</td><td>-26.80 ± 0.68</td><td>-27.81</td><td>-24.86</td><td>5.07 ± 1.08</td><td>2.89</td><td>7.12</td></tr><tr><td rowspan="2">Pardosa spp.</td><td rowspan="2">59</td><td>Abd</td><td>-27.30 ± 0.90</td><td>-29.84</td><td>-24.19</td><td>4.12 ± 1.38</td><td>1.17</td><td>6.55</td></tr><tr><td>Leg</td><td>-26.54 ± 0.93</td><td>-30.77</td><td>-24.64</td><td>4.55 ± 1.37</td><td>1.95</td><td>7.41</td></tr><tr><td rowspan="2">Piratula hygrophila</td><td rowspan="2">16</td><td>Abd</td><td>-27.39 ± 1.12</td><td>-30.47</td><td>-26.16</td><td>4.12 ± 0.81</td><td>2.92</td><td>5.78</td></tr><tr><td>Leg</td><td>-26.38 ± 0.34</td><td>-26.85</td><td>-25.74</td><td>4.49 ± 0.60</td><td>3.62</td><td>5.60</td></tr><tr><td rowspan="2">All species</td><td rowspan="2">339</td><td>Abd</td><td>-27.19 ± 0.98</td><td>-30.57</td><td>-23.32</td><td>4.97 ± 1.52</td><td>1.17</td><td>9.64</td></tr><tr><td>Leg</td><td>-26.62 ± 0.62</td><td>-30.77</td><td>-24.64</td><td>4.75 ± 1.28</td><td>1.33</td><td>7.45</td></tr></tbody></table>

Note: *Pardosa* spp. group represent juvenile and subadults in the *Pardosa* genus.
Abbreviation: Abd, abdomen.

<table><caption>TABLE 7 Slope and intercept coefficients for linear mixed model regression of abdomen δ¹³C and δ¹⁵N isotope values as a function of leg isotope values of individual field-collected Lycosid spiders and all species combined</caption><thead><tr><th>Species</th><th>n</th><th>Isotope</th><th>Slope</th><th>H<sub>0</sub>: a = 0, p<sup>a</sup></th><th>H<sub>0</sub>: a = 1, p<sup>a</sup></th><th>Intercept</th><th>H<sub>0</sub>: b = 0, p<sup>a</sup></th><th>R<sup>2</sup></th></tr></thead><tbody><tr><td rowspan="2">Pardosa amentata</td><td>122</td><td>C</td><td>0.96 ± 0.17</td><td><strong>&lt;0.001</strong></td><td>0.83</td><td>-1.31 ± 4.45</td><td>0.768</td><td>0.21</td></tr><tr><td>122</td><td>N</td><td>1.17 ± 0.11</td><td><strong>&lt;0.001</strong></td><td>0.14</td><td>-0.46 ± 0.63</td><td>0.464</td><td>0.47</td></tr><tr><td rowspan="2">Pardosa lugubris</td><td>100</td><td>C</td><td>0.62 ± 0.23</td><td><strong>0.009</strong></td><td>0.10</td><td>-10.76 ± 6.0</td><td>0.078</td><td>0.06</td></tr><tr><td>100</td><td>N</td><td>0.63 ± 0.07</td><td><strong>&lt;0.001</strong></td><td><strong>&lt;0.01</strong></td><td>1.84 ± 0.3</td><td><strong>&lt;0.001</strong></td><td>0.41</td></tr><tr><td rowspan="2">Pardosa prativaga</td><td>31</td><td>C</td><td>0.95 ± 0.21</td><td><strong>&lt;0.001</strong></td><td>0.81</td><td>-1.97 ± 5.54</td><td>0.724</td><td>0.40</td></tr><tr><td>33</td><td>N</td><td>1.03 ± 0.13</td><td><strong>&lt;0.001</strong></td><td>0.99</td><td>-0.05 ± 0.68</td><td>0.940</td><td>0.59</td></tr><tr><td rowspan="2">Pardosa spp.</td><td>54</td><td>C</td><td>0.46 ± 0.12</td><td><strong>&lt;0.001</strong></td><td><strong>&lt;0.01</strong></td><td>-15.16 ± 3.1</td><td><strong>&lt;0.001</strong></td><td>0.21</td></tr><tr><td>54</td><td>N</td><td>0.7 ± 0.1</td><td><strong>&lt;0.001</strong></td><td><strong>&lt;0.01</strong></td><td>0.99 ± 0.45</td><td><strong>0.032</strong></td><td>0.50</td></tr><tr><td rowspan="2">Piratula hygrophila</td><td>14</td><td>C</td><td>1.01 ± 0.92</td><td>0.290</td><td>-</td><td>-0.76 ± 24.2</td><td>0.975</td><td>NA</td></tr><tr><td>15</td><td>N</td><td>0.82 ± 0.29</td><td><strong>0.014</strong></td><td>0.56</td><td>0.41 ± 1.3</td><td>0.700</td><td>0.32</td></tr><tr><td rowspan="2">All species</td><td>345</td><td>C</td><td>0.66 ± 0.08</td><td><strong>&lt;0.001</strong></td><td><strong>&lt;0.01</strong></td><td>-9.7 ± 2.13</td><td><strong>&lt;0.001</strong></td><td>0.17</td></tr><tr><td>350</td><td>N</td><td>0.9 ± 0.04</td><td><strong>&lt;0.001</strong></td><td><strong>0.02</strong></td><td>0.74 ± 0.21</td><td><strong>&lt;0.001</strong></td><td>0.57</td></tr></tbody></table>

Note: Bold indicates a significance at *p* < 0.05.

the field and the limited ability to identify molts to lower taxonomic levels, especially with cursorial spiders. With proper experimental set up, SIA of field-collected spider molts can give a high taxonomic level (Araneae) or a specific community level (i.e., web-building or cursorial species) snapshot of spider trophic position, for example, before a common seasonal event (i.e., flooding) or after a large-scale disturbance (i.e., recolonization after a fire). This type of investigation is well suited for web-building or burrowing spiders that often discard molts on the web

periphery or just outside of a burrow. Additionally, laboratory studies can use spider molts to create robust feeding trials focusing on isotopic changes over long life cycles while requiring fewer live specimens.

## Discrimination values using ¹³C and ¹⁵N

Our results show that discrimination values of spiders for ¹⁵N are considerably higher than previously reported.

<footnote>ªModels were tested for significance against a null hypothesis (H₀) of slope (a) = 0. If model was significant we tested it against a 1:1 fit line of slope = 1.
Intercept (b) coefficient tested against the null hypothesis of intercept = 0.</footnote>
<figure><img src="image_5.png" /><figcaption>**FIGURE 4** δ¹³C and δ¹⁵N isotopic values of abdomens as a function of leg tissue in: (a) *Pardosa amentata*, (b) *P. lugubris*, (c) *P. prativaga*, (d) *Pardosa* spp., and (e) *Piratula hygrophila*. Solid lines represent the fitted regression line. Each plot shows a 1:1 line for comparison (dashed line). Fitted line is shown only in significant relationships (see Table 7). Each tissue tested is represented by red-highlighted body parts</figcaption></figure>

However, these values are within a common range of other studied arthropods such as locust (5.1‰), woodlice (5.7‰), and mites feeding on *Chlorella* spp. (9.8‰)

(Heethoff & Scheu, 2016; Post, 2002; Rothe & Gleixner, 2000; Webb et al., 1998). Discrimination values of ¹⁵N averaged 4.66‰ in the terrestrial group and 6.82‰
in the aquatic group and were different from prey in all tissues. ¹⁵N discrimination values were not significantly different between spider legs and whole bodies suggesting that nonlethal samples of spider legs can be used to determine ¹⁵N discrimination values without using an offset. Discrimination values of ¹³C were only significantly different from prey in legs and prosomas averaging 1.49‰ and 1.24‰ in the terrestrial and aquatic feeding groups, respectively. ¹³C discrimination values in legs were significantly different from whole bodies in both feeding groups by an average of 1.67‰ and 0.84‰ in the terrestrial and aquatic feeding groups, respectively. The discrimination values between spider legs and prey support other studies showing that ¹³C can be used to show trophic shifts in arthropods (Heethoff & Scheu, 2016; Oelbermann & Scheu, 2002; Rothe & Gleixner, 2000; Webb et al., 1998). We believe that the exclusion of the abdomen from analysis removes the possibility of isotopic signatures partially reflecting leftover prey thus revealing the trophic shift using ¹³C discrimination values. Nonlethal samples of spider legs can be used to determine ¹³C discrimination, but it may require a discrimination value offset of ~1.5‰ to estimate the whole-body values.

## δ²H in spiders

In contrast to δ¹³C and δ¹⁵N in laboratory-tested spiders, δ²H signatures and discrimination values in spider legs are not reliable substitutes for whole-body spider δ²H isotopic values. Our results showed no linear relationship and consequently no predictive power of spider legs to proxy other spider body tissues. In addition, the δ²H values in our study revealed no difference between the aquatic and terrestrial cockroach groups nor any significant indication of a shift in trophic position between cockroaches and spider whole bodies. Other studies suggest that δ²H signatures can be used to indicate trophic position or differentiate between autochthonous and allochthonous primary food sources (Doucett et al., 2007; Finlay et al., 2010; Vander Zanden et al., 2016). The difference between our results and previous studies may be linked to water source. In our experiment, all cockroaches and spiders were provided the same water source, which is a primary driver of δ²H values (Hobson et al., 1999; Solomon et al., 2009). Furthermore, past investigations showing significant separation in basal food sources using δ²H (Doucett et al., 2007; Finlay et al., 2010) were carried out in arid locations that have significant effects on plant evaporation and transpiration, two metabolic processes that greatly influence δ²H isotopic values and isotopic fractionation (Vander Zanden

et al., 2016). If δ²H isotope signatures are necessary or if water source is not accounted for in an analysis, we suggest using the entire spider body to help control for the high variation in isotopic values between tissues.

## Conclusions

Our study shows that spider legs and molts are reliable proxies of whole-body δ¹³C and δ¹⁵N stable isotope values and should be considered as an alternative to lethal whole-body sampling in practical situations in both laboratory and field settings. However, future researchers using nonlethal tissues need to have clear understanding of the isotopic time reflected by each tissue. We also found that nonlethal spider leg samples are not reliable substitutes for δ²H isotopic value in whole bodies, although the drivers of δ²H values require much more research. Our study has also highlighted the varying discrimination values found in spiders and spider tissues and how they can affect nonlethal sampling in stable isotope investigations. The demonstrated nonlethal sampling technique is a critical step to study food webs or diet sources of rare or endangered spider species (Wilhelm & Nelson, 2012). Nonlethal sampling is especially well suited for threatened species such as *Dolo medes plantarius* (Monsimet et al., 2020), which has a body size and life history suitable for such a method. Appendage loss (autotomy) in spiders may influence mating, foraging, or locomotion (Fraser et al., 2020), and concerns about removing legs of live specimens are warranted. However, juvenile and subadult spiders regrow legs between molts and most spiders can generally live full lives with fewer than eight legs (Brueseke et al., 2001; Foelix, 2010). Using legs in laboratory settings may be even more valuable than whole bodies as spider leg regeneration allows for robust stable isotope feeding studies with fewer spiders needed for time series analysis. Additionally, using only spider legs for isotope analysis may be more advantageous as the longer tissue turnover time in spider legs (Belivanov & Hambäck, 2015) reflects the diet over a longer period compared to the abdomen. When coupled with other methods to study spider diet, such as DNA barcoding, an investigator can produce a conclusive model of spider diets over longer periods (Hambäck et al., 2016). Stable isotope research using spiders should continue to focus on nonlethal sampling techniques and understanding the factors that influence isotopic discrimination values.

## ACKNOWLEDGMENTS

The authors thank Karlstad University and FORMAS for financial support. The authors also thank Lutz Eckstein
for constructive and significant comments on this manu-
script. The authors would also like to thank Frida Sjöborg
and Johanna Ekman for the maintenance of cockroach
colonies and spider room upkeep and Robin Andreasson
for providing the spiders.

CONFLICT OF INTEREST

The authors declare no conflict of interest.

DATA AVAILABILITY STATEMENT

Data are available from Zenodo (http://doi.org/10.5281/
zenodo.5075207).

ORCID

Jeffery Marker https://orcid.org/0000-0002-6011-8540

Denis Lafage https://orcid.org/0000-0002-6205-611X

Eva Bergman https://orcid.org/0000-0003-2220-1615

Rachel E. Bowes https://orcid.org/0000-0002-5614-069X

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**How to cite this article:** Marker, Jeffery, Denis Lafage, Eva Bergman, and Rachel E. Bowes. 2022. "Greater than the Sum of Your Parts: Nonlethal Stable Isotope Sampling Methods in Spiders." *Ecosphere* 13(1): e3903. https://doi.org/10.1002/ecs2.3903