Species composition of shoreline wolf spider communities vary with salinity, but their diets vary with wrack inflow

Abstract Wolf spiders are typically the most common group of arthropod predators on both lake and marine shorelines because of the high prey availability in these habitats. However, shores are also harsh environments due to flooding and, in proximity to marine waters, to toxic salinity levels. Here, we describe the spider community, prey availabilities, and spider diets between shoreline sites with different salinities, albeit with comparatively small differences (5‰ vs. 7‰). Despite the small environmental differences, spider communities between lower and higher saline sites showed an almost complete species turnover. At the same time, differences in prey availability or spider gut contents did not match changes in spider species composition but rather changed with habitat characteristics within a region, where spiders collected at sites with thick wrack beds had a different diet than sites with little wrack. These data suggest that shifts in spider communities are due to habitat characteristics other than prey availabilities, and the most likely candidate restricting species in high salinity would be saline sensitivity. At the same time, species absence from low‐saline habitats remains unresolved.

& McLachlan, 2013). Species diversity on shorelines may therefore be poor, particularly on marine shorelines where communities often consist of a range of habitat specialists that can endure high salinity levels (Cheng, 1976;Irmler et al., 2002).
Despite these general patterns, there is a lack of understanding on how physical processes and prey availability interact in shaping coastal arthropod communities (Hyndes et al., 2022). In fact, the spatial variability of arthropod communities in these habitats is poorly documented compared with inland habitats. For instance, what differences in the species composition between limnic and marine shorelines are due to direct effects from a saline environment and what differences are rather due to differences in prey communities?
Prey communities on limnic shorelines are often dominated by midges and a range of other taxa (Benke, 1998;Delettre & Morvan, 2000;Salvarina et al., 2017), whereas prey communities on marine shorelines are more dominated by species developing in rotting wrack beds (Hyndes et al., 2022;Schlacher et al., 2017). Similarly, what is the relative importance of the inflow of dead organic matter versus prey that developed in the water for shoreline predators? Previous studies suggest that the importance of these different resources for spiders and other shoreline predators may vary both between sites, between life stages, and over time (Mellbrand et al., 2011;Paetzold et al., 2008;Verschut et al., 2019). The diet analysis of spiders across the season by Verschut et al. (2019) indicated that adult wolf spiders during early summer on marine shorelines feed largely on terrestrial dipterans such as dung flies, which have developed in wrack beds, whereas juvenile wolf spiders later in season had fed more on aquatic dipterans such as chironomids, where the larvae had fed on algae or detritus in the water.
To approach these questions, we studied prey communities, spider diets, and spider community structure between regions with different salinity along the Swedish coast. The salinity changes continuously from freshwater (<1‰) in the inner parts of the Bothnian Bay to oceanic conditions (>30‰) on the western coastlines, which allow us to explore effects from comparatively small salinity differences. In this study, we included two coastal regions with 5 ‰ and 7 ‰, respectively, where previous studies have indicated the shifting dominance of spider species (Hambäck et al., 2016;Verschut et al., 2019). We focus our attention on wolf spiders because these typically dominate the shoreline predator community in the area (Mellbrand & Hambäck, 2010). To account for the role of marine inflow, we aimed to include sites with and without thick wrack beds in each region. We also needed to control for climatic effects because the salinity gradient for our study is also a latitudinal gradient. For this reason, we used a similarly collected data set of spider communities on shores by inland waters along the same latitudinal gradient and with similar climate ( Figure A1). Finally, to examine the role of a changing prey community and spider diet, we estimate prey densities using SLAM traps and collected spiders for gut metabarcoding in the same sites. Prey densities and spider diets were estimated two times, to cover seasonal changes in prey availability and diet differences between adult and juvenile spiders (cf. Verschut et al., 2019).

| ME THODS
Study sites: The coastal regions included in the study were (a) Uppland north of Stockholm with the lowest salinity (≈5‰, northern region) and (b) Kalmar and Öland in southeastern Sweden with somewhat higher salinity (≈7‰, southern region) ( Figure 1, Table A1). The numbers of coastal sites were 13 (Uppland) and 7 (Kalmar). Among these, two sites, respectively, had thick wrack beds, and the other sites were similar but without thick wrack beds and often with short-cut grass due to grazing. The thick wrack beds had a thickness of more than 20 cm with a considerable extension (several 10 s of meters).
The nonwrack sites either lacked wrack almost completely (as in the Uppland region) or that wrack occurred in scattered patches and never so thick as to provide a suitable habitat for detritivores (as in the Kalmar region). The inland regions included 15 and 23 shoreline sites in Uppland and southern Halland (same latitude as Kalmar), respectively ( Figure 1; Table A1), as part of a broader study focusing on both insect and spider communities in wetlands. Inland wolf spider communities were sampled during June 2020 from sites between a few km to more than 100 km inland. These times were chosen because wolf spiders are then adults or subadults, which simplifies species identification, and abundances are quite constant until the end of June when reproduction is finished and adults die off. The interannual differences are also small, as indicated by multiannual trapping campaigns in some of the included sites (Hambäck unpubl. data). Captured spiders were placed in 70% ethanol and brought to the laboratory for identification. Spiders from the inland sites were identified by R. Vicente and those from coastal sites by M. Langbak and A. Hoffmann, with assistance from R. Vicente for complicated cases (mainly involving Pardosa agrestis/ agricola/monticola).

F I G U R E 1
Map showing the location of regions (northern sites = Uppland, southern coastal sites = Kalmar, southern inland sites = Halland). For site information, see SI Table A1.
Spiders used for diet analyses were only collected from 13 sites, six from Uppland and seven from Kalmar, including all wrack sites. Spiders were individually collected by hand (30 per site), to reduce contamination risk, at two times (June and August 2019) and directly transferred to 95% ethanol. In the lab, samples were placed in a freezer (−20°C) until DNA extraction and further processing. Finally, prey densities were estimated by placing one SLAM (Sea Land Air Malaise) trap for two nights at the same time when collecting spiders for diet analyses. SLAM trap catches were placed in 70% ethanol, and brought to the laboratory for sorting to family or order level.
Diet analyses: To metabarcode prey content of the handcollected spiders, DNA was extracted from either a dissected abdomen (larger spiders) or the whole specimen (small spiders). To reduce the DNA yield of the focal spiders, we used a forward primer designed not to amplify wolf spider DNA (NoSpi2, Lafage et al., 2020) in combination with a general reverse primer (fwhR2n, Vamos et al., 2017) to amplify a section within the Folmer region of COI (Folmer et al., 1994). Procedures for PCR amplification and library building follow Hambäck et al. (2021), and sequencing of the spider samples was performed in one batch on the Illumina MiSeq3 platform at SciLifeLab in Stockholm. To detect individual samples after sequencing, a dual tagging approach was used where the 5′-end of both primers included an 8 base-pair tag (Binladen et al., 2007).
Illumina-adaptors bearing unique indices were then ligated to the phosphorylated amplicons without a PCR step to preclude tagjumping errors (Bohmann et al., 2022). Due to problems with low DNA content, we had to change the strategy and add a second PCR step with a low cycle number (6). Because this additional step increases the risk of tag-jumping errors, we built libraries separately for each site, using SMARTer ThruPLEX DNA-seq library preparation kit excluding fragmentation of DNA (Takara Bio), as tag jumps between spiders within the site do not affect the results due to pooling at this level before analysis. In each library, we also included at least 25% empty combinations to estimate tag-jumping errors (which was about 6%). After sequencing, we used ObiTools (Boyer et al., 2016) within the Galaxy Platform (Jalili et al., 2020) to assemble pairedend sequences of high quality (score > 40), trim primers, clean sequences using "obiclean," and demultiplex resulting sequences to individual samples using "NGSFILTER" after filtering for size. These procedures resulted in a data set of 367 spider individuals and about 384,600 prey sequences that were grouped based on 97% similarity and where representative sequences were taxonomically assigned using BoLD (Ratnasingham & Hebert, 2007) before further analyses.
Statistical analyses: Spider communities were modeled as the abundance of each spider species per site in a multivariate analysis with region, inland/coast, wrack, and the region-by-inland/coast interactions as independent variables using the command manyglm (package: mvabund, Wang et al., 2012) with a negative binomial error distribution. Prey communities were similarly modeled as the abundance of major groups in a multivariate analysis with manyglm between regions with wrack as an independent variable and a negative binomial error distribution, but these tests additionally included season (June and August) as an independent variable. Finally, the proportional number of prey sequences (logit-transformed) of major groups were pooled for each species within site and season and was modeled using adonis2 (package: vegan, Oksanen et al., 2019). To compare diet composition between spider species, we also compared gut contents while controlling for the effects of the region.
To examine model assumptions, we used plot.manyglm and all tests showed no pattern in errors, which confirms the model appropriateness. Significant relationships were further explored using ANOVA with adjusted p-values, to identify which groups that explained the variation. In all these tests, prey communities and spider diets were included at the level of family or higher taxonomic unit and not at a species level.
To study prey diversity and diet consistency within and among species, we first calculated individual diets using the dynamic threshold model in Cirtwill and Hambäck (2021). We then compared species accumulation curves in spider guts using specaccum with spider individual as a sampling unit (package: vegan, Oksanen et al., 2019) and then estimated diet consistency by calculating the Jaccard similarity index between diets of individual spiders' prey species and prey families, first between pairs of all spider individuals and then between individual pairs of the same species. Diet similarity was compared between region, wrack, and their interaction, firstly, depending on if pairs included all spider individuals or were restricted to within species comparison and, secondly, depending on if diets were based on prey species or prey family. If the interaction terms did not contribute, models were re-fit without the interaction. We then tested for pairwise differences between region-wrack combinations using a Tukey's HSD test applied to the analysis of variance of the above linear models, including the interaction term between region and wrack. All tests were performed using R 3.6.3 (R Core Team, 2020).

| RE SULTS
The analysis of spider communities included 3931 spider individuals separated into 16 taxa ( Figure 2). The variation in community composition was explained by a region-by-inland/coast interaction (Wald statistics = 8.3, p < .001) and not by the presence or absence of a thick wrack bed (Wald statistics = 4.7, p > .1). The region-byinland/coast interaction arose because of a larger difference between southern and northern coastal sites compared with southern and northern inland sites ( Figure 2). When comparing abundances at the species level (Table 1), four species (Pardosa agrestis, P. agricola, Arctosa leopardus, and Alopecosa cuneata) were found almost exclusively at southern coastal sites and three taxa (Pardosa prativaga, P. amentata, and Pirata spp. [mainly P. piraticus]) almost never occurred in these sites but were abundant elsewhere ( Figure 2). In addition, one species (P. monticola) was mainly coastal whereas another species (Pardosa palustris) occurred mainly inland, irrespective of region.
The number of prey items encountered in the gut of spider individuals varied between one and 15, with an average of 3.9. The dominant order in the guts was Diptera, both Brachycera (60%) and Nematocera (18%), with minor amounts of other groups; Homoptera (10%, mainly Cicadellidae and Delphacidae), Collembola (4%), other flying prey (3%, Hymenoptera and Lepidoptera), Formicidae (2%), Acari (2%), and Heteroptera (1%) (Figure 4, Table A2). The diet contents varied considerably among sites and were mainly explained by wrack (Lawley-Hotelling trace statistics = 6.1, p < .001) and season  region. Because of the almost significant region-by-wrack interaction on gut contents, we repeated the analysis for sites with or without wrack separately. In this analysis, the region was significant for sites without wrack (p < .03) but not for sites with wrack (p > .2).
The species accumulation curves indicated that prey diversity was higher in southern sites and in sites with no wrack compared with northern sites and wrack sites ( Figure 5). When comparing F I G U R E 3 Relative abundances of prey catches in SLAM traps, separated by wrack occurrence, region (south = Kalmar, north = Uppland), and season (A = August, S = July). Detritivore flies include Sepsidae, Sphaeroceridae, and Coelopidae. Other flying prey include Hymenoptera and Lepidoptera but also a range of terrestrial Diptera.

F I G U R E 4
Relative contents of spider guts from sites with or without wrack and in the northern (Uppland) or southern (Kalmar)  consistency than those from southern nonwrack sites ( Figure 6).
These diet similarities were larger when performed for pairs of the same spider species (Figure 6), but patterns were otherwise similar

| DISCUSS ION
The spider community showed large regional changes along the Baltic Sea seashore despite comparatively small salinity differ- High salinity has several negative impacts on spiders and other arthropods, by reducing both survival and reproduction (Foucreau et al., 2012;Pétillon et al., 2011;Puzin et al., 2011). Even though none of the species found on the Baltic shorelines can be considered true halophilic and are usually not found on more marine seashores (Pétillon et al., 2008), it seems reasonable to assume that species F I G U R E 5 Species accumulation curves (±SD) relative to the number of sampled spiders for northern sites (Uppland) and southern sites (Kalmar), with or without wrack accumulation.

F I G U R E 6
Individual diet similarity estimated as Jaccard similarity index (±SE) separated for the region (N=Uppland, S=Kalmar) and wrack presence. The diet similarity was estimated between all pairs of individuals (red) or between pairs of the same species (white) and when prey were included at the species ( To summarize, our study indicates that quite a small difference in salinity caused the species composition of wolf spider communities to change almost completely. The mechanism underlying this community shift is less obvious, why species disappear either in the high salinity or in the low salinity ends, but we can conclude that prey availability or differences in the trophic niche between species is likely not involved. Investigation (equal); writing -review and editing (supporting).

ACK N OWLED G M ENTS
This work was supported by the National Genomics Infrastructure (NGI) and Uppmax through the Science for Life Laboratory, which is funded by the Knut and Alice Wallenberg Foundation and the Swedish Research Council Vetenskapsrådet.

CO N FLI C T O F I NTE R E S T
The authors have no competing financial or personal interests that would conflict with the content of this paper.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are openly available in Dryad at http://doi.org/10.5061/dryad.gxd25 47qk (Hambäck et al., 2023).

TA B L E A 1
Site information, where spider communities were quantified in all sites and where diet data and prey availabilities were collected from a subset of sites