Where three snail species attach while emersed in relation to heterogenous substrate temperatures underneath intertidal boulders

Sampling location

Two intertidal boulderfields along South Australia’s Gulf St Vincent were sampled, grey siltstone at Marino Rocks (35°02′ S, 138°30′E) and quartzite at O’Sullivan Beach (35°07′ S, 138°28′E). Both seashores have a westerly aspect and are located just 20 km apart on the south-eastern side of Gulf St Vincent, a large inverse estuary with a geographic setting that protects the coastline from swell. While both seashores have similar oceanic and environmental conditions such as low wave exposure, a relatively flat shore slope, and are free from shading by cliffs, localised variations in wave splash and wind speed/direction may occur. Janetzki, Benkendorff & Fairweather (2021) showed that grey siltstone was consistently one of the hottest rock types sampled from this region, while quartzite was one of the coolest (Appendix Fig. A1). Hence, these two rock types were selected as they represented the upper and lower extremes of surface temperature variability among seashore rock types for this region.

Sampling design

Janetzki, Benkendorff & Fairweather (2021) identified three qualitative patterns of temperature difference: mosaics, gradients and limited heterogeneity (Appendix Fig. A2). Limited temperature heterogeneity consisted of only small temperature differences <5 °C between the warmest and coolest areas on a boulder surface (Janetzki, Benkendorff & Fairweather, 2021, Appendix Fig. A2a). Temperature gradients consisted of a gradual decrease in temperature from the boulder side nearest the sun to the side opposite, with a temperature difference between the warmest and coolest gradient areas ≥5 °C (Janetzki, Benkendorff & Fairweather, 2021, Appendix Fig. A2b). Temperature mosaics consisted of heterogeneous temperatures across the entire boulder surface, with a temperature difference between the warmest and coolest mosaic areas ≥5 °C (Janetzki, Benkendorff & Fairweather, 2021, Appendix Fig. A2c).

Two observations regarding the development of temperature gradients and mosaics on boulder surfaces were noted during the common-garden experiment: (1) boulders must be exposed to sunlight to develop temperature differences ≥5 °C; and (2) even small-sized boulders (maximum dimension ≤ 30 cm) still take at least 4 hours to approach their thermal maxima when exposed to uninterrupted sunshine (Janetzki, Benkendorff & Fairweather, 2021; Appendix Fig. A1). Consequently, only sunny days with little to no cloud cover were sampled during this study. Boulders were sampled approximately one hour either side of predicted daytime low tides (low tide ≤ 0.40 m Australian Height Datum), as pilot studies showed boulders had been emersed for at least four hours by this time. Uninterrupted sunshine (i.e. no cloud cover) and daytime low tides are typical for the Gulf St Vincent during summer.

All sampling was completed between December 2016 and February 2017 during the austral summer to target days of extreme aerial and substratum temperatures. Air temperature measurements (1955–2017 inclusive) from the weather station nearest the sampled seashores (Adelaide Airport) showed this region of the coast experiences a mean summer maximum air temperature of approximately 28 °C (Commonwealth of Australia BoM, 2017). Therefore, sampling was completed on four days at each seashore that had maximum air temperatures at the time of low tide above this summer average (i.e. hotter days), and on four days that had maximum air temperatures at or below this summer average (i.e. cooler days) (Table 1). Field studies were approved by Primary Industries & Regions South Australia (PIRSA) under S115 Ministerial Exemption Number 9902638.

Pilot studies found the grazing snails Nerita atramentosa (Reeve, 1855), Diloma concameratum (W. Wood, 1828), and Bembicium nanum (Lamarck, 1822) to be most abundant, with sampling subsequently targeting these three species. McMahon (1990) showed D. concameratum had the lowest heat tolerance of these species, reporting a heat coma temperature (HCT) of 35.6 °C in heated seawater. A HCT of 38.9 °C was identified for N. atramentosa in heated seawater (McMahon, 1990). Bembicium nanum likely has the highest heat tolerance of these three species, although no HCT was identified in the published literature. However, experiments testing the heat tolerance of a congener, B. vittatum, identified a HCT of 41.0 °C in heated seawater (McMahon, 1990). Given B. nanum is also a higher-shore littorinid (Underwood, 1975), and occurs in similar sub-tropical and temperate habitats as its congener B. vittatum, it is likely to have a similar heat tolerance.

Nerita atramentosa and D. concameratum generally had a mean shell length ≥15 mm, and were hence identified as adults, with N. atramentosa reaching reproductive maturity at a mean shell length of 13.5 mm (Underwood, 1975). The highest abundances of N. atramentosa and D. concameratum were recorded lower on the seashore, approximately 5–15 m shoreward of the low-tide mark (Fig. 1). In contrast, B. nanum generally had a mean shell breadth ≤5 mm, with individuals identified as juveniles, with this species reaching reproductive maturity at a mean shell breadth of 11 mm (Underwood, 1975). The highest abundances of B. nanum occurred higher up the seashore, approximately 15–25 m shoreward of the low-tide mark (Fig. 1). These species-specific seashore distributions mandated that sampling be completed in two shore-parallel zones, with each zone having an across-shore width of 5 m and an along-shore length of 80 m (Fig. 1). One zone, Zone A, was established approximately 5 m shoreward of the low-tide mark to sample adult N. atramentosa and D. concameratum, while Zone B was established 15 m shoreward of the low tide mark to sample juvenile B. nanum (Fig. 1).

Randomly-selected boulders were only sampled if their lower surfaces were inhabited by an individual from one or more target species, with each sampled individual having to occur as an independent organism. In this study, an independent organism was defined as any individual that had no direct physical contact with conspecifics. Consequently, organisms clustered in groups (predominantly N. atramentosa and D. concameratum) were only sampled if the individual organisms constituting each cluster were not in physical contact with one another, so as to constrain their choice. We chose not investigate any associations between gastropod aggregation and substrate temperature, as these have been investigated previously (e.g. Chapperon, Le Bris & Seuront, 2013; Chapperon, Studerus & Clavier, 2017). Only one individual, from each target species, per lower surface was sampled, with sampled individuals being randomly selected. Multiple target species per surface were sampled if surfaces were cohabited.

To sample lower surfaces, boulders were flipped upside down and shaded from direct sunlight. Boulders were only sampled if snails remained attached during the flipping process. Where possible, 30 under-boulder surfaces inhabited by each target species were sampled at each seashore on each day (Table 1). Target snail species exhibited a patchy and over-dispersed distribution (Chapman & Underwood, 1996; Chapman, 2005), with many surfaces unoccupied by any target species. Zone A was always sampled before Zone B to ensure all boulders were sampled before they were submersed by the incoming tide (Fig. 1).

Imagery was used to capture associations between snails and substrate temperatures in situ. For every sampled surface (N = 1152), a digital photograph and a thermal image were recorded, and archived images were subsequently processed in the laboratory. Digital photographs were captured using an Olympus Tough TG-820 digital camera, and were used as a reference to identify individual snails (Fig. 2). Thermal images were captured using a Ti20 Fluke thermal imaging camera, using the same camera settings described by Janetzki, Benkendorff & Fairweather (2021). The thermal resolution of this camera was ≤0.2 °C at 30 °C, with an accuracy to 2% or 2 °C, whichever was greater (Janetzki, Benkendorff & Fairweather, 2021). Default camera settings were employed, including emissivity, which was set at 0.95 (Janetzki, Benkendorff & Fairweather, 2021). As detailed in Janetzki, Benkendorff & Fairweather (2021), this default emissivity was applied, even though emissivity may have differed within and among the rock types sampled, as previous thermography studies in the long-infrared range (9–14 µm) have shown that the emissivity of dry rock generally ranges between 0.95 and 1 (Rivard, Thomas & Giroux, 1995; Danov, Vitchko & Stoyanov, 2007; Cox & Smith, 2011; Lathlean, Ayre & Minchinton, 2012). To avoid measuring inaccurate temperatures from increased amounts of thermal energy being reflected by wet surfaces (Lathlean, Ayre & Minchinton, 2012; Lathlean & Seuront, 2014; Seuront, Ng & Lathlean, 2018), boulders from wet habitats (i.e. rock pools or wet sediment) were not sampled. As boulders had been emersed for at least 4 h before being sampled, and previous research has confirmed these types of boulders take no longer than 5–10 min to dry once emersed as water rarely permeated their surfaces (Janetzki, Benkendorff & Fairweather, 2021), differential rates of drying among individual boulders or rock types was not a confounding variable.

Due to their large size and shell temperatures generally somewhat cooler than the substrate, N. atramentosa and D. concameratum were easy to locate in archived thermal images (Fig. 2). In contrast, the smaller B. nanum generally had shell temperatures more similar to the substrate, and were often difficult to locate on the image (Fig. 2). To rectify this, empty N. atramentosa shells were chilled on ice and were then placed approximately 1 cm to the lower right of B. nanum individuals when recording thermal images (Fig. 2). As the chilled shells were much cooler than the substrate, they acted as an easily-identifiable reference point, making it easier to locate B. nanum in archived thermal images (Fig. 2).

Archived thermal images were processed using the InsideIR version 4.0 software (Fluke Corporation, Everett, WA, USA). For each image of lower surfaces, maximum, minimum and average boulder temperatures were quantified by tracing around each boulder. A temperature range (i.e. maximum − minimum = range) for each surface was then quantified (daily n = 62–80, Fig. 2). A target temperature was recorded for each sampled snail, and, for purposes of standardisation, was measured to the immediate left of each snail (Fig. 2). The maximum, minimum, and average temperatures of each lower surface were subtracted from each target temperature, to determine how snails were associated with substratum temperatures. Due to the low probability that individual snails will be randomly recorded only in areas with the maximum (or minimum) substratum temperature, comparisons between snail target temperatures and average substrate temperatures were also completed to better test the hypothesis that snails occupy cooler areas on boulder surfaces.

Within each traced boulder perimeter, a transect was drawn from the centre of the boulder side facing the sun to the centre of the side opposite (Fig. 2). This was done to verify whether the three qualitative patterns of temperature heterogeneity (gradients, mosaics, limited heterogeneity) described by Janetzki, Benkendorff & Fairweather (2021) also occur for boulders on the seashore. Transects were drawn for 20 randomly-selected images for three days per seashore, spanning the range of maximum daily air temperatures sampled. This subset of boulders was deemed sufficient to quantify the frequency of occurrence for these temperature patterns, as Janetzki, Benkendorff & Fairweather (2021) showed that their occurrence was primarily influenced by the presence/absence of cloud cover, which was not a factor here as only sunny days were sampled.

Statistical analyses

Analyses were completed using either PRIMER v7/PERMANOVA+ (PRIMER-e, Plymouth, UK) or SYSTAT v13 (Systat Software Inc) statistical software, with significance set at α = 0.05, unless otherwise noted. To establish whether boulders on the seashore developed heterogenous temperatures (H1), frequencies of occurrence (%) for each temperature pattern (gradients, mosaics, limited heterogeneity) were tallied separately for 20 boulders per seashore, on each day sampled. Temperature pattern tallies were then pooled across the three days sampled per rock type, with a Chi-square two-way contingency table analysis testing (α = 0.05) for any associations between rock type and temperature patterns using these pooled tallies.

To test whether temperature characteristics differed between rock types (H2), means for boulder temperature range and maxima on each sampling day were used in separate univariate one-factor PERMutational ANalyses of COVAriance (PERMANCOVA). Analyses were completed using their untransformed means, with Euclidean distance resemblance matrices prepared separately for each temperature characteristic as dependent variables. To test whether mean temperature characteristics were related to air temperature, maximum daily air temperatures were used as the co-variate in PERMANCOVA models. Permutations of residuals were completed using a reduced model with 9,999 permutations.

To test whether snail target temperatures were significantly different from the maximum or average temperature of boulders (H3), paired t-tests were completed for each target species on each day sampled for each rock type. As the same target temperatures were used in comparisons against maximum and average temperatures, a Bonferroni correction was applied to reduce the likelihood of making a Type I error. Consequently, a corrected significance level of α = 0.025 was applied. Means were then calculated from the replicate boulders on each day for the difference between target temperatures and boulder maxima, and between target temperatures and boulder minima. Mean differences were also used in one-factor PERMANCOVA, as described above (with air temperature as the covariate), to test for differences between grey siltstone and quartzite in how each snail species was associated with the temperature range on lower surfaces (H4). As the same mean target temperature was used to calculate each temperature difference measure for each species, a Bonferroni correction (corrected α = 0.025) was applied.

Emersed boulder lower surfaces have a heterogenous surface temperature

The lower surfaces of grey siltstone and quartzite boulders generally had a heterogeneous surface temperature. The mean range of this heterogeneous temperature differed by >5 °C on each day sampled at each seashore (Fig. 3). The type of qualitative temperature pattern identified on lower surfaces differed between rock types (Pearson Chi-square p-value = <0.001), with siltstone generally developing gradients (Fig. 3A) whereas quartzite developed mosaics (Fig. 3B). Quartzite (60%) had an overall higher frequency of mosaics versus siltstone (none), while siltstone (76.7%) had more gradients than quartzite (15%) when data were pooled across all days analysed (Table 2, Appendix Fig. A3).

Overall occurrences of limited temperature heterogeneity were similar between grey siltstone (23.3%) and quartzite (25%) when data were pooled across all days analysed. Occurrences of limited heterogeneity were low (15%) for both rock types on the two cooler days sampled at each seashore, where gradients dominated on siltstone (85%) and mosaics on quartzite (≥70%, Table 2, Appendix Fig. A3). However, on the hottest day sampled for both rock types, occurrences of limited heterogeneity increased to 40–45% of all boulders sampled (Table 2), with a corresponding decrease in the occurrence of gradients and mosaics, especially mosaics on quartzite (Table 2, Appendix Fig. A3).

Grey siltstone has a hotter maximum than quartzite

The mean temperature range on boulders across sampling days did not significantly differ between grey siltstone and quartzite (univariate PERMANCOVA p-value = 0.77, Figs. 3 & 4A, Appendix Table A1). Temperature range was not related to the daily maximum air temperature (PERMANCOVA covariate p-value = 0.64, Fig. 4A, Appendix Table A1). For grey siltstone, the smallest temperature range for an individual surface was 2.3 °C, while the largest temperature range was 18.5 °C. For quartzite, the smallest temperature range was 3.2 °C, while the largest temperature range was 16.9 °C.

Mean maximum temperature differed between rock types (PERMANCOVA p-value = 0.0073, Fig. 4B, Appendix Table A1), with grey siltstone (overall grand mean = 41.1 ± 0.2 °C) having significantly hotter maxima than quartzite (overall grand mean = 39.3 ± 0.2 °C). The mean maxima were significantly correlated with the maximum air temperatures on the day of sampling (PERMANCOVA covariate p-value = <0.0011, Appendix Table A1), with hotter maxima recorded on days with hotter air temperatures (Fig. 4B). The maximum temperature of each replicate boulder was generally hotter than the air temperature at sampling (Figs. 4C–4D). For grey siltstone, the coolest temperature measured was 20.1 °C, while the hottest temperature was 53.6 °C. For quartzite, the coolest temperature was 18.5 °C, while the hottest temperature was 52.2 °C.

Target temperature differs from the maximum or average boulder temperature

Target temperatures, immediately adjacent to snails, were significantly cooler than the maximum temperature of boulders, on each day sampled for both rock types, for all three species (all paired t-test p-values = <0.001, Table 3, Figs. 5 & 6). Somewhat larger differences between target and maximum temperatures were recorded for areas of the rock surface occupied by D. concameratum and N. atramentosa than for B. nanum (Fig. 5, Table 1). For B. nanum, mean target temperatures were 4.1–5.7 °C and 4.0–4.8 °C cooler than mean maxima on each day sampled for grey siltstone and quartzite, respectively (Table 1). For D. concameratum mean target temperatures were 5.0–8.0 °C cooler on siltstone and 5.0–5.7 °C cooler on quartzite than mean maxima, while for N. atramentosa, mean target temperatures were 4.9–7.9 °C cooler on siltstone and 4.9–5.9 °C cooler on quartzite than mean maxima on each day sampled (Table 1). The hottest individual target temperatures measured immediately adjacent to snails for each species were 50.7 °C for B. nanum, 49.1 °C for D. concameratum and 46.3 °C for N. atramentosa. The association between target temperature and boulder maxima developed during the first hour that boulders were emersed, and was maintained during the four hour low-tide period. The position of snails on the boulders and associated target temperatures was not related to the amount of surface moisture retained on boulders (see Appendix for additional hypotheses tested and results).

For both D. concameratum and N. atramentosa, target temperatures were always cooler than average boulder temperatures on each day sampled for both rock types, with differences of up to 1.81 °C (Table 3). When observations are pooled across rock type, this difference was significant on 13 out of 16 days sampled for D. concameratum and 11 out of 16 days for N. atramentosa. In contrast, B. nanum target temperatures were generally similar (always <1 °C difference) to mean boulder temperatures on each day sampled for both rock types (Table 3). Target temperatures, adjacent to the snails, were significantly warmer than the minimum temperature of boulders, on each day sampled for each rock type, for all three species (paired t-test p-values = <0.001, Table 3, Fig. 5).

Differences between target temperature and either thermal maxima or minima on boulders differed between grey siltstone and quartzite

Target temperatures were further from the maxima, nearer to the minima, and cooler relative to the average, on grey siltstone versus quartzite for all three snail species (Appendix Table A2). For D. concameratum, significantly larger negative mean differences were detected for grey siltstone than quartzite for target—maximum substrate temperatures (PERMANCOVA p-value = 0.0014, Appendix Table A1), while significantly smaller positive mean differences were detected for grey siltstone than quartzite for target—minimum temperatures (PERMANCOVA p-value = 0.0030, Fig. 5, Appendix Table A1). For N. atramentosa, significantly larger negative mean differences were identified for grey siltstone than quartzite for target—maximum temperatures (PERMANCOVA p-value = 0.0026, Appendix Table A1), although the smaller positive mean differences for siltstone than quartzite for target - minimum temperatures were not significantly different after Bonferroni correction (PERMANCOVA p-value = 0.0380, Fig. 5, Appendix Table A1). For B. nanum, this rock-related difference was not significant for mean target—maximum substrate temperatures (PERMANCOVA p-value = 0.12, Appendix Table A1), although significantly smaller positive mean differences were recorded on siltstone than quartzite for target—minimum substrate temperatures (PERMANCOVA p-value = 0.0019, Fig. 5, Appendix Table A1).

These mean differences between snail target temperature and the maxima and minima of boulders, for each rock type, were not strongly influenced by the air temperature at sampling for any species (smallest PERMANCOVA covariate p-value = 0.0329 for target—minimum temperatures for D. concameratum, which was not significant after Bonferroni correction, Appendix Table A1).