Seasonal changes in the thermal regime and gastropod tolerance to temperature and desiccation stress in the rocky intertidal zone

Journal of Experimental Marine Biology and Ecology 488 (2017) 83–91

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Seasonal changes in the thermal regime and gastropod tolerance to temperature and desiccation stress in the rocky intertidal zone William B. Stickle a,⁎, Emily Carrington b, Hilary Hayford b a b

Louisiana State University, Department of Biological Sciences, Baton Rouge, LA 70803-1715, USA Department of Biology and Friday Harbor Laboratories, University of Washington, Seattle, WA, USA

a r t i c l e

i n f o

Article history: Received 23 September 2016 Received in revised form 5 December 2016 Accepted 8 December 2016 Available online xxxx Keywords: Seasonal thermal regime High emersion temperature tolerance Freezing temperature tolerance Desiccation tolerance Rocky intertidal zone Gastropod species

a b s t r a c t The upper end of a species distribution in the rocky intertidal zone is determined by abiotic stress tolerance. Hobo probes recorded intertidal temperatures throughout the rocky intertidal zone every 5 min from March 2015 through May 2016. Probes were placed at +2.0 m, +1.5 m, +1.0 m, +0.5 m and 0 m above mean lower low water. Emersion duration and temperature increased as a function of probe height for the eastern and southern transect probes from May through August 2015 but were not significantly different between spring and neap tide periods. The maximum emersion temperature of intertidal probes during the summer of 2015 was 31.65 °C and the minimum air temperature was −3.42 °C at +1.5 and 2.0 m during the winter. Duration of exposure to freezing also increased with intertidal height. High temperature, freeze temperature and desiccation emersion tolerance of Nucella lamellosa; N. canaliculata; N. ostrina; Littorina sitkana; and L. scutulata were studied to examine the idea that upper distribution limits are set by abiotic stressors and different species along the intertidal gradient should exhibit differential sensitivities. Snail tolerances varied directly with their intertidal position. Emersion temperatures during the summer were well below species high temperature LT50 values for the Littorines and N. lamellosa but near those of N. canaliculata and N. ostrina; these latter two species may be physiologically stressed and utilize behavioral avoidance strategies. Emersion temperatures below freezing occurred in November and December 2015 and January 2016 at the 1.0, 1.5 and 2.0 m levels. Minimum freezing air temperatures in the intertidal zone were well above the freeze tolerance of low intertidal N. lamellosa, and high intertidal L. sitkana and L. scutulata but within the freeze tolerance range of mid intertidal N. canaliculata and N. ostrina indicating that they may be stressed and behavioral avoidance strategies may be operative. © 2016 Elsevier B.V. All rights reserved.

1. Introduction The upper end of a species vertical range in the rocky intertidal zone is frequently determined by the species tolerance of emersion temperature and desiccation (Connell, 1970, 1972; Wolcott, 1973; Menge and Sutherland, 1987) and by biotic interactions at the lower end of the species range (Paine, 1966, 1969). The latitudinal and vertical patterning of species distributions in the intertidal zone commonly reflects gradients or discontinuities in emersion temperatures (Somero, 2002, 2005). Low tides in the Inside Passage from Puget Sound, WA to Skagway, AK produce extreme emersion temperatures because low spring tides occur during the day in the summer and at night in the winter. Biota in the upper regions of the intertidal zone are exposed to a longer duration of emersion during neap tides in the mixed semidiurnal tidal pattern which is characteristic of Southeast Alaska. Here the vertical distribution of three common species of rocky intertidal snails, Nucella lamellosa, N. ⁎ Corresponding author. E-mail address: [email protected] (W.B. Stickle).

http://dx.doi.org/10.1016/j.jembe.2016.12.006 0022-0981/© 2016 Elsevier B.V. All rights reserved.

lima, and Littorina sitkana, was directly related to their high emersion temperature and freeze tolerance but was not related to desiccation tolerance, perhaps because southeast Alaska is found in the largest temperate rain forest in the world (Stickle et al., 2016). Modifying factors such as regional differences in the timing of low tides can overwhelm large-scale climatic gradients (Mislan et al., 2009). The thermal regime of the mid intertidal on the outer Pacific coast of the continental Unites States is ameliorated because spring low tides occur at night in the summer and during the day in the winter (Helmuth et al., 2002, 2006a, 2006b). Helmuth et al. (2002, 2006a, 2006b) also reported mid-tidal emersion data for two locations at the southern end of the inside passage on San Juan Island, WA. They noted significantly more freezing events at those locations than occurred on the outer coast. A number of studies have documented the effects of emersion temperature on the acute and chronic performance of rocky intertidal fauna throughout their latitudinal and intertidal thermal zones on the continental Pacific coast of the United States as discussed in Stickle et al. (2016). Among the physiological traits related to intertidal vertical

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zonation are thermal tolerance, heart function, mitochondrial respiration, membrane fluidity, action potential generation, protein synthesis, heat-shock protein expression and protein thermal stability (Somero, 2002). It is important to link patterns and mechanisms of physiological adaptation to global climate change (Somero, 2010) in order to make accurate predictions of future climatic effects on species' performance and distribution. Earlier studies reporting thermal responses of species or congeneric species as a function of their intertidal zonation or their latitudinal distribution were summarized by Stickle et al. (2016). All intertidal gastropods are freeze tolerant (Sinclair et al., 2004). Freeze tolerances are strongly correlated with latitudinal distributions across many taxa (Sunday et al., 2014) and freeze tolerances of intertidal snails from Southeastern Alaska as a function of their vertical intertidal distribution has also been documented by Stickle et al. (2010, 2015). Recent studies have documented the effects of emersion temperature throughout the year on the freeze tolerance (Stickle et al., 2010, 2015), high temperature tolerance and desiccation tolerance of common intertidal gastropods at a site at the north end of the inside passage (Stickle et al., 2016). This companion study has two objectives (1) document the thermal regime of rocky intertidal gastropods throughout their vertical ranges from a location at the south end of the inside passage of the west coast of North America and (2) document the temperature and desiccation tolerance of five species of rocky intertidal gastropods; the high intertidal Littorina scutulata and L. sitkana, mid-intertidal Nucella ostrina and N. canaliculata, and low intertidal N. lamellosa. 2. Materials and methods 2.1. Seasonal change in thermal conditions Two vertical transects of ProV2 Hobo temperature probes covered with protective sleeves were deployed on March 27, 2015 through May 31, 2016 at the University of Washington's Friday Harbor Laboratories Biological Preserve (FHL) on San Juan Island in the Salish Sea (28.546° N, 123.008° W). Probes recorded ambient temperature every 5 min just off the rocky substrate, representing environmental temperatures that may be experienced by benthic intertidal organisms at each discrete tidal elevation. The two transects were separated by several m, with one transect having a southern aspect and the other an eastern aspect. Probes were deployed at +2 m, +1.5 m, +1.0 m, +0.5 m and 0 m above mean low low water (MLLW). Some probes were lost during the deployment period, only the 0 m and 2.0 m probes of both transects were present at the end of the study period. Morphology is important to estimating body temperature (Helmuth and Hofmann, 2001; Gilman et al., 2015), therefore we chose aerial probes to estimate the environmental stress that may be experienced by several species. Biomimetic temperature probes constructed of epoxy-filled N. ostrina shells and thermocouples (see Gilman et al., 2015 for details) already deployed on a nearby shoreline were referenced to ground truth environmental temperatures determined with Hobo probes to snail body temperature. The hours of emersion for each probe height during 2015 for all spring and neap tide dates were computed as the number of hours the water level was below that height with two emersion events per lunar day based on the NOAA/CO-OPS observed water levels at the Friday Harbor Harmonic station (48.2983°N 134.0150°W; mixed semidiurnal tidal pattern). The CO-OPS Data Retrieval API was used to obtain the hourly water levels across all dates of the study period, with MLLW as the 0 tide level. The water levels for the spring and neap tide dates were extracted from these data. The spring and neap tide events were determined by the dates of the primary moon phases, 25 spring tides and 24 neap tides, obtained via the data services of the Astronomical Application Department of the U.S. Naval Observatory. Welch's correction of the t-test was used to compare neap and spring tide events at

each tidal height with significant differences between means given at the p b 0.05 level. Five monthly temperature measures of the records were extracted throughout the study period from the data set as described by Helmuth et al. (2006b) and Stickle et al. (2016). The average monthly temperature is the average of all emersion and immersion temperatures during the month. The average daily maximum and minimum is the average of all daily maxima and minima recorded during the month. Maximum and minimum data for each month are emersion data as determined by a temperature at least 1° above or below the previous 5 min temperature recording. Days when precipitation occurred were recorded from a Weather Source database at the Friday Harbor Airport (https://Weathersource. com). The airport is 8.0 km north of the collecting site and 25.0 km north of the probe site. Temperature profiles were constructed using a script written in Python 3.4 with the support of multiple third-party modules. For each probe, the temperature records were read in by the script, and methods from the MatPlotLib and NumPy modules were used to plot those records in a stacked bar chart (Hunter, 2007). In the bar charts, each month has three stacked bars representing temperature ranges. The solid black bar shows the range from the monthly minimum to the average daily minimum temperature. The solid white bar shows the range from the average daily minimum to the average daily maximum, and finally the white bar with diagonal hatch marks shows the range from the average daily maximum to the monthly maximum temperature. The average monthly temperatures were plotted as a line between the minimum and maximum bars. Summer emersion temperatures were calculated beginning with the first temperature at least 1 °C above the surface seawater temperature until the temperature fell below the 1 °C above the surface seawater temperature. The neap and spring tide ± one day emersion temperatures were calculated for the south transect probes from May 3, 2015 through August 30, 2015. Probes were installed at tidal elevations that bounded the vertical distribution of the study species, 0–2.0 m above MLLW 2.2. Snail zonation, collection and maintenance On San Juan Island, WA the relative vertical ranges of the five species of gastropod from lowest on shore to highest are: N. lamellosa, N. canaliculata, N. ostrina, L. sitkana, and L. scutulata (Littorina spp.: Yamada and Boulding, 1996, Hohenlohe, 2003; Nucella spp.: Connell, 1970, Bertness and Schneider, 1976). These five species of gastropods were collected during June 2015 and February 2016 at Cattle Point on San Juan Island, WA and placed in a running seawater table at the Friday Harbor Laboratories. June 2015 intertidal range of the species were determined in reference to probe heights at the FHL Biological Preserve site and visually estimated at the Cattle Point site were L. scutulata - 0.5–2.0 m; L. sitkana - 0.5–1.7 m; N. ostrina – 1.0– 1.5 m; N. canaliculata – 0.5–1.5 m; and N. lamellosa - − 0.5–0.4 m. The February intertidal range of L. scutulata had contracted to 0.5–1.6 m. Nucella lamellosa was lower than the lowest February spring tide so this species was not collected at that time. Snails were maintained at near ambient surface temperature and salinity and used for thermal and desiccation studies within one week of collection. 2.3. Desiccation tolerance Snails of each species, collected in June 2015, were placed into a desiccator filled with Drierite at room temperature (20–22 °C) and the desiccator was sealed with silicone grease. Ten individuals of each species were removed from the desiccator every 24 h and placed aperture up in a pyrex dish filled with 30PSU seawater at 8.5–10 °C for 1 h. Snail activity was assessed by scoring each snail's condition as a 1.0 if the snail was righted and the foot attached to the dish, 0.5 if the foot was irritable

W.B. Stickle et al. / Journal of Experimental Marine Biology and Ecology 488 (2017) 83–91

and extended from the aperture, or 0.0 if the foot was not irritable to the touch and the snail was deemed dead after 1 h. Snail shell length for all individuals of all species was measured with a Vernier caliper to the nearest 0.1 mm from the base of the aperture or siphonal canal to the tip of the spire. The desiccation experiment was continued until all 10 snails assayed for that day had an activity score of 0.0. Lt50 values estimate days until 5 of the 10 snails sampled had an activity score of 0.0 and is given in days of desiccation. Lt50 values were determined by the method of PoloPlus v. 2.0 (LeOra Software Company; Petaluma, CA) and desiccation tolerance differences between Littorina scutulata, L. sitkana, Nucella ostrina, N. canaliculata and N. lamellosa were determined by non-overlap of 95% confidence intervals of LT50 values.

2.4. High temperature tolerance Snails of each species were subjected to 5 h emersion exposures in a recirculating water bath at a series of 4–5 emersion temperatures. A 5 h emersion temperature was chosen since it represents an average emersion period at the mid tidal level (Stickle et al., 2010). All experimental temperatures exposed 15 individuals of each of the five species. Emersion temperatures were chosen experimentally to bracket the LT50 temperature so that one temperature yield 0% mortality, two temperatures yielded partial mortalities, and on temperature yielded 100% mortality. Emersion temperatures were determined to be the asymptote temperature (within 1 °C of the final temperature) with a Hobo ProV2 probe that read the emersion bottle temperature once per minute as was done in freeze tolerance studies on the species of snails from Southeast Alaska (Stickle et al., 2010, 2015). The time from introduction of the bottle into the water bath until the bottle reached the asymptote temperature varied from 14 to 79 min out of the 300 min (5 h) emersion exposure accounting for 6–26% of the 5 h exposure time. Snail condition was determined as in the desiccation tolerance experiments. LT50 temperatures were determined by the Spearman Karber test (Silverstone, 1957) of data from each species. The treatments used with this analysis require one 0% mortality temperature, two partial percent mortality temperatures and one 100% mortality emersion temperature. Emersion temperature tolerance differences between Littorina scutulata, L. sitkana, Nucella ostrina, N. canaliculata and N. lamellosa were determined by non-overlap of 95% confidence intervals.

2.5. Low temperature freeze tolerance Procedures used to determine 5 h high temperature tolerance were replicated for the freeze tolerance experiments. Summer freeze tolerance of the five species of gastropods collected at Cattle Point was compared for snails collected on June 3–6, 2015. The 5 h freeze tolerance of 15 Littorina scutulata, L. sitkana, Nucella ostrina, N. canaliculata and N. lamellosa per treatment from the intertidal zone was determined by exposing separate groups of snails to five temperatures below freezing. Winter freeze tolerances of all species but N. lamellosa, which was below the lowest tide emersion during the study period, were determined between February 3–8, 2016. The freeze tolerance of each species was determined by procedures described in Stickle et al. (2010, 2015) over the 5 h were used in calculations of LT50 values. There was no significant difference in the 2 and 5 h freeze tolerance of the low intertidal N. lamellosa or in the 5 and 10 h freeze tolerance of the mid-high intertidal Littorina sitkana in the southeast Alaska study (Stickle et al., 2015) so a 5 h time period was used in this study for all species. Linear and second degree polynomial non-linear analysis of the temperature tolerance of species as a function of the midpoint of their intertidal zonation on the shore was used to fit the pattern of their tolerance as a function of their distribution with the Graphpad statistical package (GraphPad Software, Inc., San Diego, CA).

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3. Results 3.1. Seasonal thermal regime The thermal regime from March 27, 2015 through May 31, 2016 includes a high intertidal probe at +2.0 m which gave information on aerial temperature (Fig. 1A and F). Probe temperature at 2.0 m from May 1 to August 31, 2015 indicated that maximum monthly temperature and average daily maximum temperature per month was slightly higher for the southern than the eastern transect. Monthly maximum temperatures for the + 2.0 m probe of the southern transect were 42.6, 45.6, 46.2, and 43.9 °C compared with 41.7, 44.9, 45.1, and 46.9 °C for the eastern transect. Average daily maximum temperature per month were 37.1, 38.6, 39.0, and 36.6 °C for the southern transect compared with 33.7, 37.6, 38.3, and 35.2 °C for the eastern transect. The average daily maximum temperature per month of the southern transect varied between 83.4 and 87.1% of the monthly maximum while those values for the eastern transect varied between 75.06 and 84.96% of the monthly maximum. There was no algal cover over any of the probes. Traces (b0.0254 cm/day) of rain fell at the Friday Harbor Airport from May through August on 26, 10, 16, and 29% of the days per month during those months.

3.2. Tidal pattern at Friday Harbor The mixed semidiurnal tidal pattern at the Friday Harbor Harmonic Station exhibited an amplitude of 2.37 m between mean high high water and mean low low water in 2015. Emersion duration, calculated as the number of hours the water level was below the height of each logger with two emersion events per lunar day based on the NOAA/CO-OPS observed water levels, increased with tidal height and was significantly longer during spring tides at the 0 and + 1 m tidal heights and was significantly longer during neap tides at the + 1.5 and + 2.0 m tidal heights (t-test comparisons). The minimum emersion duration averaged 0.097 h during neap tides at 0 m and the maximum duration was 19.79 h during neap tides at + 2.0 m (Fig. 2). Emersion duration and temperature increased with intertidal height of both the eastern and southern transect probes from May through August 2015 but was not significantly different between spring and neap tide periods for the southern transect (Table 1). Duration of exposure to elevated emersion temperature increased with probe intertidal height position on the shore in all 9 spring tide series and in 6 out of 8 neap tide series during the summer. Emersion temperature increased with intertidal probe height in 7 of 9 spring tide series and 6 of 8 neap tide series. The timing of the tides and increased wave activity will have an influence on the duration and amplitude of elevated emersion temperature events. The maximum emersion temperature of intertidal probes of the southern transect over the emersion period during the summer of 2015 was 31.65 °C during a Spring tide series at the + 2 m probe on July 31. A biomimetic snail model at + 2.0 m on an adjacent south-facing shoreline approximately 20 m from probe site logged a maximum temperature of 34.72 °C on the same day. Emersion temperatures below freezing occurred in November and December 2015 and January 2016 at the 1.0, 1.5 and 2.0 m levels (Fig. 1). Snails were exposed to freezing emersion temperatures for tidal periods on 0 days at 0 m, 2 days at + 1 m, and 11 days at + 1.5 and + 2 m. Duration of exposure to freezing increased from 1.5 h at 1 m, to 5.53 h at 1.5 m, and 7.20 h at 2 m. The minimum probe air temperature at + 1 m was − 0.90 °C, and was − 3.42 °C at + 1.5 and 2.0 m. Biomimetic snail models on a south-facing shoreline approximately 20 m away from probe site registered minimum temperatures of − 0.66 at 1.0 m and − 0.93 °C at 1.5 m during the same time period.

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Fig. 1. Temperature measurements were recorded every 5 min along two transects of the rocky intertidal zone at Friday Harbor, WA between March 27, 2015 and May 31, 2016. Probes were deployed at +2.0 m (A, F), at +1.5 m (B, G), at 1.0 m (C, H), at +0.5 m (D, I) and at 0 m (E, J). The upper (+2.0 m; C, H) and lower edges (+ 1.0 m; D, I) of barnacles, and at the 0 m tide level (E, J). Temperature data for the east transect probes are shown in the left hand vertical bank of panels (A–E) and for the south transect is shown in the right hand bank (F–J). Four mathematical measures of the temperature records were 1) the monthly maximum which is the highest daily maximum recorded during the month, upper end of the hatched bar in each month; 2) the average daily maximum which is the average of all daily maxima recorded during the month, lower end of the hatched bar for each month; 3) the average daily minimum was the average lowest temperature recorded for each day in a month, upper end of the black bar for each month; and 4) the monthly minimum is the lowest daily minimum recorded during the month, lower end of the black bar for each month. The average monthly temperature is shown by the line which connects the monthly bars.

3.3. Desiccation and thermal tolerance Abiotic factor tolerance of the five species of snails studied varied directly with their intertidal zonation range at Cattle Point. Desiccation tolerance of the three species of Nucella were significantly lower than the two species of Littorina (Fig. 3A) and were directly related to snail shell length. Desiccation tolerance was 2.32 (1.45–3.14; 95% confidence range) days for N. ostrina, 2.67 (2.13–3.12) days for N. canaliculata, and 3.79 (3.29–4.26) days for N. lamellosa. Snail length ranges for N. ostrina were 15.1–25.6 mm, for N. canaliculata were 26.1–41.6 mm, and for N. lamellosa were 21.6–45.3 mm. Significantly longer desiccation tolerances were exhibited for L. sitkana; 5.21 (4.55–5.81) days and L.

scutulata; 6.78 (5.22–9.08) days. Desiccation tolerance for the low to mid intertidal species of Nucella were significantly lower than for the mid to high intertidal species of Littorina (Fig. 3A). A second degree curve best fit the desiccation tolerance of the five species of snails as a function of intertidal height (r2 = 0.1799). Summer high emersion temperature tolerance (5 h) of the five species of intertidal snails ranged from 30.94 °C for N. lamellosa to 41.47 °C for L. scutulata and varied directly with their intertidal zonation range (Fig. 3B; Table 2). The highest average emersion temperature exposure of N. lamellosa within its vertical range is 99.7% of its 5 h LT50, 99.1% for N. canaliculata, 91.1% for N. ostrina, 77.2% for L. sitkana, and 76.7% for L. scutulata. The three species of Nucella are at a greater risk of severe

W.B. Stickle et al. / Journal of Experimental Marine Biology and Ecology 488 (2017) 83–91

Fig. 2. The hours of emersion for each probe height during 2015 for all Spring and Neap tide dates were computed as the number of hours the water level was below that height with two emersion events per lunar day based on the NOAA/CO-OPS observed water levels at the Friday Harbor Harmonic station (48.2983°N 134.0150°W; mixed semidiurnal tidal pattern). The spring and neap tide events were determined by the dates of the primary moon phases. There were 25 spring tides and 24 neap tides in 2015. Welch's correction of the t-test was used to compare Neap and Spring tide events at each tidal height with significant differences between means given at the p b 0.05 level.

sublethal stress or mortality than the two species of Littorina on San Juan Island, assuming no behavioral avoidance strategies are used. A second degree curve best fit the 5 h high temperature tolerance of the five species of snails as a function of intertidal height (r2 = 0.5203). Freeze tolerance of the five species of intertidal snails (5 h) ranged from − 1.71 °C for N. lamellosa in summer to − 11.64 °C for L. sitkana in winter and varied seasonally with higher freeze tolerance (lower freeze temperature) in the winter than in the summer, except for N. ostrina (Fig. 3C: Table 3). Freeze tolerance varied directly with the species intertidal zonation range. A second degree curve best fit the winter freeze tolerance of the five species of snails as a function of intertidal height (r2 = 0.6857). No N. lamellosa were collected in February 2016. Seasonal adaptation to freezing emersion temperatures occurs in the other four species of rocky intertidal snails (Table 3). 4. Discussion This study extends our understanding of the thermal regime of the rocky intertidal zone of the Pacific coast of North America to the southern end of the inside passage where the timing of spring and neap low tides maximizes the effects of emersion temperature extremes and also documents variability in emersion temperatures throughout the vertical extent of the intertidal zone. In a study of biomimetic body temperatures of Nucella ostrina, N. lamellosa, and Balanus glandula at the same site at Friday Harbor labs, temperatures of all three species differed significantly among intertidal height-habitats where high shore sun exposed biomimetic models were warmer than mid- and lowshore sun exposed biomimetics. Likewise there was about a 0.5 °C difference between south- and east-facing sun exposed habitats. The relative rank of the two sun exposed habitats changed monthly in our study and that of Gilman et al. (2015). Aerial temperature probes were cooler than adjacent biomimetics. We did not test the mechanism leading to this difference, but hypothesize that probe temperature is lowered by the relatively low contact with the rock surface and higher surface area exposed to wind cooling. Aerial temperature does not perfectly correlate to body temperature (Helmuth, 1998), however, our aerial probes indicated relative environmental differences between tidal elevations that our multiple study species might experience, and matched known thermal patterns of biomimetics (Gilman et al., 2015).

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Although maximum temperatures were similar at the Friday Harbor Laboratories Biological Preserve on San Juan Island, WA and Bridget Cove, AK, the average daily temperature each month during the summer of 2015 varied between 75 and 87% of the monthly maximum temperature at the Friday Harbor site, in part due to no quantitatively measurable precipitation. Only traces of rain occurred on days when it rained which may not be enough to alleviate desiccation stress and sublethal snail stress responses during emersion. In contrast, it is likely that the expression of sublethal snail responses is less likely at the Bridget Cove, AK site where the average daily maximum temperature dropped from May through August representing 84%, 67%, 60% and 62% of the monthly maximum temperature with considerably more rain and cloud cover as the summer progressed (Stickle et al., 2016). In contrast to minimal traces of rain at Friday Harbor during the 2015 summer (b 0.025 cm per day), rain at Lena Point near Bridget Cove, AK totaled 1.3 cm in May, 11.7 cm in June, 19.2 cm in July and 23.0 cm in August (WeatherSource data). Southeast Alaska lies in a temperate rain forest which ameliorates the number of summer sunny days during the photophase of low tide emersions. Salinity stratification was found to be inversely related to temperature stratification at Sunshine Cove, Alaska (Stickle and Denoux, 1976) and Thais (Nucella) lapillus was found to be a poor volume regulator at reduced salinity (Stickle et al., 1985). Waterlogging at low salinity potentially prolongs desiccation tolerance of snails at Sunshine Cove but not at Friday Harbor. Minimal salinity stratification occurs at the Friday Harbor Lab site of the seawater intake system at −1.7 m with average monthly minimum salinity of 29.7PSU occurring in August and a maximum of 30.7PSU in October between 1931 and 1935 as a result of variation in outflow from the Columbia River (Phifer and Thompson, 1937) with surface acute salinities falling as low as 21PSU in 2014 (Bashevkin et al., 2016). In contrast, the Bridget Cove and Sunshine Cove sites in southeast Alaska are highly salinity stressed during the summer due to meltwater flowing north from the Eagle and Herbert rivers (Stickle and Denoux, 1976). Tidal cycle salinity at + 1 m varied between 7.9 and 25.1 PSU, at + 0.6 m it varied between 13.5 and 24.9 PSU, at − 0.9 m it varied between 11.0 and 28.2 PSU, and subtidally at −3.7 m it varied between 14.1 and 28.0 PSU during spring tidal cycles. The desiccation tolerance of the five species of rocky intertidal fauna at Cattle Point, WA was directly related to their intertidal vertical distribution and modified somewhat by species differences in size. As previously noted a second degree curve fit the species data better, with an R2 of 0.1799, than did a linear regression. Previous studies at Monterey, CA demonstrated a correlation between vertical stratification of three species of gastropods (Tegula funebralis, Littorina scutulata, and L. planaxis: Hewatt, 1937) and at Point Arago, OR in Calliostoma ligatum, T. funebralis, and L. scutulata (Emerson, 1965) and their desiccation tolerance. In contrast, the desiccation tolerance of the three species studied in southeast Alaska, Nucella lamellosa, N. lima, and Littorina sitkana (Stickle et al., 2016), was not related to their intertidal distribution and was of shorter duration than at Friday Harbor which may be influenced by the fact that this site lies within the largest temperate rain forest in North America that extends 4023 km in a narrow coastal corridor from the far edge of Prince William Sound in southcentral Alaska to northern California (US Forest Service, 2012). Interestingly, in Washington all species of the larger genus (Nucella) had a lower desiccation tolerance than species of the smaller genus (Littorina). This inverse relationship between volume and desiccation tolerance contrasts with findings of individuals within species (Foster, 1971; Kennedy, 1976) and counters predictions that a lower surface area to volume ratio should result in better water retention even across species. This suggests that another factor, such as evolutionary history of range, is driving observed differences in desiccation tolerance. The high tide mark is thought to represent a major physiological barrier on rocky shores in part because desiccation stress is minimal below the high tide mark and maximal above it (McMahon, 1990).

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Table 1 Average emersion duration and temperature of south transect probes during Neap ±1 day and Spring ±1 day from May–August 2015. X1 = Emersion on only 1 day in that 3 day period and X2 = emersion on 2 of 3 days during that period. X3 = No emersion in this 3 day period. The average MMMW-MLLW tidal range is 2.37 m. Only the lowest low tide is given for each date. 0m

1m

Month/Type

Date

Time

H (m)

Hours

Aver. °C

May/Spring

3 4 5 10 11 12 17 18 19 24 25 26

11:19 11:50 12:23 16:05 17:04 18:06 10:56 11:36 12:36 15.51 16.42 17.37

0.00 −0.12 −0.24 −0.12 0.06 0.30 −0.40 −0.49 −0.52 0.27 0.52 0.76

X1 X1 1.42

11.03

1 2 3 8 9 10 15 16 17 23 24 25

10:48 11:21 11:57 15:37 16:30 17:29 10:37 11:21 11:54 15:50 16:36 7:32

−0.30 −0.43 −0.52 0.0 0.30 0.67 0.52 −0.43 −0.52 0.64 0.91 0.64

X2 4.30

1 2 3 7 8 9 15 16 17 23 24 25 30 31

10:57 11:36 12:17 15:37 16:30 17:29 10.58 11:34 12:10 5:34 6:10 7:04 10:33 11:15

−0.58 −0.64 −0.64 0.00 0.91 0.67 −0.40 −0.34 −0.20 0.79 0.55 0.34 −0.55 −0.55

1 6 7 8 13 14 15 21 22 23 28 29 30

11:58 4:43 5:45 6:45 10:39 11:15 11:50 4:13 5:04 6:45 10:39 11:15 11:50

−0.49 0.43 0.24 0.06 −0.09 −0.03 −0.09 0.58 0.43 0.06 −0.09 −0.03 −0.09

Neap

Spring

Neap

June/Spring

Neap

Spring

Neap

July/Spring

Neap

Spring

Neap

Spring

August Neap

Spring

Neap

Spring

X3

2.85

19.08

X3

22.35

X3

4.40

20.71

X3

4.30

20.71

X3

4.40

20.71

X3

1.5 m

2.0 m

Hours

Aver. °C

Hours

Aver. °C

Hours

Aver. °C

5.11

18.18

6.58

18.70

9.58

22.14

4.78

16.03

7.87

18.58

13.83

18.52

6.10

23.81

7.46

26.79

9.39

27.18

3.08

13.98

6.72

20.67

14.92

24.69

5.67

16.41

7.84

17.09

9.85

17.73

10.72

16.95

17.28

20.35

21.41

21.85

6.33

24.13

8.61

21.93

10.86

27.02

5.77

12.24

8.28

14.29

14.92

24.69

7.11

22.90

8.53

28.37

12.45

29.37

4.02 X2

14:52

3.53

17.52

13.31

20.62

6.33

24.13

7.53

27.40

9.83

28.20

6.50

13.91

13.51

17.76

X3

4.39

20.72

4.39

18.54

7.17

28.37

9.53

31.65

X3

X1

4.02

14.62

1.80

14.68

20.80

15.26

X3

4.97

19.01

6.36

18.71

11.53

19.55

X3

X3

17.67

13.60

20.11

15.91

X3

3.70

5.48

18.82

8.08

21.47

Nucella lamellosa, N. canaliculata, and N. ostrina are restricted to the eulittoral zone (0 and + 1.0 m) while Littorina scutulata and L. sitkana have ranges that extend into the eulittoral fringe (+1.5 and +2.0 m). Spring tide emergence is of longer duration than Neap tide emergence at 0 and + 1.0 m while Neap tide emergence duration is longer at +1.5 and +2.0 m. All three species from Southeast Alaska and five species from San Juan Island, WA have life histories with crawl away juveniles emerging from egg capsules (Strathmann, 1987). Natural selection likely is a selective factor increasing desiccation tolerance on San Juan Island but not in Southeast Alaska since there is not increased desiccation stress in the upper rocky intertidal zone there. Climate differences between San Juan Island, WA and sites in Southeast and South Central Alaska

18.46

(Stickle et al., 2016) can have a profound influence on thermal and desiccation stress on emersed intertidal fauna and flora. Minimal differences exist in the high temperature emersion tolerance of snails at the same relative intertidal height at Sunshine Cove, Alaska (Stickle et al., 2016) and Cattle Point, WA. Likewise, comparison of high emersion temperature tolerance of sub tidal to mid tidal Lunella smaragda collected from six locales near Auckland New Zealand exhibited similar thermotolerances (LT50s) even though snails from the three sites on west coast were exposed to aerial temperatures up to 10 °C cooler than three sites on the east coast because of the timing of low spring tides (Mortensen and Dunphy, 2016). Snail emersion temperatures from the east coast sites were exposed to ambient emersion temperatures which exceed their LT50 values which suggests that these

W.B. Stickle et al. / Journal of Experimental Marine Biology and Ecology 488 (2017) 83–91

89

Table 2 Summer 2015 5 h high temperature emersion tolerance of intertidal snails from Cattle Point, WA and Sunshine Cove, AK (Stickle et al., 2016). All data are presented as Mean ± 95% confidence intervals. Significant differences exist if there is non-overlap of 95% confidence intervals. N = 15 snails per treatment. * Significant differences by site and the same intertidal position. Site

Intertidal height

Species

Mean ± 95%CI in °C

Cattle Point, WA

Low Mid

Nucella lamellosa Nucella canaliculata Nucella ostrina Littorina sitkana Littorina scutulata

30.94:30.56–31.32⁎ 31.00:30.32–31.70 33.83:33.56–34.11⁎ 41.01:40.64–41.38 41.47:40.97–41.98

Nucella lamellosa Nucella lima Littorina sitkana

29.63:29.46–29.80 31.39:31.18–31.60 41.51:40.59–42.44

High

Sunshine Cove, AK

Low Mid High

the high shore zone 14.5% of the summer days but in all lower habitats the frequency was 3.8% or less (Gilman et al., 2015). A similar difference exists for low intertidal N. lamellosa: Sunshine Cove, AK - 29.63 (29.46– 29.80) °C; Cattle Point, WA - 30.94(30.56–31.32) °C. The body temperature of biomimetic N. lamellosa exceeded the 4 h LT50 of 30 °C, determined by Bertness and Schneider (1976) 33.8% of the days in the summer in the sun exposed high-intertidal, 14.6% of the days in the sun exposed mid-intertidal, 2.6% of the days under Fucus mid-intertidal and 4.7% of the days in the sun exposed low-intertidal (Gilman et al., 2015). High emersion temperature tolerance of rocky intertidal snails is not significantly different in Southeast Alaska and the southern end of the inside passage on San Juan Island, WA. Sorte and Hofmann (2005) have determined that the south-latitude N. emarginata and the mid-latitude N. ostrina (see Collins et al., 1996; Marko et al., 2003 and Sorte and Hofmann, 2004) are more thermo-tolerant than the mid-latitude low-intertidal N. canaliculata and N. lamellosa and the north-latitude high intertidal N. lima (Sorte and Hofmann, 2005). Their and our results suggest that temperature plays a role in determining the tidal height and latitudinal distributions of these species of Nucella. High temperatures in the range of LT50 for N. canaliculata and N. ostrina suggest that these species are physiologically stressed or use behavior to avoid stress. N. ostrina forages in high shore, sun-exposed locations on the days of the tidal cycle with the shortest daytime emersion, retreating into cracks and low shore refuges on days of the tidal cycle with longer sun exposure, and reducing the number of stressful temperatures experienced (Spight, 1982; Hayford et al., 2015).

Fig. 3. Desiccation (A), 5 h high temperature (B), and summer and winter freezing emersion temperature tolerance (C) of low intertidal Nucella lamellosa, low-mid intertidal N. canaliculata, mid intertidal N. ostrina, and mid-high intertidal Littorina sitkana and L. scutulata. Data are plotted as the average ±95% confidence interval in days. Significant differences between species are shown by non-overlap of 95% confidence ranges between species.

populations of snails have less of a safety margin and are more susceptible to climate change than the west coast populations. High temperature emersion tolerance varied directly with the intertidal height range of the species at both locations. Mid to high intertidal Littorina species at both locations exhibited emersion tolerances that were not significantly different from each other and ranged from 41.01 °C to 41.51 °C. In contrast, the LT50 of the mid-intertidal Nucella species exhibited LT50 values that were considerably lower than the littorines (Table 2). In a separate study, N. ostrina biomimetic temperatures exceeded their 4 h LT50 of 34 °C (Bertness and Schneider, 1976) in

Table 3 Site and seasonal freeze tolerance of intertidal snails from Cattle Point, WA and Sunshine Cove, AK published in Stickle et al. (2015). All data are presented as Mean ± 95% confidence intervals. Average 5 h freezing temperatures were used for the Sunshine Cove, Alaska data and 5 h asymptote temperature for the Cattle Point Data. Significant differences exist if there is non-overlap of 95% confidence intervals. N = 15 snails per treatment. * = Significant differences by season at the same site and ** by site for winter sampling times.

Site

Intertidal height Species

Cattle Point, WA

Low Mid

Nucella lamellosa Nucella canaliculata Nucella ostrina

High

Littorina sitkana Littorina scutulata

Sunshine Cove, Low AK Mid High

Season

Mean ± 95%CI in °C3

Summer Summer Winter Summer Winter Summer Winter Summer Winter

−1.71:−1.23–2.38 −2.54:−2.21–2.38 −3.47:−3.27–3.69* −2.58:−1.87–3.57 −3.64:−3.51–3.71 −5.73:−4.89–6.72 −10.31:−9.65–11.01* −5.17:−4.39–6.09 −8.35:−7.91–8.82*

Nucella lamellosa Summer Winter Summer Nucella lima Winter Littorina sitkana Summer Winter

−4.43:−4.06–4.85** −5.45:−4.75–6.24 −5.10:−4.65–5.60 −8.58:−8.18–8.98* −9.73:−9.42–10.04 −11.64:−10.83–11.88*

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The five species of rocky intertidal snails that we studied on San Juan Island exhibited freeze tolerances that increased as a function of their zonation height and exhibited seasonal adaptation to increased freeze tolerance temperature in the winter. Low to mid intertidal Nucella lamellosa, N. canaliculata, and N. ostrina were much less freeze tolerant than mid to upper intertidal Littorina scutulata and L. sitkana (Fig. 3C). Freezing emersion air temperatures and duration of freezing emersion were much lower than the freeze tolerance of the low intertidal N. lamellosa and the mid-high intertidal L. sitkana and L. scutulata on San Juan Island while the minimum emersion temperatures observed fell within the tolerance range of the mid-intertidal N. canaliculata and N. ostrina. Behavioral freeze avoidance mechanisms such as crawling into crevices, under boulders, and into particulate matter in the southeast Alaska snails may also be operative at Cattle Point, WA (Stickle et al., 2010, 2015). Nucella lamellosa, N. lima, and Littorina sitkana from Sunshine Cove, AK were exposed to a freezing emersion temperature gradient which increased in frequency and duration as a function of intertidal height which was more frequent, and much colder than occurred with the snails at Cattle Point (Stickle et al., 2010, 2015). Freeze tolerance of the three species increased directly with their intertidal vertical range. The summer freeze tolerance on N. lamellosa from Sunshine Cove, AK was 2.59 times lower than the same species at Cattle Point, WA (Table 3 and Stickle et al., 2015). The winter freeze tolerances of N. lima from Sunshine Cove, AK was 2.47 lower than N. canaliculata and 2.31 times lower than N. ostrina from Cattle Point, WA (Table 3). The winter freeze tolerances of Littorina sitkana from Sunshine Cove, AK was 1.13 times lower than the same species from Cattle Point, WA and not significantly different (Table 3, Stickle et al., 2015). The winter freeze tolerance of Littorina scutulata was −8.35 (−7.91 to −8.82 °C). The freeze tolerance of the low and mid-intertidal Nucella species from southeastern Alaska was greater than in snails from the same intertidal range on San Juan Island, WA 3360 Km to the south and they were exposed to more frequent and colder freezing emersion temperatures. The freeze tolerance of the high intertidal L. sitkana from the same locales was not significantly different. Local variation in the freeze tolerance of populations of Littorina brevicula from Japan was correlated with the lowest local temperature at the sampled locales over a latitudinal range of 1584 km (Chiba et al., 2016). Our results of the freeze tolerance of Nucella spp. from the mid-intertidal of Southeast Alaska and San Juan Island agree with the findings of Chiba et al. (2016) but not for L. sitkana. Dennis et al. (2014) found that northern range limits of closely related temperate species of the pulmonate genus Melampus on the US Atlantic coast are not governed by differences in freeze tolerance alone. Differences in the supercooling point and the free amino acids taurine, alanine, and total free amino acids of intertidal snails are freezing adaptations in Southeastern Alaska (Stickle et al., 2010, 2015). The thermal regime in the rocky intertidal zone was determined with an east facing and a south facing transect of probes located at one supratidal and four intertidal locations on San Juan Island, WA at the southern end of the inside passage ranging along the coastline of Washington, British Columbia and southeast Alaska. Within the intertidal zone maximum and minimum emersion temperatures increased with intertidal height. Tolerance of desiccation stress, high and freezing emersion temperatures increased with the intertidal zonation range of Nucella lamellosa, N. canaliculata, N. ostrina, Littorina sitkana, and L. scutulata. While this study is taxonomically constrained to two genera, it is interesting to note that temperature and desiccation tolerances of each intra-generic species matched its relative vertical range. In the parallel comparison study at the northern end of the inside passage at Sunshine and Bridget Cove Alaska (Stickle et al., 2016) there is no significant difference in the high emersion tolerance of the common mid and high intertidal snails from Cattle Point, WA and Sunshine Cove, AK. The latitudinal distance between Bridget Cove, AK (Stickle et al., 2010, 2015, 2016) and our San Juan Island study site in

the inside passage is 3360 km. The high temperature tolerance of the low intertidal N. lamellosa and mid intertidal N. ostrina was significantly higher than their ecological equivalents at Sunshine Cove, AK. Snails are significantly less tolerant of freezing than those at the same relative intertidal height at Sunshine Cove, AK and they are not exposed to such extreme freezing events. Desiccation tolerance of Cattle Point snails varies directly with species intertidal ranges compared with no such intertidal desiccation tolerance-intertidal height relationship at Sunshine Cove, AK which lies in a temperate rain forest.

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