Latitudinal clines in body size, but not in thermal tolerance or heat-shock cognate 70 (HSC70), in the highly-dispersing intertidal gastropod keenae (: )

HYUK JE LEE1* and ELIZABETH G. BOULDING Department of Integrative Biology, University of Guelph, Guelph, ON, N1G 2Wl, Canada

Natural populations of widely-distributed often exhibit dinal variation in phenotypic traits or in allele frequencies of a particular gene over their geographical range. A planktotrophic intertidal snail, Littorina keenae is broadly distributed along the north-eastern Pacific coast through a large latitudinal range (24°50'N-43°18'N). We tested for latitudinal dines in two complex phenotypic traits - thermal tolerance and body size - and one single locus trait - heat shock cognate 70 (HSC70) - in L. keenae along almost its entire geographical range. We found only weak evidence for a latitudinal dine in the thermal tolerance and no evidence for a dine in allele frequencies at HSC70. However, as predicted by Bergmann's rule, we detected a strong latitudinal dine that accounted for 60% of the variance in body size (R2 = 0.598; P < 0.001). In contrast, body size did not significantly affect thermal tolerance. HSC70 showed no genetic differentiation among the populations, supporting our previous mitochondrial gene-based estimate of high gene flow during this snail's free-swimming larval stage. Given that L. keenae experiences panmixia along its range, the observed size dine may be partially or entirely caused by a phenotypically plastic response to local thermal environments rather than by genetic divergence in body size among populations in response to locally optimizing natural selection.

ADDITIONAL KEYWORDS: Bergmann's rule - gene flow - intertidal snail - local adaptation - phenotypic plasticity - planktotrophic larvae - stabilizing selection.

INTRODUCTION intertidal invertebrate species (Vernberg, 1962; Hoch­ achka & Somero, 2002). Many experimental studies of Temperature plays a pivotal role in determining the intertidal invertebrates have found that more south­ geographical distributions of organisms (Brown & ern species have higher thermal tolerances than more Gibson, 1983). Temperature is particularly critical for northern species (e.g. Fraenkel, 1968; McMahon, 2001; marine invertebrates inhabiting intertidal habitats Sorte & Hofmann, 2005). Surprisingly, however, only a because they are periodically exposed to elevated tem­ few studies of intertidal invertebrates have compared perature and desiccation stress while being emersed the thermal tolerances of geographically separated during low tides. Therefore, the levels of thermal stress populations of a single species (e.g. Kuo & Sanford, in intertidal habitats have been considered to be the 2009). key determinant of the latitudinal distributions of Temperature also has a significant influence on the adult body size of animals. The body size of endothermic animals, such as terrestrial birds and *Corresponding author. lCurrent address: Chair in Zoology mammals, often shows thermal dines that are posi­ and Evolutionary Biology, Department of Biology, University of Konstanz, Universitatsstrasse 10, D-78457 Konstanz, tively correlated with latitude. This pattern is Germany. E-mail: [email protected] referred to as Bergmann's rule (Bergmann, 1847). 494 495

The latitudinal trend in body size has also been found because gene flow is higher relative to stabilizing in ectothermic animals like marine invertebrates selection for local optima (Garcia-Ramos & Kirk­ [e.g. the black turban snail Tegula funebralis (Frank, patrick, 1997; Barton, 1999). Therefore, local adapta­ 1975)], although several exceptions to this pattern tions are expected to be rare or absent in species with have been reported [e.g. the deep-sea gastropod extensive gene flow, such as planktotrophic marine Troschelia berniciensis (Olabarria & Thurston, 2003); species like L. keenae, unless local optimizing selec­ and 915 species of marine bivalves (Roy & Martien, tion is strong (reviewed by Conover et al., 2006; Boul­ 2001)]. Schmitt (1993) found that Littorina keenae ding et al., 2007). snails from Bodega Bay (38°15'N, 123°05'W) in north­ The heat shock cognate 70 (HSC70) gene is a ern California had a larger mean body size than those member of the heat shock protein 70 (HSP70) gene from Santa Catalina Island (33°27'N, 118°20'W) in family of molecular chaperones, and is therefore southern California, USA. Yet, his observation was likely to be involved in the snails' response to thermal only based on two geographical locations. The present stress (e.g. dilorio et al., 1996; Fangue, Hofmeister & study extends his findings and tests for the latitudi­ Schulte, 2006; Hemmer-Hansen et al., 2007). The nal dine in body size of this snail species from 13 function of HSC70 is considered to be similar to that locations along almost its entire geographical range of HSP70, except that the HSC70 genes are primarily (",,2000 km), using moderately large sample sizes. constitutively expressed (Yamashita, Hirayoshi & There have been far fewer studies on dinal variation Nagata, 2004). However, several studies have shown in body size in marine animals than in terrestrial significant increases in the levels of HSC70 expres­ ones [e.g. the fruit flies Drosophila melanogaster sion in response to heat shock in hybrids of the (van't Land et al., 1999) and Drosophila subobscura livebearing fishes Poeciliopsis (dilorio et al., 1996), (Gilchrist et al., 2004), the copepod crustacean Scot- and in the common killifish Fundulus heteroclitus tolana canadensis (Lonsdale & Levinton, 1985), and (Fangue et al., 2006). This upregulation of HSC70 in the venomous snake dades Viperidae and Elapidae response to heat shock may be correlated with whole­ (Terribile et al., 2009)]. organismal thermal tolerance (dilorio et al., 1996; Body size can affect the thermal tolerance of an Fangue et al., 2006). Further, Fangue et al. (2006) organism of intertidal gastropods (Vermeij, 1973; detected a single highly conserved amino acid substi­ McMahon, 1990). This is because gastropod size influ­ tution in only HSC70, but not in HSP70 between ences the rate at which organisms achieve equilib­ northern and southern populations of F. heteroclitus rium body temperature in the face of thermal stress along the north-western Atlantic coast (although the (Helmuth, 1998). Smaller snails often have a higher­ function of this amino acid change was unknown). spired shell that presents a smaller projected area to Hemmer-Hansen et al. (2007) recently found evidence incident solar radiation, which can reduce thermal for adaptive divergence at the intron of HSC70 stress (Vermeij, 1973; McMahon, 1990). Moreover, between Baltic Sea and North Sea populations of the smaller gastropods often have smaller foot surface European flounder, Platichthys flesus L., occurring in areas or hang from mucus threads, which can cause different temperature regimes, despite its high gene significantly lower rates of heat flux from the sub­ flow. In this study we assess variation in an exon of stratum, resulting in increased thermal tolerance HSC70 to test for local adaptation at this locus among (e.g. Clarke, Mill & Grahame, 2000a). However, eight geographical populations of L. keenae inhabiting several studies have also revealed no significant effect different thermal environments. of gastropod body size on thermal tolerance (e.g. Littorina keenae Rosewater, 1978 (Gastropoda: Lit­ Clarke, Mill & Grahame, 2000b; Miller, 2008). torinidae; formerly Littorina planaxis) occurs in the In ectotherms, latitudinal dines in complex pheno­ high intertidal or splash zone above the high tidal typic traits, like thermal tolerance or body size, may mark on wave-exposed rocky shores (Reid, 1996; H.J. result from genetic divergence in the traits among Lee, pers. observ.). The present documented geo­ populations caused by natural selection (Endler, graphic range of this species is from Cape Arago, 1977), from the phenotypically plastic response of Oregon (43°18'N) (Behrens Yamada, 1977) to Laguna populations to local thermal environments (e.g. body Manuela in Baja, California (28°15'N) (H.J. Lee, pers. size; Atkinson, 1994; Atkinson & Sibly, 1997), or from observ.) (Fig. 1). There are correctly identified histori­ the combined effect of both. Natural selection can cal collections of L. keenae from slightly further south permit local genetic adaptation at an enzyme locus of (24°50'N in Reid, 1996; Fig. 1). This species lays different populations of a species living along an benthic egg masses that hatch into a free-swimming environmental gradient (Panova & Johannesson, (planktotrophic) larval phase. The duration of the 2004). However, theory suggests that populations of larval period for L. keenae is unknown, but our pre­ species with high dispersal may be constrained from vious genetic study (Lee & Boulding, 2007) suggests evolving to the local optima for quantitative traits that dispersal of this species is high. Mitochondrial 496

body size, and allele frequencies at HSC70 among geographically separated populations of the plank­ totrophic intertidal gastropod L. keenae, along the north-eastern Pacific coast; (2) investigate if body size had a significant effect on thermal tolerance. Based on the results, we discuss three hypotheses to explain the observed clines in these complex traits for this snail along its geographical range.

MATERIAL AND METHODS Northern limit CA SAMPLE COLLECTION AND LABORATORY ACCLIMATION (43.18N) Littorina keenae snails were collected from nine geo­ CC graphical locations along almost its entire geographi­ cal range on the north-eastern Pacific coast between July 2005 and September 2006 (Fig. 1; Table 1). The selected sampling sites ranged from Laguna Manuela, Mexico (LM, 28°14'50"N, 114°05'OI"W) in central BO Baja, California, to Cape Arago, Oregon, USA (CA, 43°18'24"N, 124°24'02"W) (Fig. 1; Table 1). All sampled snails were placed in insulated plastic containers, containing seawater-soaked paper towels, which were kept cold by placing them inside an insu­ lated plastic cooler, and were immediately trans­ ported to the Hagen Aqualab at the University of Guelph in Ontario, Canada. Upon arrival, 20-30 snails were placed together in separate plastic con­ tainers (10 cm x 10 cm x 9 cm) that had openings (of 4 cm x 4 cm) cut in three of its six sides. The open­ ings, which were covered with I-mm-square nylon mesh that was attached with hot-melt glue, permitted water flow through the container. The containers holding the snails were then floated in seawater tanks at a common temperature of 10°C, with con­ stant 34%0 artificial running seawater, on a 12-h light) 12-h dark cycle. The duration of acclimation was at Southern limit least 4 weeks for all the samples to minimize envi­ 100 km (24.5N) ronmental acclimatization effects caused by different sampling dates or seasons. Figure 1. Geographical distribution and sampling locali­ ties of Littorina keenae along the north-eastern Pacific coast. The latitude and longitude of each location and the DETERMINATION OF UPPER THERMAL location codes are shown in Table 1. TOLERANCE LIMIT Upper thermal tolerance limits for L. keenae snails were determined using a protocol slightly modified DNA (mtDNA) variation at ND6 and Cyt b genes from that used by McMahon (1990). The heat-coma (762 bp) showed no genetic differentiation among the temperature (HCT) in gastropods has been defined as populations of L. keenae throughout its geographical the temperature at which normal nervous function is range along the north-eastern Pacific coast (Lee & lost, manifested by the cessation of locomotion, a Boulding, 2007). However, that study was performed ventral medial curling of the lateral edges of the foot, with a selectively neutral mtDNA marker. If gene flow and eventually the inability to remain attached to is high, genetic differentiation will only be seen with substrata (McMahon, 1990). Heat coma is defined as 'selected' markers showing local genetic adaptation a 'nonlethal' reversible condition (McMahon, 1990). In (reviewed by Lenormand, 2002). this study, we measured HCT by recording either (1) The objectives of this study were to: (1) examine if the temperature at which snails were no longer able there were latitudinal clines in thermal tolerance, to remain attached to the sides of the test tube, or (2) 497

Table 1. Sampling localities and dates, latitude and longitude, location code, sample size (N), and mean and standard deviation (SD) of heat coma temperatures (HCTs) for each sampled population of the planktotrophic intertidal gastropod Littorina keenae

Location Sample Mean Sampling localities Sampling dates Latitude, longitude code size (N) HCT±SD (OC)

Laguna Manuela, Mexico September 2006 2so14'50"N; 114°05'01"W LM IS 3S.2 ± 1.4 Ensenada, Mexico March 2006 31°51'44"N; 116°39'60"W EN 30 37.0 ± 1.3 La Fonda, Mexico September 2006 32°09'13"N; 116°53'5S"W LF 29 37.1 ± 2.3 Carlsbad, California February 2006 33°0S'13"N; 117°20'12"W CB 29 36.2 ± 1.1 San Pedro, California October 2005 33°25'26"N; l1so09'50"W SP 30 36.1 ± 1.1 Monterey, California October 2005 36°37'0l"N; 121°54'54"W MO 30 37.2 ± 1.9 Bodega, California October 2005 3so1S'3S"N; 123°03'39"W BO 60 36.5 ± 1.9 Crescent City, California July 2005 41°47'00"N; 124°15'OS"W CC 25 35.S ± 1.7 Cape Arago, Oregon July 2005 43°1S'24"N; 124°24'02"W CA 10 36.1 ± 1.0

the temperature at which snails completely withdrew and snail tissue temperatures negligible (Broekhuy­ the foot inside their shell. The second criterion was sen, 1940). For each trial, we placed only ten tubes needed because many individuals of L. keenae never into the experimental tank so that all tested snails fell to the bottom of the tube because they had could be simultaneously watched during the course of securely attached themselves to the side of the tube the experiment. For each HCT determination obser­ using transparent mucus films while withdrawing vations began at 10°C, the acclimation temperature, their foot entirely into their shell. Dangling from and continued until every snail showed one of the rocks by mucus threads is a natural behavioural criteria used for the diagnosis of heat coma described response used by high intertidal gastropods to mini­ above. Water temperatures in the test tubes were mize their contact with the rock substratum (Miller, monitored using a digital dial thermometer (Fisher 2008). Scientific). We recorded the water temperature at Prior to HCT measurements, the columella length which each snail first showed heat coma criteria. (CL; Grahame & Mill, 1989) of all tested snails were Immediately after each HCT determination was com­ measured with digital calipers (Mitutoyo Corp.) to pleted, all the tubes were removed from the experi­ the nearest 0.01 mm. Each snail was then placed mental tank, partially decanted, and allowed to cool into a separate 50-mL plastic tube filled with 40 mL to room temperature (15°C). This allowed us to deter­ of seawater, which allowed them to attach to the mine whether the snails recovered from heat coma tube walls and to actively crawl. Porous foam plugs and became active again. None of the snails had died blocked the tube openings at the water surface to from heat stress. After HCT determination, all snails keep the snails immersed (R.F. McMahon, pers. were frozen whole at -80°C for subsequent genetic comm.). It may have been more appropriate if we studies. had measured HCT for L. keenae in air rather than in water, because these snails are unlikely to be submerged in nature, even during high tides. SEQUENCING OF A CANDIDATE HSC70 GENE However, the measurements of HCT of snails in Genomic DNA was isolated using a chelex protocol their submerged conditions have been typically used with 6% InstaGene Matrix (Bio-Rad), as described in in many previous studies of different species (e.g. Lee & Boulding (2009). A nuclear HSC70 gene was Fraenkel, 1968; McMahon, 1990, 2001; Clarke et al., amplified by polymerase chain reaction (PCR) with 2000a, b). Therefore, our measured HCT data can our newly designed primers for L. keenae, based on be directly compared with previous studies. There is the published DNA sequences for Littorina scutulata direct evidence that HCT in water is positively cor­ (GenBank accession nos AF191824-AF191826) and related with HCT in air for littorinid gastropods for Littorina plena (AF191827-AF191830): LKEEN­ (Sandison, 1967). HSC70F1 (forward), 5'-GGCACCTTTGACGTGTC The tubes containing the snails were then placed AGTCC-3' and LKEEN-HSC70R1 (reverse), 5'-CC in a metal test-tube rack inside a water-filled TAGCACGAGTGATGCTTGTG-3'. PCR amplification, temperature-controlled aquarium that was heated at purification of the amplified PCR products, and DNA a rate of approximately 1 °C per 5 min period. This sequencing were carried out as described in Lee & rate of increase makes the lag between tube water Boulding (2009). The DNA sequences obtained were 498

Table 2. Multiple regression analysis of latitude and body size (i.e. columella length) on heat coma temperature (HeT) in nine populations of Littorina keenae

Collinerity statistics

Source d.f. SS MS F p R2 Tolerance VIF

Model 2 42.228 21.114 7.164 0.001 0.053 Latitude 1 -2.987 (t ratio) 0.003 0.425 2.353 Body size 1 0.749 (t ratio) 0.455 0.425 2.353 Residual 258 760.377 2.947 Total 260 802.606 Predicted equation: HCT = 40.586 - 0.124 x latitude + 0.037 x body size d.f., degrees of freedom; MS, mean of square; SS, sum of square; VIF, variance inflation factor. edited using Chromas v2.01. Alignment of sequences 1998). Our calculated value of the VIF = 2.353 sug­ was conducted using ClustalW v1.83, and then veri­ gests no significant multicollinearity effects in the fied by eye. model (Table 2). As body size had not significantly The samples that were used for measuring HCT contributed to our model (P = 0.455; Table 2), we per­ were largely overlapping, but not identical to samples formed additional linear regression analysis to test that were used for sequencing HSC70. For example, for the significant relationship between HCT and we did not sequence HSC70 for the LF (La Fonda, latitude only. Mexico) samples. As a result, the number of samples We carried out linear and second-order polynomial used in HSC70 sequence analysis, HCT measure­ regression analyses to determine which model better ments, and body size measurements differed for logis­ fitted the body size data, using latitude as a pre­ tical reasons. dictor. We did both linear and quadratic regression analyses because body size decreased slightly at the highest latitude (Cape Arago, CA, USA; 43°18'N). STATISTICAL ANALYSES Lastly, we conducted nine different linear regression HEAT-COMA TEMPERATURES (HCTs) analyses to test for a significant relationship All statistical analyses were performed using SPSS between HCT and body size within the same popu­ vI6.0. A Kruskal-Wallis (K-W) test was used to lations. The specimens used for body size data had examine whether there was significant variation in been randomly sampled from 13 locations through­ HCT among nine geographical populations of L. out nearly the entire geographical range of L. keenae. We used a nonparametric (K-W) test for this keenae, and included snails for which HCT was not analysis because the variances were heterogeneous determined. among the groups, even after a log transformation. Non-parametric multiple comparisons were then per­ formed manually as recommended by Zar (1984), HEAT SHOCK COGNATE 70 (HSC70) using Microsoft Excel. The gametic phase of the HSC70 exon alleles was We performed multiple regression analysis at an determined with the Excoffier-Laval-Balding (ELB) individual level to examine the relative contribution algorithm (Excoffier, Laval & Balding, 2003), as of each of the two independent variables (predictors), implemented in ARLEQUIN 3.11 (Excoffier, Laval & such as latitude and body size, to the HCT data. Schneider, 2005). The ELB algorithm was incapable Collinearity statistics [e.g. tolerance and variance of resolving the phase of an HSC70 fragment longer inflation factor (VIF)] were calculated to test for mul­ than 105 bp. Therefore, we only used 105 bp of the ticollinearity between the two independent variables. sequence from the 5' end of the coding strand of the The inherent correlation between independent vari­ HSC70 exon to genotype all 139 L. keenae samples, ables (i.e. multicollinearity) would result in the false even though longer reads of 204 bp of the exon frag­ exclusion of significant predictor variables during ment were produced by the designed primers. The model creation (Freund & Wilson, 1998). A commonly determined alleles were given specific allele numbers given rule of thumb is that multicollinearity is only as follows: HSCKEE1-HSCKEE69 (GenBank acces­ typically severe at VIFs > 10 (Freund & Wilson, sion numbers: GU562894-GU562962) (Appendix SI). 499

44 Nuclear allelic DNA diversity within eight geo­ (A) graphical populations of L. keenae was estimated as 42 .,b the number of alleles (N.), substitutions, polymorphic .,b E • b sites, expected heterozygosity (HE), observed het­ 2! 40 • erozygosity (Ho), and nucleotide diversity (It) using .a b !!' 0) ARLEQUIN. Deviations from the Hardy-Weinberg 0. 38 E equilibrium (HWE) were estimated for each popula­ 0) I- 36 tion by calculating Weir & Cockerham's (1984) '"E ), 0 inbreeding coefficient f (equivalent to Wright's F IS as () 34 $e~9~6~ implemented in GENEPOP 4.0 (Rousset, 2008). m 0) ~~ Departures from HWE expectations were assessed :r: 32 • • using exact tests (Guo & Thompson, 1992), with sta­ 30 tistical significance determined by 10 000 dememo­ LM EN LF CB SP MO BO CC CA rizations, with 10 000 iterations of a Markov chain SOUTH Populations NORTH method. The significance levels for every exact test for HWE were adjusted, using a Bonferroni correction. To 44 test the selective neutrality of distributions of HSC70 (B) alleles for each population as well as for the entire Y =-0.1X + 40.191 (R2 = 0.051, P< 0.001) 42 pooled population of L. keenae, the Ewens-Watterson E • I • homozygosity method (Ewens, 1972; Watterson, 1978) 2! 40 :J • I was used with 10 000 permutations of the data. 1§ • 0) The degree of population differentiation for the 0. 38 E eight geographical populations was estimated by pair­ 0) I- 36 wise FST and

singletons). The most common allele (HSCKEE4) con­ 3.3 : 1. The overall value of 1t for the entire HSC70 sisted of 53 individuals (38% of total samples), and data set was 0.0240 ± 0.0143 (Table 3). was detected in all of the eight geographical popula­ All geographical populations of L. keenae tested tions (Appendix S2). Twenty-eight polymorphic sites conformed to HWE expectations (Table 3). In addition, were found, and the observed 34 point mutations the Ewens-Watterson tests of selective neutrality were all synonymous substitutions. There was sub­ showed that the observed HSC70 allele distributions stantial genetic diversity in 105 bp of the exon of follow the expected distributions of selectively neutral HSC70 among the 69 alleles [the average pairwise alleles in all of the populations. All P values of the sequence divergence (n) among the 69 alleles was Watterson F statistic for each population were inside 4.0 ± 0.20% (± Sm]. In the complete data set, the the top and bottom 2.5% (i.e. 0.025-0.975), suggesting transition to transversion ratio was approximately neutral distributions of the HSC70 alleles [Watterson F statistic, P = 0.3270-0.9730 (CC-CA)]. All the pairwise estimates of Fsr and

Table 3. Summary of the level of genetic diversity in eight geographical populations of Littorina keenae at HSC70. Sample size (N), observed number of alleles (N.), number of substitutions, number of polymorphic sites, P-values of exact tests for deviations from the Hardy-Weinberg equilibrium (HWE test; Guo & Thompson, 1992), expected (HE) and observed (Ho) heterozygosity, and nucleotide diversity (n) within a given population are provided. No cases of significant departure from the HWE were observed after a Bonferroni correction for multiple testing. Location codes are given in Table 1

No. of No. of polymorphic HWE Nucleotide Sample N N. substitutions sites test (P) HE Ho diversity (n)

LM 16 18 10 9 0.2210 0.9133 0.8125 0.0228 ± 0.0141 EN 17 22 19 17 0.0315 0.9679 0.8235 0.0286 ± 0.0170 CB 20 25 16 13 0.4969 0.9538 0.9000 0.0254 ± 0.0154 SP 17 17 9 9 0.2603 0.9216 0.8235 0.0219 ± 0.0137 MO 17 16 10 9 0.3134 0.9091 0.9421 0.0201 ± 0.0128 BO 16 16 10 8 0.6244 0.9375 0.9375 0.0206 ± 0.0131 CC 18 21 17 15 0.7107 0.9619 0.9444 0.0283 ± 0.0168 CA 18 19 16 14 0.9037 0.9095 0.8889 0.0237 ± 0.0145 Total 139 69 34 28 0.9361 0.8849 0.0240 ± 0.0143 501 of the range without LM there is no latitudinal cline temperature data from eight weather stations located in the thermal tolerance of L. keenae. More extensive on the north-eastern Pacific coast, which have similar sampling, particularly at intermediate sites between latitudes and longitudes to our eight sampling sites LM and Ensenada (EN), as well as at more southern (Appendix S4), suggest that there is a linear increase sites than LM, would be required to confirm whether in thermal stress during emersion with decreasing a step cline exists between our two most southern latitude. Despite this negative linear correlation sites, LM and EN. between air temperature and latitude, recurrent The lack of a linear latitudinal cline in thermal immigrant L. keenae larvae with alleles from else­ tolerance of L. keenae could be explained by three where (i.e. high gene flow) could constrain local adap­ different hypotheses: (1) its evolutionary response to a tation to a given thermal environment (reviewed by nonlinear latitudinal gradient in thermal stress; (2) Boulding et al., 2007). gene flow constraining local thermal adaptation; and A third hypothesis is that the weak clinal variation (3) phenotypic plasticity. The first hypothesis is that in thermal tolerance of L. keenae may result from the levels of thermal stress along the north-eastern environmental acclimatization (i.e. phenotypic plas­ Pacific coast do not increase linearly with decreasing ticity). The duration of time required for fulllabora­ latitude, and therefore southern L. keenae popula­ tory acclimation is fairly variable depending upon the tions will not be expected to evolve significantly species being studied (e.g. Clarke et al., 2000a; Sorte higher thermal tolerances than more northerly popu­ & Hofmann, 2005). Clarke et al. (2000a) found that lations. Previous monitoring of temperatures using the duration of acclimation did not have an effect on the low intertidal mussel Mytilus californianus as a HCT for Littorina littorea held at 12 QC from 0 (i.e. model has shown that local environmental factors, without acclimation) to 15 days. However, Sorte & such as wave splash and timing of low tide in Hofmann (2005) observed significant increases in summer, can overwhelm latitudinal gradients so that thermal tolerance between the 7 -week and the 5-day levels of thermal stress along the north-eastern acclimation groups for five different species of Pacific coast do not decrease linearly with latitude Nucella. We acclimated every L. keenae snail in the (Helmuth et al., 2002). This nonlinear environmental common laboratory conditions for a minimum of 4 gradient hypothesis is supported for the direct­ weeks. Ifthe duration ofthis time period was not long developing lower intertidal snail, Nucella canalicu- enough for full acclimation (Sorte & Hofmann, 2005), lata. Populations from southern central California then the observed weak latitudinal cline in thermal were less tolerant to thermal stress than populations tolerance could be partially or entirely caused by from northern Oregon; furthermore, this inter­ residual effects of acclimatization to the air tempera­ regional difference is likely to be genetically based tures at different latitudes. The lack of evidence for because the snails had been reared in a common population structure at HSC70, coupled with no laboratory environment for two generations (Kuo & deviations from the Hardy-Weinberg equilibrium, Sanford, 2009). Moreover, Sagarin & Somero (2006) suggests panmixia of L. keenae, making it plausible found the nonlinear latitudinal gradient in levels of that the most clinal variation that we observed in HSP70 expression for the intertidal mussel M. cali- HCT may be partially or entirely the result of phe­ fornianus, and also for the snail Nucella ostrina. notypic plasticity. However, the nonlinear environmental gradient In addition, our different time of sampling could hypothesis seems unlikely to be true for L. keenae bias the observed patterns of HCT in L. keenae. As because of its high vertical distribution on the shore described in Table 1, samples for the HCT were col­ above the high-tide mark on wave-exposed open lected mostly in summer within a 2-year period. Dif­ coasts, and thus they are not directly affected by local ferent time of sampling could bias our HCT data if the tidal cycles, even during summer when the surf is less 4 weeks of our laboratory acclimation were not long (H.J. Lee, pers. observ.). enough to eliminate the environmentally acquired A second hypothesis to explain the absence of a thermal tolerances of L. keenae. Seasonal differences cline in thermal adaptation for L. keenae is that in thermal tolerance have been reported in some although there may be selection for an increase in Littorina species, with HCT being higher in samples thermal tolerance with decreasing latitude, high gene collected in summer than in winter (Clarke et al., flow overwhelms the effects of selection, and prevents 2000b). This hypothesis of seasonal effects could be the populations from evolving to the local thermal tested by comparing the thermal tolerances among optima (Garcia-Ramos & Kirkpatrick, 1997; Barton, different seasonal samples from the 'same' population. 1999). Local adaptations are expected to be limited in Our analysis of HSC70 shows no evidence for a species with extensive gene flow, such as plank­ genetic signature of natural selection among the eight totrophic marine species like L. keenae (reviewed by geographical populations of L. keenae inhabiting ther­ Conover et al., 2006). Historical average monthly air mally varying environments. The Ewens-Watterson 502

tests suggest that all of the genetic variation that we found an increase in body size with latitude in the observed was selectively neutral. In addition, all of herbivorous intertidal gastropod T. funebralis from the 34 point mutations that we observed at the Carmel (36.5°N), California, USA, to Bamfield HSC70 exon were synonymous substitutions. This (48.8°N), British Columbia, Canada. However, Ola­ makes it difficult to think of a mechanism that would barria & Thurston (2003) observed no significant cause these mutations to have an effect on the fitness relationship between body size and latitude in the ofthis snail when under thermal stress. Furthermore, deep-sea gastropod T. berniciensis. our estimate of population differentiation at HSC70 There could be two different hypotheses that are revealed that there are no differences in allele fre­ not mutually exclusive to explain the intraspecific quencies among the eight populations, thus providing latitudinal cline in body size for L. keenae: (1) genetic further evidence that local adaptation at this locus is differences in body size among populations arise by unlikely. natural selection (Endler, 1977; Partridge & Coyne, A better understanding of the functional signifi­ 1997); and (2) phenotypic plasticity occurs in response cance of HSC70 in thermal adaptation would be aided to environmental temperature (Atkinson, 1994; by examining the variation in HSC70 gene expression Atkinson & Sibly, 1997). The first hypothesis of the and/or enzyme activity of HSC70 among the popula­ genetic origin of the relationship between body size tions of L. keenae. Fangue et al. (2006) found that and latitude has been supported by common garden southern populations of the common killifish F. het- experiments in some ectotherms. By rearing different eroclitus exhibited a significant increase in HSC70 geographical populations at constant temperature in expression levels in response to increasing tempera­ the laboratory, several studies of ectothermic species ture, but no change was detected in northern popu­ have demonstrated that latitudinal variation in body lations. This upregulation of HSC70 expression by size has a genetic basis (at least partially), although southern killifish in response to heat shock may rep­ the underlying mechanisms are unknown [e.g. the resent a significant role for HSC70 in rescuing copepod crustacean S. canadensis (Lonsdale & protein damage associated with daily fluctuations in Levinton, 1985); the fruit fly D. melanogaster (van't environmental temperatures in southern populations Land et al., 1999)]. Genetic divergence may be (Fangue et al., 2006). It would also be interesting to present in genes affecting body size among the popu­ explore more functionally relevant sites of HSC70, or lations of L. keenae. However, our population genetic an inducible isoform of HSP70, such as the promoter data using two molecular markers [mtDNA ND6 and region or the entire coding sequence of these genes. Cyt b (Lee & Boulding, 2007); HSC70, this study] Differences in HSC70 or HSP70 function can be suggest high gene flow, which could impede the caused by variation in sequences in the regulatory genetic differentiation in body size. region and/or primary sequences of amino acids The second hypothesis is that the observed latitu­ (Hochachka & Somero, 2002). dinal cline in body size may result from a phenotypic response to local temperature. This hypothesis includes phenotypic plasticity of body size itself to EFFECT OF LATITUDINAL CLINE IN BODY SIZE developmental temperature (Atkinson, 1994; Atkin­ ON THERMAL TOLERANCE son & Sibly, 1997: 'developmental temperature size Interestingly, our data shows a strong positive linear rule') as well as other temperature-related factors correlation between body size and latitude in L. [e.g. recruitment (Frank, 1975), food supply (Frank, keenae, which is consistent with Bergmann's rule. 1975), and predation (Fawcett, 1984)] that can influ­ However, body size decreases at the highest latitude ence the adult body size of L. keenae. The former has (CA, 43°18'N) so that a second degree of polynomial been well supported for a variety of ectothermic curve provides a slightly better fit. This positive qua­ taxa. Atkinson (1994) reviewed the data on dratic relationship between body size and latitude has the effect of temperature on body size in ectotherms also been shown in the fruit flies D. subobscura (Kari and found increases in body size of 83% in 109 studies & Huey, 2000) and D. melanogaster (de Jong & Boch­ of diverse ectothermic species, including protists, danovits, 2003). Indeed, latitude accounts for 59.8% of flies, and vertebrates that were grown at lower tem­ the variance observed in the body size in 1105 indi­ peratures. Van Voorhies (1996) proposed that Berg­ vidual L. keenae along its geographical range. mann size clines in ectotherms might be simply Although latitudinal patterns in body size have caused by developmental plasticity that allows cells to long been of great interest to biologists, only a few grow larger at lower environmental temperatures. To studies have focused on intraspecific latitudinal test the 'genetic differences in body size' and 'pheno­ trends in body size of marine invertebrates over a typic plasticity' hypotheses directly common garden or large geographical scale (e.g. Frank, 1975; Olabarria reciprocal transplant experiments for multiple gen­ & Thurston, 2003). Similar to our study, Frank (1975) erations must be performed. 503

Although we do not know the mechanisms causing E.G.B. All experiments complied with the current the latitudinal dine in body size in L. keenae, we laws of Canada. argue that these differences in body size are likely to be adaptive, not random (Partridge & Coyne, 1997). There is ample evidence that plasticity of phenotypic REFERENCES characters can itself respond to natural selection. For Atkinson D. 1994. Temperature and organism size - a bio­ example, van't Land et al. (1999) showed that plas­ logical law for ectotherms? Advances in Ecological Research ticity of body size in response to temperature was 25: 1-58. heritable in D. melanogaster. Atkinson D, Sibly RM. 1997. Why are organisms usually Our data suggests that the effect of body size on the bigger in colder environments? Making sense of a life thermal tolerance of L. keenae was not significant. We history puzzle. 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