Results of covariance analysis corresponding to the linear regressions drawn in Figure 1 plus Antarctic data.

<p>P-values are indicated for the comparison between slopes and intercepts for each pair of environments. NHWTSH: Northern Hemisphere Warm Temperate environments in Shallow Water (<2 m), NHWT: Northern Hemisphere Warm Temperate environments in deeper water, SHWT: Southern Hemisphere Warm Temperate environment, CT: Cold Temperate environment and A: Antarctic environment.</p>*<p>: statistically significant.</p

Log-log relationship for upper temperature limit (UTL) versus rate of temperature change.

<p>Only marine subtidal species from experiments done in summer are used in this figure. Data for the <i>dynamic method</i> were combined with those from the <i>static method</i> after transformation using the Kilgour and McCauley <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034655#pone.0034655-Kilgour1" target="_blank">[17]</a> model. Black triangles: species from Northern Hemisphere Warm Temperate environments (NHWTSW) in the South of France and the West coast of the United States of America in very Shallow Water (<2 m); grey triangles: species from Northern Hemisphere Warm Temperate environments (NHWT) in the South of France and the West coast of the United States of America in deeper water (>2 m); open triangles: species from a Southern Hemisphere Warm Temperate environment (SHWT) on the Peru coast; and black squares: species from a Cold Temperate environment (CT) in Scotland. For each ecosystem, a linear regression is fitted. Black triangles: y = −0.01×+3.6 R² = 0.57, grey triangles: y = −0.02×+3.5 R² = 0.75, open triangles: y = −0.07×+3.3 R² = 0.86, black squares: y = −0.03×+3.3 R² = 0.99.</p

Correlation between upper temperature limit (UTL) and rate of temperature change.

<p>Black triangles: Northern Hemisphere Warm Temperate environments in Shallow Water (<2 m; NHWTSW), grey triangles: Northern Hemisphere Warm Temperate environments in deeper water (NHWT), open triangles: Southern Hemisphere Warm Temperate environment (SHWT), black squares: Cold Temperate environment (CT) and black diamonds: Antarctica (A). For each environment, the non linear model described in the methods is fitted.</p

Correlation between Slope and Intercept (from

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034655#pone-0034655-g001" target="_blank"><b>Figure 1</b></a><b>+Antarctic environment).</b> Each diamond represents an environment. NHWTSW: Northern Hemisphere Warm Temperate environments in the South of France and the West coast of the United States of America in very Shallow Water (<2 m); NHWT: Northern Hemisphere Warm Temperate environments in the South of France and the West coast of the United States of America in deeper water (>2 m); SHWT: Southern Hemisphere Warm Temperate environment on the Peru coast; CT: Cold Temperate environment in Scotland; A: Antarctic environment. Linear regression SHWT excepted: y = 0.07×−0.27, R² = 0.99, p-value: 0.0003.</p

Gene expression analysis of RNAi-feeding in PaDAW1.

<p>(A) qPCR of <i>gfp(RNAi)</i>-treated (control) <i>Pd-rps-2(RNAi)</i>, <i>Pd-dhc(RNAi)</i> and <i>Pd-duox-42(RNAi)</i> treated PaDAW1 (B) qPCR of <i>Pd-rps-2(RNAi)</i> after a feeding period of 12 h, 24 h, 36 h and 48 h. The expression of RNAi treated samples is shown relative to control samples, which are normalized to a value of 1. Each value represents the mean±s.d. of three or four biological replicates. No significant differences were found in these data (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166228#pone.0166228.s004" target="_blank">S3 File</a>).</p

Results of covariance analysis corresponding to the linear regressions drawn in Figure 2 A and B.

<p>P-values are indicated for the comparison between slopes and intercepts for each season and species.</p>*<p>: statistically significant.</p

Phenotypic analysis of three different RNAi-soaking techniques.

<p>(A-C) Micrographs showing the FITC uptake in PaDAW1 using three different soaking techniques: soaking-only (A), octopamine-enhanced soaking (B) and desiccation-enhanced soaking (C). s = stylet, p = pharynx, m = median bulb, l = lipids and v = vulva. Scale bar, 0.1 mm. (D) Survival rate of nematodes for soaking-only, octopamine-enhanced soaking and desiccation-enhanced soaking. Each value represents the mean±s.d. of 4 biological replicates and an analysis of variance showed no significant differences among conditions (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166228#pone.0166228.s005" target="_blank">S4 File</a>).</p

Micrograph showing a mass of coiled PaDAW1 desiccated for 24 h at 98% relative humidity.

<p>Scale bar, 1 mm.</p

Embryonic lethal phenotypes in <i>C</i>. <i>elegans</i> and PaDAW1 after feeding <i>E</i>. <i>coli</i> expressing dsRNA.

<p>(A) Larval hatching of <i>gfp(RNAi)</i>-treated, <i>Pd-rps-2(RNAi)</i>-treated and <i>Pd-dhc(RNAi)</i>-treated PaDAW1 (left y-axes) and <i>C</i>. <i>elegans</i> (right y-axes) 48 h after removal of adults from the plates. Each value represents the mean±s.d. of 6–18 biological replicates (three nematodes per biological replicate). For PaDAW1 the experiment was replicated three times (six biological replicates per experiment) and the values represent the mean±s.d. of all 18 biological replicates. (B) Larval hatching of <i>gfp(RNAi)</i>-treated, <i>Ce-rps-2(RNAi)</i>-treated and <i>Ce-dhc-1(RNAi)</i>-treated PaDAW1 (left y-axes) and <i>C</i>. <i>elegans</i> (right y-axes) 24 h after removal of adults from the plates. Each value represents the mean±s.d. of 6 biological replicates. Conditions where a significant decreases in larval hatching was assessed by multiple comparison of means by Dunnett Contrasts [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166228#pone.0166228.ref030" target="_blank">30</a>] are indicated with a <i>p</i> value (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166228#pone.0166228.s003" target="_blank">S2 File</a>).</p

Log-log relationship for upper temperature limit (UTL) versus rate of temperature change.

<p>This is shown for four different species from the Mediterranean Sea in different seasons: A) <i>Cardium tuberculatum</i>, B) <i>Cardium glaucum</i>, C) <i>Donax trunculus</i> and D) <i>Donax semistriatus</i>. Black diamonds: summer, open black diamonds: winter, grey diamonds in a and b: autumn and spring, grey diamonds in c and d: autumn and open grey diamonds: spring. For each season and each species a linear regression is fitted. A) Black diamonds: y = −0.034×+3.5 R² = 1, grey diamonds: y = −0.034×+3.4 R² = 1, open black diamonds: y = −0.04×+3.4 R² = 1; B) Black diamonds: y = −0.024×+3.6 R² = 1, grey diamonds: y = −0.032×+3.5 R² = 1, open black diamonds: y = −0.034×+3.5 R² = 1; C) Black diamonds: y = −0.032×+3.5 R² = 1, grey diamonds: y = −0.038×+3.5 R² = 1, open grey diamonds: y = −0.039×+3.5 R² = 1, open black diamonds: y = −0.048×+3.5 R² = 1; D) Black diamonds: y = −0.023×+3.5 R² = 1, grey diamonds: y = −0.037×+3.4 R² = 1, open grey diamonds: y = −0.034×+3.4 R² = 1, open black diamonds: y = −0.035×+3.4 R² = 0.99.</p