The Effects of Submergence on the Thermal Function of Pinniped Fur

Notes

The effects of submergence on the thermal function of pinniped fur

NEHA SHARMA Department of Biology, Adelphi University, 1 South Avenue, Garden 1 City, New York 11530-0701, U.S.A. AND HEATHER E. M. LIWANAG, Department of Biology, Adelphi University, 1 South Avenue, Garden City, New York 11530-0701, U.S.A. and Department of Biological Sciences, California Polytechnic State University, 1 Grand Ave- nue, San Luis Obispo, California, 93407-0401, U.S.A.

As endothermic homeotherms, mammals derive their body temperature from internal metabolism and achieve balance by regulating core temperature around a set point that is independent from the environment (Scholander et al. 1950). Ther- moregulation is especially challenging for endotherms in marine environments, because heat loss in water occurs 25 times faster than in air at the same temperature (Dejours et al. 1987). As a result, most marine mammals have evolved physiological and morphological mechanisms for reducing heat loss to the environment (Pabst et al. 1999). Across marine mammal lineages, species have developed specialized insulation in the form of modiﬁed fur and/or blubber (Scholander et al. 1950, Pabst et al. 1999, Liwanag et al. 2012a, b). The most recent lineages to reinvade the marine environment, sea otters and polar bears, still rely on fur for their primary insulation (Pabst et al. 1999, Berta et al. 2006). The more derived lineages, cetaceans and sireni- ans, have secondarily lost the characteristic mammalian fur and instead rely on blubber as their primary insulator (Hart and Fisher 1964, Ling 1970, Berta et al. 2006). Pinnipedia is the only extant marine mammal lineage that has retained both forms of insulation. Among the pinnipeds, phocids (true seals) and odobenids (walrus) rely on blubber for their thermal insulation. Otariids (eared seals), however, retain body heat through two distinct mechanisms: fur seals have dense, waterproof fur along with a moderate blubber layer, whereas sea lions have nonwaterproof fur and primarily rely on their thicker blubber to keep warm (Liwanag et al.2012a, b). Pinnipeds are unique among marine mammals because they forage and migrate in the water but breed and rest on land. Heat loss on land can take place through conduction, convection, radiation, and evaporation, whereas in an aquatic medium the more likely avenues of heat loss are conduction and convection (Perrin et al. 2009).

1Corresponding author (e-mail: [email protected]).

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The amphibious lifestyle of pinnipeds could result in trade-offs with regard to how well their insulation performs in each medium. The insulating properties of fur are dependent on its ability to sustain a layer of air trapped between the hairs (Kvad- sheim and Aarseth 2002). There are two primary types of hairs that characterize mammalian fur: short, fine underhairs, which act to trap air against the animal’s skin, and longer, coarse guard hairs, which cover and protect the underhairs (Ling 1970). Fur has been described as the superior insulator over blubber, as long as the air layer is maintained (Kvadsheim and Aarseth 2002, Liwanag et al.2012b). When the animal is submerged, however, the air layer can become compromised and the fur may no longer serve as effective insulation. Because blubber is a subcutaneous tissue, its effectiveness as insulation is not compromised by submergence (Schmidt-Nielsen 1997, Liwanag et al. 2012b). Change in fur function during submergence is likely variable among pinniped groups; fur seals have the densest fur and can maintain a layer of air between the underhairs while submerged, whereas sea lion and phocid pelts become saturated with water upon submergence (Liwanag et al. 2012a). Though several previous studies have measured the thermal properties of marine mammal integument and/or blubber (Kvadsheim and Aarseth 2002; Dunkin et al. 2005; Bagge et al. 2012; Liwa- nag et al. 2012b;Pearsonet al.2014a, b;Horganet al. 2014), very few have directly measured the effects of submergence on those properties, particularly across groups (Scholander et al. 1950, Frisch et al. 1974, Kvadsheim and Aarseth 2002). The objec- tive of this study was to quantify the extent to which submergence in water reduces the effectiveness of pinniped fur as an insulator. This was achieved by comparing the thermal conductivity (k;Wm–1°C–1) and thermal resistance (R;m2°CW–1)ofpin- niped pelts in air and in water. We hypothesized that fur seal pelts would undergo a minimal loss of thermal function in water compared to those of sea lions and phocids, based on differences in fur density and ability to maintain a layer of air within the fur. Sculp (fur, skin, and blubber) samples were collected postmortem, from the dor- sum of five fur seal species (n = 17), one sea lion species (n = 20), and three phocid species (n = 9; Table S1). Fresh tissue samples were collected only from good condition carcasses, as determined by tissue color. Because samples were acquired oppor- tunistically from stranded or harvested animals, age classes were variable and included pups, yearlings, subadults, and adults. Young mammals typically exhibit similar fur characteristics to adult conspecifics (Meyer et al. 2002), and this is particularly true for pinnipeds that have shed the initial lanugo coat (Pearson et al. 2014a, Gmuca et al. 2015); thus, pup pelts were only included if they did not have lanugo. Samples included in this study did not show any signs of molt. All samples were stored in plastic food wrap and heavy-duty freezer bags to prevent desiccation, and kept frozen (–20°C) until use. Samples were cut into squares (approximately 10 9 10 cm), rinsed under cold, distilled water, and then dried using a hairdryer on the cool setting to restore the naturally existing air layer between the hairs (Williams et al. 1988, Kvadsheim and Aarseth 2002, Liwanag et al. 2012a). Conductivity measurements in air were made on these samples in a previous study, with pelt and blubber conductivity determined simultaneously but distinctly for each layer (Liwanag et al. 2012a, b). For the present study, the blubber layer was carefully cut away from each sample, leaving the pelt (fur and skin). Skin, dry fur (before trial), and wet fur (after trial) thicknesses were measured to the nearest 0.001 mm using digital calipers (Absolute Digimatic caliper 500 series, Mitutoyo, Aurora, IL) three times on each side, and the mean values were used for calculations. NOTES 613

Thermal conductivity (k) was measured in a heat flux chamber (162 quart Igloo Marine ice chest, Igloo Commercial) using the standard material method (Kvadsheim et al. 1994; Kvadsheim and Aarseth 2002; Dunkin et al. 2005; Liwanag et al. 2012a, b). The chamber consisted of an insulated lower compartment heated to 37°C with a circulating water bath (Lauda RM20, Brinkmann Instruments or Isotemp 6200, Fisher Scientific) and a chilled upper compartment filled with ice packs (Liwa- nag et al. 2012a, b;Pearsonet al.2014b). The entire setup was surrounded by foam insulation to maintain a stable thermal gradient and to ensure unidirectional heat flow. An elastomer (Plastisol vinyl, Carolina Biological Supply, Burlington, NC) was used as the standard material (k = 0.119 0.017 Wm–1°C–1). The standard material was placed directly against the heat source. On top of the standard material, the pelt sample was placed skin-side down with the edges carefully adjusted into a cup-like formation to prevent water from leaking below the sample. Cold water (5.88°C 3.49°C, approximately 1 cm height) was evenly poured on top of the fur to simulate submergence. Three copper-constantin (Type T) thermocouples (Physitemp Instruments, Inc., Clifton, NJ) were placed between each layer: between the heat source and standard material, between the standard material and pelt, and on top of the pelt. Each thermocouple was connected to a Fluke Hydra data logger (Model 2625A, Fluke Inc., Everett, WA), which recorded outputs every 6 s onto a desktop computer. Trials were conducted for a minimum of 2 h to ensure a steady thermal gradient, such that the water reached a slightly warmer, but steady, temperature. Data collected during the final 30 min of each trial were used for analysis. Thermal conductivity was calculated across the pelt using the Fourier equa- tion (Kreith 1958):

H ¼ k A DT L1 ð1Þ where H is heat transfer (J/s), k is thermal conductivity (Wm–1°C–1), A is the area (m2) through which the heat is moving, DT is the temperature differential (°C) across the material, and L is the thickness of the material (m). Assuming equal rates of heat transfer across the standard material and pelt sample, the equations for both materials can be set equal and solved for thermal conductivity of the pelt sample. Note that thermal conductivity is a material property, and describes the quality of the insulation independent of thickness (Kvadsheim et al. 1994). To take thickness of the insulation into account, thermal resistance (R;m2°CW–1) was calculated as: R ¼ L=k ð2Þ

To determine thickness (L) during submergence, the height of the wet pelt was measured with digital calipers upon completion of each conductivity trial. Within-group comparisons between measurements in air and in water were analyzed using paired t-tests, and comparisons among groups (fur seal, sea lion, phocid) were analyzed using 1-way ANOVA followed by the Tukey honestly significant difference test (JMP Pro 11.2.1, SAS, Cary, NC). Upon analysis, we observed that the northern elephant seals (subfamily Monachinae) showed a different pattern from the other phocid species (subfamily Phocinae); therefore, we also conducted 1-way ANOVA to compare subgroups (fur seal, sea lion, phocine, monachine). Thermal conductivity of the pelt was significantly different among groups, both in air (F2,44 = 3.33,P= 0.046) and in water (F2,44 = 10.81,P< 0.001). In air, thermal conductivity 614 MARINE MAMMAL SCIENCE, VOL. 33, NO. 2, 2017 of phocid pelts was significantly higher when compared to fur seal pelts (P = 0.045). There was no significant difference in thermal conductivity between fur seal and sea lion pelts (P = 0.915) or between sea lion and phocid pelts (P = 0.085). When analyzed according to subgroups (F3,44 = 33.27, P < 0.001; Fig. 1), there was no significant difference in thermal conductivity between fur seal and sea lion pelts in air (P = 0.903), but thermal conductivity was significantly lower for phocine pelts (P < 0.030) and significantly higher for monachine pelts (P < 0.001). In water, the thermal conductivity of fur seal pelts was significantly lower when compared to that of both sea lion (P = 0.002) and phocid pelts (P < 0.001); thermal conductivities of sea lion and phocid pelts were not significantly different in water (P = 0.406). When compared among subgroups (F3,44 = 7.08, P < 0.001; Fig. 1), the thermal conductivity of fur seal pelts in water was significantly lower compared to sea lion (P = 0.005), phocine (P = 0.011), and monachine pelts (P = 0.014), with no significant difference among the remaining subgroups (P ≥ 0.662). When comparing values within groups, the thermal conductivity of fur seal pelts was slightly but not significantly higher in water compared to air (P = 0.057), whereas the conductivity of sea lion pelts was significantly higher in water compared to air (P < 0.001). There was no significant difference in conductivity for phocid pelts in water compared to air (P = 0.105). However, the thermal conductivity of phocine pelts was significantly higher in water compared to air (P = 0.036), whereas there was no significant difference in conductivity for monachine pelts in water compared to air (P = 0.965). Thermal resistance was significantly different among the three groups in air (F2,44 = 9.40,P< 0.001) and in water (F2,44 = 21.92,P< 0.001). In air, the thermal resistance of fur seal pelts was significantly higher when compared to that of sea lion

Figure 1. Thermal conductivity (mean SEM) for fur seal, sea lion, phocine, and monachine pelts in air and in water. Asterisks indicate a statistically signiﬁcant difference in thermal conductivity between air and water within that group. Different letters indicate statistically signiﬁcant differences among groups (upper case in air, lower case in water). NOTES 615

(P < 0.001) and phocid pelts (P < 0.001). There was no significant difference in thermal resistance between sea lion and phocid pelts in air (P = 0.916). When analyzed according to subgroups (F3,43 = 9.85, P < 0.001; Fig. 2), the thermal resistance of fur seal pelts in air was significantly higher compared to that of sea lion pelts (P = 0.002) and monachine pelts (P < 0.001), and there was no significant difference in thermal resistance between sea lion and monachine pelts (P = 0.120). The thermal resistance of phocine pelts in air was not significantly different from that of fur seal (P = 0.686), sea lion (P = 0.746), or monachine pelts (P = 0.088). In water, the thermal resistance of fur seal pelts was significantly higher compared to that of sea lion pelts (P < 0.001) and phocid pelts (P < 0.001). There was no significant difference in thermal resistance between sea lion and phocid pelts in water (P = 0.923). When compared among subgroups (F3,44 = 15.12, P < 0.001; Fig. 2), the thermal resistance of fur seal pelts in water was significantly higher compared to that of sea lion (P < 0.001), phocine (P < 0.001), and monachine pelts (P = 0.005). Thermal resistance in water did not differ significantly among the remaining subgroups (P ≥ 0.682). Fur seal and sea lion pelt resistance decreased significantly in water compared to air (P < 0.001), whereas there was no significant difference in thermal resistance between air and water for phocid pelts (P = 0.068). Thermal resistance did decrease significantly in water compared to air for phocine pelts (P = 0.023), whereas there was no significant difference between air and water for monachine pelts (P = 0.622). The differences in thermal function among pinniped pelts are likely due to differences in fur density and morphology. For otariids and phocines, thermal function (i.e., resistance) decreased under conditions of submergence (Fig. 2). This suggests

Figure 2. Thermal resistance (mean SEM) for fur seal, sea lion, phocine, and monachine pelts in air and in water. Asterisks indicate a statistically significant difference in thermal conductivity between air and water within that group. Different letters indicate statistically significant differences among groups (upper case in air, lower case in water). 616 MARINE MAMMAL SCIENCE, VOL. 33, NO. 2, 2017 that the air layer that is normally maintained within the fur may be compromised under water. Fur seal pelts exhibited the highest thermal resistance in both air and water (Fig. 2), indicating that they were most effectively able to prevent heat loss through the fur in both media. This can be credited to the high fur density and unique cuticular scale patterning of the hairs, both of which facilitate the trapping of air and thus help to conserve heat (Liwanag et al. 2012a). Most of the reduced thermal resistance of fur seal pelts in water resulted from flattening of hairs (i.e., the hairs lying closer to the body) during submergence (Table 1), as there was no significant difference in conductivity between media (Fig. 1). For all subgroups, pelt thickness decreased significantly when wet (paired t-tests, P ≤ 0.040; Table 1). This flattening effect can reduce drag while the animal is swimming and diving, and those activities may further alter fur position in relation to the body (Fish 2000). Sea lion and phocid pelts performed equivalently to each other while submerged, with high conductivity and low resistance values, which indicate higher rates of heat loss associated with a thin, nonwaterproof fur layer (Fig. 1, 2). These groups are able to compensate for a lower density fur layer by having a much thicker blubber layer than fur seals (Liwanag et al. 2012b), which allows them to maintain thermal insulation during a dive. Fur is not the optimal insulation type for deep diving because the air layer becomes compressed at depth and is then lost upon ascent (Liwanag et al. 2012a), whereas blubber maintains its thickness and insulative function even at depth (Liwanag et al.2012b). Sea lions and phocids are generally deeper divers than fur seals, and it is possible that increases in dive depth could have contributed selec- tive pressure to shift to blubber as the primary insulator over evolutionary time (Schreer and Kovacs 1997, Kooyman and Ponganis 1998, Perrin et al. 2009, Bestley et al. 2015, Kooyman 2015). Indeed, the monachine species in this study, the northern elephant seal (Mirounga angustirostris, Gill), is one of the deepest diving seals and appears to be among the most derived with regard to shifting to blubber as the primary insulator (Berta et al. 2006, Perrin et al. 2009). Monachine pelts functioned as equally poor insulators in air and in water (Fig. 1, 2), likely due to distinctive morphological characteristics. The pelts of monachine species, including the northern elephant seal, lack underhairs, which specifically function to trap a still layer of air against the skin (Scheffer 1964, Ling 1970); this lack of underhairs may explain the low thermal function of monachine pelts in air, even when compared to phocines. In addition, northern elephant seal guard hairs are particularly short in length and remarkably flat in cross section, two morphological characteristics associated with a reduced reliance on fur as an insulator (Liwanag et al.2012a). In contrast, the presence of underhairs in phocine pelts appears to have maintained the thermal function of those pelts in air, whereas the nonwaterproof nature of phocine pelts allowed a drastic reduction of thermal function in water (Liwanag et al. 2012a; Fig. 1, 2). As the current study includes only one monachine species, more research is required to further investigate differences in the form and function of phocine and monachine pelts; this could elucidate whether reduction in pelt quality shows a greater correlation with dive depth or with phylogeny among the phocids. Phylogenetic relationships among otariid species are still under extensive debate. Classic studies supported by morphological data suggested that fur seals and sea lions were monophyletic groups within the otariids (King 1983, Wyss 1988, Berta and Wyss 1994), whereas more recent studies using mitochondrial gene sequences suggest the groups are polyphyletic (Wynen et al. 2001, Higdon et al. 2007, Yonezawa et al. 2009). Despite the ambiguity, it has been established that the northern fur seal Table 1. Thicknesses of pinniped pelts (fur and skin) when dry and when wet. Values indicate means 1 SD for individual species and indicated subgroups. Pelt thickness decreased significantly when wet for all subgroups.

Family Subgroup Species Dry pelt thickness (mm) Wet pelt thickness (mm) Otariids Fur seals Arctocephalus gazella 12.57 0.49 11.00 1.97 6.71 0.64 6.41 1.15 Arctocephalus pusillus pusillus 9.77 1.57 6.79 1.13 NOTES Arctocephalus tropicalis 9.82 1.19 7.01 1.11 Arctocephalus townsendi 10.89 0.85 5.80 0.10 Callorhinus ursinus 13.28 1.29 5.18 0.72 Sea lion Zalophus californianus 7.55 1.47 5.76 1.17 Phocids Monachine Mirounga angustirostris 7.70 1.42 6.66 1.36 Phocines Phoca vitulina 6.86 1.92 6.74 1.58 4.67 0.33 4.44 0.54 Pusa hispida 6.38 0.76 3.73 0.18 617 618 MARINE MAMMAL SCIENCE, VOL. 33, NO. 2, 2017

(Callorhinus ursinus, Linnaeus) is the earliest diverging species of the extant otariids (Wynen et al. 2001, Berta et al.2006,Higdonet al. 2007, Yonezawa et al. 2009). After divergence from the common ancestor with the northern fur seal, there have likely been multiple transitions to the “sea lion” condition, as sea lion species appear on multiple branches within the otariid tree (Wynen et al. 2001, Higdon et al. 2007, Yonezawa et al. 2009, Liwanag et al. 2012a). Differences in pelage morphology therefore suggest that a dense, waterproof fur layer is a trait that sea lions have secondarily lost through multiple convergent events (Liwanag et al. 2012a). The loss of thermal function observed in sea lion fur under conditions of submergence aligns with previous evidence of convergent morphological evolution with phocids. This is consistent with results from previous studies showing similarities in fur morphology and reduction in fur density in sea lion and phocid fur (Scheffer 1964, Liwanag et al. 2012a). These changes are associated with the evolutionary transition to a thicker blubber layer (Liwanag et al.2012b). Note, however, that quality and quantity of blubber can vary seasonally (Samuel and Worthy 2004, Budge et al. 2008) and may be dependent on animal body size (Fay 1981, Liwanag et al. 2012b), condition (Dunkin et al. 2005), reproductive stage, and diet (Pabst et al.1999, Struntz et al. 2004). Despite these ﬂuctuations, blubber appears to be the superior insulator for more aquatic species, due to the hydrodynamic factors associated with submergence, swimming, and diving (Fish 2000, Fish et al. 2002, Liwanag et al. 2012b). The results of this study indicate an important difference in the thermal function of the pelt among pinniped groups, conﬁrming the reliance of fur seals on their thicker fur and the reliance of sea lions on their blubber layer during submergence. These changes may be associated with differences in dive behavior and dive depth, which may be related to the evolutionary shift from fur to blubber in pinnipeds.

Acknowledgments

Postmortem tissue samples were collected under U.S. Department of Commerce permit number 960-1528-00/PRT-017891. We would like to thank J. Arnould, D. Casper, M. Goe- bel, T. Goldstein, M. Gray, D. Guertin, B. McDonald, L. Leppert, B. Long, M. Miller, L. Pola- sek, P. Robinson, S. Simmons, C. Stephens, P. Tuomi, D. Verrier, S. Villegas, E. Wheeler, and especially J. Burns and P. Morris for their assistance with obtaining samples for this project. We also thank Natalia Gmuca and Candice Marcos for assistance with data collection. We thank Leslie Liwanag for his invaluable assistance with ﬁgure preparation. Carey Kuhn and three anonymous reviewers contributed valuable feedback on the manuscript. This project was funded by a Horace McDonell Summer Research Fellowship to NS.

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Received: 10 January 2016 Accepted: 28 August 2016

Supporting Information The following supporting information is available for this article online at http:// onlinelibrary.wiley.com/doi/10.1111/mms.12372/suppinfo. Table S1. Species and age classes of pinniped sculp samples included in the present study.