Gill Morphometrics of the Thresher Sharks (Genus Alopias): Correlation of Gill Dimensions with Aerobic Demand and Environmental Oxygen

Home , Bigeye thresher, Fish gill, Pelagic thresher

JOURNAL OF MORPHOLOGY :1–12 (2015)

Thomas P. Wootton,1 Chugey A. Sepulveda,2 and Nicholas C. Wegner1,3*

1Center for Marine Biotechnology and Biomedicine, Marine Biology Research Division, Scripps Institution of Oceanography, University of California San Diego, La Jolla, CA 92093 2Pﬂeger Institute of Environmental Research, Oceanside, CA 92054 3Fisheries Resource Division, Southwest Fisheries Science Center, NOAA Fisheries, La Jolla, CA 92037

ABSTRACT Gill morphometrics of the three thresher related species that inhabit similar environments shark species (genus Alopias) were determined to or have comparable metabolic requirements. As examine how metabolism and habitat correlate with such, in reviews of gill morphology (e.g., Gray, respiratory specialization for increased gas exchange. 1954; Hughes, 1984a; Wegner, 2011), fishes are Thresher sharks have large gill surface areas, short often categorized into morphological ecotypes water–blood barrier distances, and thin lamellae. Their large gill areas are derived from long total filament based on the respiratory dimensions of the gills, lengths and large lamellae, a morphometric configura- namely gill surface area and the thickness of the tion documented for other active elasmobranchs (i.e., gill epithelium (the water–blood barrier distance), lamnid sharks, Lamnidae) that augments respiratory which both reflect a species’ capacity for oxygen surface area while limiting increases in branchial uptake (i.e., the diffusion capacity of the gills). resistance to ventilatory flow. The bigeye thresher, Alo- Currently at least six groups are recognized: (1) pias superciliosus, which can experience prolonged fast-swimming oceanic species, (2) marine fishes of exposure to hypoxia during diel vertical migrations, intermediate activity, (3) sluggish marine species, has the largest gill surface area documented for any (4) freshwater fishes, (5) air-breathers, and (6) elasmobranch species studied to date. The pelagic hypoxia-dwellers (Wegner, 2011). thresher shark, A. pelagicus,awarm-waterepi-pelagic species, has a gill surface area comparable to that of While these six morphological ecotypes are gen- the common thresher shark, A. vulpinus, despite the erally broad, they encompass the breadth of fish latter’s expected higher aerobic requirements associ- diversity and gill morphology based primarily on ated with regional endothermy. In addition, A. vulpinus fish metabolic requirements and the availability of has a significantly longer water–blood barrier distance oxygen in their respective habitats. Fast oceanic than A. pelagicus and A. superciliosus, which likely species have high oxygen demands associated with reflects its cold, well-oxygenated habitat relative to the elevated capacities for aerobic performance and two other Alopias species. In fast-swimming fishes thus possess large gill surface areas and short gill (such as A. vulpinus and A. pelagicus) cranial stream- diffusion distances to facilitate oxygen uptake lining may impose morphological constraints on gill (Muir and Hughes, 1969; Emery and Szczepanski, size. However, such constraints may be relaxed in hypoxia-dwelling species (such as A. superciliosus)that 1986; Wegner, 2010a, b). Most fishes comprising are likely less dependent on streamlining and can this group (tunas, Scombridae; lamnid sharks, therefore accommodate larger branchial chambers and Lamnidae; billfishes, Istiophoridae and Xiphiidae) gills. J. Morphol. :1–12, 2015. VC 2015 Wiley Periodicals, are capable of regional endothermy (the ability to Inc. conserve metabolically produced heat to warm cer- tain regions of the body), which results in KEY WORDS: gill surface area; elasmobranch; hypoxia; enhanced physiological function (Dickson, 1995; aerobic metabolism; regional endothermy; diffusion capacity

*Correspondence to: Nicholas C. Wegner, National Marine Fish- INTRODUCTION eries Service, Southwest Fisheries Science Center, 8901 La Jolla Shores Dr., La Jolla, CA 92037. Fish gill morphology correlates with metabolic Email: [email protected] demand and habitat (Gray, 1954; Hughes, 1966; Hughes and Morgan, 1973; De Jager and Dekkers, Received 18 April 2014; Revised 17 December 2014; 1975; Hughes, 1984a; Palzenberger and Pohla, Accepted 2 January 2015. 1992; Chapman, 2007; Wegner, 2011). Despite the Published online 00 Month 2015 in extreme diversity of ﬁshes, noticeable similarities Wiley Online Library (wileyonlinelibrary.com). in respiratory structure occur in even distantly DOI 10.1002/jmor.20369

VC 2015 WILEY PERIODICALS, INC. 2 T.P. WOOTTON ET AL. Brill, 1996; Bernal et al., 2001; Korsmeyer and vating the temperature of the slow-twitch aerobic Dewar, 2001; Dickson and Graham, 2004; Sepul- (red) muscle (Bernal and Sepulveda, 2005; Sepul- veda et al., 2007). Marine fishes of intermediate veda et al., 2005) and is thus expected to have activity have relatively standard gill morphologies short gill diffusion distances and a large gill sur- due to their more “typical” activity levels, lack of face area like lamnid sharks. (2) The pelagic endothermy, and generally normoxic habitats thresher, A. pelagicus, lacks the capacity for red- (Gray, 1954; Hughes, 1966; Hughes, 1970; Hughes, muscle endothermy (Sepulveda et al., 2005, Pat- 1984a). Sluggish marine species, freshwater fishes, terson et al., 2011) and is thus hypothesized to and air-breathers all tend to have reduced gill sur- have lower oxygen requirements and hence a more face areas and thick water–blood barrier distances, “standard” gill morphology (i.e., marine fish of although the environmental and physiological fac- intermediate activity). (3) The bigeye thresher, A. tors influencing these dimensions appear to differ superciliosus, is a deep-diving shark (Nakano between groups. For sluggish marine species, a et al., 2003; Weng and Block, 2004; Musyl et al., reduced gill area and thicker epithelium are asso- 2011) that in many parts of its range experiences ciated with low metabolic demands (Gray, 1954; prolonged exposure to hypoxia in the oceans’ oxy- Hughes and Gray, 1972; Hughes and Morgan, gen minimum zones (OMZs) and is thus hypothe- 1973, Hughes and Iwai, 1978), while most fresh- sized to have a large gill area with short diffusion water fishes likely have a generally reduced gill distances to facilitate oxygen uptake in a low- diffusion capacity due to the relatively higher oxygen environment. availability of oxygen in freshwater (air-saturated Because the three thresher shark species differ freshwater contains 15–20% more dissolved oxy- in life history and habitat and appear to fall into gen than seawater depending on temperature; Pal- three different ecological morphotypes, they com- zenberger and Pohla, 1992). For air-breathing prise an ideal system for testing how gill dimen- fishes, an increased reliance on oxygen absorption sions correlate with high oxygen demands (as from the air results in smaller and thicker gills expected for the regionally endothermic A. vulpi- (Graham, 1997; Graham et al., 2007; Graham and nus) and low oxygen availability (as expected for Wegner, 2010). Although they are often sedentary the hypoxia-dwelling A. superciliosus) in compari- and found in freshwater, hypoxia-dwelling species son to more typical conditions (as expected for A. have large gill surface areas (and likely short pelagicus). This study thus examines the respira- water–blood barriers) to absorb sufficient oxygen tory dimensions of the gills (i.e., the gill surface to meet their metabolic requirements in an area, water–blood barrier distance, and lamellar oxygen-deficient habitat (Graham, 2006; Chapman, thickness) in the three closely related species to 2007; Mandic et al., 2009; Wegner, 2011). better understand the specific effects of metabolic Thus, while broad, these morphological ecotypes demand and dissolved oxygen availability on gill provide insight into the role of oxygen demand morphology. and availability in shaping gill morphology. How- ever, because phylogenetic assemblages tend to experience similar evolutionary pressures, closely related species usually fall into the same ecomor- MATERIALS AND METHODS photypic group. As such, conclusions drawn from Gill Collection and Preparation these broad morphological ecotypes are often made Gills were collected opportunistically from nine A. vulpinus by comparing distantly related fishes. While gen- (Bonnaterre, 1788), nine A. superciliosus (Lowe, 1841), and six eral trends in gill morphology can be assessed by A. pelagicus (Nakamura, 1935). All A. vulpinus, most A. super- such comparisons, determining the detailed effects ciliosus, and one A. pelagicus were caught by hook and line off of specific evolutionary pressures (such as metabo- the coast of southern California, Hawaii, or Mexico during other scientific studies. These sharks were euthanized by sever- lism or environmental oxygen level) on gill mor- ing the spinal cord at its articulation to the chondrocranium phology can be difficult due to the numerous other according to protocol S00080 of the University of California, selective pressures influencing each group’s evolu- San Diego Institutional Animal Care and Use Committee. tionary history. Additional insight on the correla- Three A. superciliosus and five A. pelagicus were purchased whole from drift gillnet and longline fisheries in southern Cali- tion of gill dimensions with oxygen demand and fornia and Costa Rica. For all sharks, fork length was meas- availability can be gained by comparing cases in ured and weights were estimated using length–weight which closely related species or populations have regressions [Kohler et al. (1995) for A. vulpinus and A. superci- recently diverged in life-history characteristics liosus; Liu et al. (1999) and White (2007) for A. pelagicus]. and habitat. Because gill samples were obtained opportunistically from various sources, tissue extraction and preservation method var- The thresher sharks (Alopiidae) comprise a sin- ied. Table 1 shows shark size and collection location data as gle genus containing three species, which, based well as the treatment method of each gill sample. These treat- on their life-history characteristics, should span ments were as follows: three ecological morphotypes: (1) The common A. When possible, all five gill arches were excised from both thresher, Alopias vulpinus, is a highly active spe- sides of the head immediately following euthanasia and cies (fast-swimming oceanic species) capable of ele- fixed in a 10% formalin solution buffered in seawater.

Journal of Morphology THRESHER SHARK GILL DIMENSIONS 3 TABLE 1. Thresher shark size, collection location, and gill preservation data Species Specimen FL Mass Collection Preservation (common name) # (cm) (kg) location method A. pelagicus 1 70.0 11.82 Costa Rica B (pelagic thresher) 2 91.5 21.16 Costa Rica B 3 111.0 33.61 Costa Rica D 4 135.0 51.22 Costa Rica D 5 163.0 77.77 Mexico B 6 163.0 78.16 Costa Rica B A. superciliosus 7 153.0 48.83 Southern California B (bigeye thresher) 8 161.5 57.67 Hawaii C 9 162.0 58.23 Southern California B 10 163.0 59.34 Southern California B 11 173.0 71.29 Southern California B 12 175.0 73.85 Southern California B 13 192.0 98.26 Southern California A 14 198.0 108.37 Southern California B & D 15 209.0 127.27 Southern California B A. vulpinus 16 69.0 7.91 Southern California A (common thresher) 17 76.0 10.28 Southern California B 18 82.0 12.45 Southern California B 19 87.0 14.45 Southern California A 20 104.0 22.65 Southern California C 21 116.0 29.83 Southern California A 22 146.0 53.24 Southern California A 23 157.0 63.93 Southern California A 24 181.0 91.47 Southern California A

Preservation method: A. Fixed in 10% formalin in seawater immediately following euthanasia. B. Frozen, then ﬁxed in 10% formalin. C. Perfused with microvascular casting solution. D. Frozen and ﬁxed in 25% formalin in seawater. FL 5 Fork Length.

B. When formalin was not available, gills were frozen and and Hughes (1984b) to estimate total gill surface area, A, calcu- stored until they could be fixed in 10% formalin. (All speci- lated by the equation: mens purchased from fisherman were frozen.) C. To verify that formalin fixation or freezing did not substan- A5L 32n 3A tially alter gill dimensions, gills of two sharks (#8 and 20) fil lam lam were perfused with a microvascular casting solution (Mer- (n is doubled in this equation to represent the presence of cox, Ladd Research, Williston, VT) using procedures out- lam lamellae on both sides of each gill filament). lined in Wegner et al. (2010a,b). Following euthanasia, these sharks were placed on a V-shaped cradle with their To determine total filament length, the filaments were counted ventral side up while the gills were irrigated with aerated on all five gill arches from one side of the branchial chamber. seawater. The heart was exposed by midline incision and Beginning at the dorsal margin of each hemibranch and working cannulated. Sharks were perfused with heparinized shark ventrally, the gill filaments were separated into bins of 20 (as the saline for 2–3 min, followed by microvascular casting solu- total number of filaments was rarely divisible by 20, the final bin tion. Perfusions were performed at 75–90 mmHg, which is usually contained less than 20 filaments). The medial filament of consistent with the aortic systolic pressures determined for each bin (i.e., filament number 10, 30, 50, etc.) was measured and active sharks (Lai et al., 1997). After full polymerization assumed to be representative of the mean length of a filament in (<15 min), the perfused gills were excised and later bathed its bin (each middle filament was measured from the embedded in several washes of 15% KOH solution until all gill tissue base, where filaments are partially covered by a fleshy extension was digested. Vascular casts were subsequently rinsed and of the gill arch tissue commonly called the branchial canopy, to air-dried. the tip). The length of all filaments in a bin was calculated by D. The gills of two frozen shark specimens (#3 and 4) were multiplying the length of the medial filament by the number of fil- incorrectly fixed in a 25% formalin solution. To assess the aments in the bin. Total filament length (Lfil) was determined by potential tissue shrinkage associated with the higher forma- summing all bins from the five arches and doubling this value to lin concentration, the five gill arches from one side of a account for the gills on the opposite side of the branchial chamber. third shark (#14) were fixed in 25% formalin while the gills Following determination of Lfil, each medial filament was of the other side were fixed in the normal 10%. By compar- excised and, using a dissection microscope (Zeiss, model #47 50 ing the two, the percent difference in each gill dimension 52) fitted with a digital camera (Canon Digital Rebel XT), magni- was calculated and used to correct measurements for sharks fied photographs were taken of one side of the filament base, mid- 3 and 4. dle, and tip to determine mean lamellar frequency (the number of lamellae per mm on one side of a filament) for each bin. With a scalpel, individual lamellae were then removed from each of Gill Measurement and Analysis these three locations on the filament, mounted on slides and photographed. The most complete isolated lamella from each location Total filament length, Lfil (i.e., the total length of all gill fila- was measured to determine its bilateral surface area and the ments), lamellar frequency, nlam (i.e., the average number of three measurements were then averaged to determine the mean lamellae per unit length on one side of a filament), and the bilateral surface area of a lamellae in each bin. Digital images of mean bilateral surface area of a lamella, Alam, were determined lamellar frequency and lamellar surface area were analyzed according to the procedure outlined in Muir and Hughes (1969) using NIH Image J software.

Journal of Morphology 4 T.P. WOOTTON ET AL.

To calculate the number of lamellae in each bin, the mean of After a subsequent wash of 100% tert-butyl alcohol, samples the three lamellar frequency measurements was doubled (to were frozen at 4C and freeze-dried under vacuum to sublime account for lamellae on both sides of the filament) and multi- and extract the alcohol. plied by the total filament length of that bin. The total surface Dried filaments were mounted such that lamellar cross- area in a given bin was estimated by multiplying the number sections laid perpendicular to the SEM field of view. Samples of lamellae per bin by the mean lamellar bilateral surface area were sputter coated with gold–palladium and photographed from the corresponding representative filament. Total gill sur- under the high-vacuum mode of an FEI Quanta 600 SEM (FEI face area was determined by summing the surface area of all Instruments, Hillsboro, Oregon). Twenty measurements of bins, then doubling this value to account for the gills on the lamellar thickness and the water–blood barrier distance from opposite side of the orobranchial chamber. Total gill surface each specimen were made from acquired digital images using area was then divided by the total number of lamellae in the Image J. gills to estimate average bilateral lamellar area (Alam), and the total number of lamellae was divided by the total gill filament Statistical Analysis length (Lfil) to determine the lamellar frequency (nlam) of all bins. For each species, power–law regressions were determined for For the first specimen examined from each species (#5, 10, total gill surface area (A) and each of its constituent dimensions and 22), Alam and nlam were measured on all five arches and (Lfil, nlam, Alam) versus body mass using least-squares analysis the resultant data were used to identify the gill arch for which (Figs. 1–4). For each dimension, species-specific regressions these dimensions were most representative of the entire gills. were compared using 10,000 bootstrap replications (R v.2.7.0) of For subsequent specimens, Alam and nlam were based on this the raw data (Wegner et al., 2010a). Statistical significance gill arch (fourth arch for A. vulpinus and A. pelagicus, third between two species was determined where less than 5% of the arch for A. superciliosus). resultant replicate regressions intersected over a shared weight range of the compared species. Because lamellar thickness and the water–blood barrier distance do not appear to show a dis- Lamellar Thickness and Water–Blood Barrier tinct relationship with body mass (Hughes et al., 1986, Wegner Distance et al., 2010b) species-specific means 6 standard deviation were determined for each dimension (Table 2). Species-specific meas- Ten sharks (four A. vulpinus, three A. superciliosus, three A. urements for each dimension were compared between species pelagicus) were selected, based on quality of gill preservation, using a two-level nested ANOVA (dimension measurement for analysis of lamellar thickness and water–blood barrier dis- nested within individual nested within species) and a post-hoc tance. For each shark, six regions of the representative gill Tukey test. arch (three from each hemibranch) were identified in which corresponding lamellar area and frequency best represented the mean values. From each site, a section of four gill filaments RESULTS was excised for examination with scanning electron microscopy (SEM). Gill Surface Area Fixed filament sections were rinsed in deionized water and Gill surface area to body mass regressions deter- gradually dehydrated in tert-butyl alcohol (20% increments mined in this study for the three Alopias species over 24 h). After dehydration at 80% tert-butyl alcohol, the gill filaments were cut along their long axis to provide cross sec- are shown in comparison to data for A. vulpinus tions of lamellae for measurement. While soaking in 100% tert- from Emery and Szczepanski (1986) in Figure 1. butyl alcohol, samples were kept at 32C to prevent freezing. A. superciliosus has a significantly larger gill

Fig. 1. Power–law regressions of total gill surface area (cm2) to body mass (g) for the three Alo- pias species examined in this study1 and for A. vulpinus from Emery and Szczepanski (1986)2. [Color ﬁgure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Journal of Morphology THRESHER SHARK GILL DIMENSIONS 5

Fig. 2. Power–law regressions of total filament length (cm) in relation to body mass (g) for the three Alopias speciesexaminedinthisstudy1 and for A. vulpinus from Emery and Szczepanski (1986)2. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] surface area than A. vulpinus for most of their from 0.78 to 1.03 (Fig. 1) and fall within the range shared body mass range (48.83–81.20 kg). of those determined for other fishes (Hughes, Although the gill surface area regression for A. 1972; Palzenberger and Pohla, 1992; Wegner, pelagicus lies below that of A. superciliosus and 2011). Although the scaling exponent for A. vulpi- above that of A. vulpinus, these differences are nus gill area to body mass in this study (1.03) is not statistically significant. much larger than that measured by Emery and The scaling exponents for gill surface area in Szczepanski (1986) (0.41), no significant difference relation to body mass for the three species range was evident in the gill surface areas determined in

Fig. 3. Power–law regressions of lamellar frequency (mm21) to body mass (g) for the three Alo- pias species examined in this study1 and for A. vulpinus from Emery and Szczepanski (1986)2. [Color ﬁgure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Journal of Morphology 6 T.P. WOOTTON ET AL.

Fig. 4. Power–law regressions of mean lamellar bilateral surface area (mm2)tobodymass(g) for the three Alopias species examined in this study1 and for A. vulpinus from Emery and Szcze- panski (1986)2. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] the two studies over the shared body mass range. cies are shown in Figure 2. Total filament length The low scaling exponent of A. vulpinus in Emery in A. superciliosus is significantly greater than in and Szczepanski (1986) lies outside of the range the other Alopias species and contributes to the documented for most fishes (Palzenberger and larger gill surface area of A. superciliosus in com- Pohla, 1992) and likely results from the limited parison to A. vulpinus. High total filament length body size range sampled. The 95% confidence in A. superciliosus results from significantly longer intervals of the 1.03 scaling exponent of A. vulpi- filaments and significantly more filaments than in nus in this study (0.833–1.23) are above the A. vulpinus and A. pelagicus. A. pelagicus has a expected scaling exponent of gill surface area to significantly longer total filament length than A. body mass assuming isometric growth of the gills vulpinus over most overlapping body masses [0.67: the sum of the scaling exponents of its con- (11.82–64.90 kg). This results from more filaments stituent dimensions, total gill filament length in A. pelagicus; the mean filament length does not (0.33), lamellar frequency (20.33), and lamellar differ significantly between these species. area (0.67)]. The relationship between lamellar frequency Log–log plots for total gill filament length in and body mass are plotted in Figure 3. A. vulpinus relation to body mass for the three thresher spe- has a significantly lower lamellar frequency than

TABLE 2. Lamellar dimensions (means 6 standard deviation) of the three thresher shark species Fork length Mass Lamellar Water–blood barrier Species (cm) (kg) thickness (mm) thickness (mm) A. pelagicus 70.0 11.82 12.22 6 1.47 1.53 6 0.36 A. pelagicus 91.5 21.16 13.12 6 1.44 1.47 6 0.23 A. pelagicus 163.0 77.77 12.18 6 1.28 1.83 6 0.52 X 12.51 6 0.53 1.61 6 0.19 A. superciliosus 163.0 59.34 11.49 6 0.97 1.54 6 0.27 A. superciliosus 173.0 71.29 15.44 6 1.29 1.69 6 0.39 A. superciliosus 192.0 98.26 10.56 6 1.14 1.57 6 0.27 X 12.50 6 2.59 1.60 6 0.08 A. vulpinus 87.0 14.45 13.56 6 1.61 2.25 6 0.37 A. vulpinus 116.0 29.83 15.67 6 1.22 2.86 6 0.51 A. vulpinus 146.0 53.24 13.87 6 1.27 2.67 6 0.52 A. vulpinus 181.0 91.47 14.06 6 1.48 2.41 6 0.56 X 14.29 6 0.94 2.55 6 0.27*

*Signiﬁcantly different from A. pelagicus and A. superciliosus

Journal of Morphology THRESHER SHARK GILL DIMENSIONS 7 both A. superciliosus (48.83–83.15 kg) and A. pela- those of pelagic carcharhiniform sharks (which gicus (entire comparable mass range). Lamellar have gill surface areas consistent with fishes of frequency does not differ significantly between A. intermediate activity), such as the blue shark, superciliosus and A. pelagicus. The scaling rela- Prionace glauca, and less-active benthic elasmo- tionships of mean lamellar bilateral surface area branch species (Fig. 5). Although the water–blood to body mass plotted in Figure 4 do not differ sig- barrier distances in Alopias (range: 1.60–2.55 mm) nificantly between the three species. are not as short as those of the shortfin mako (1.15 mm), they are similar to the blue shark (1.65 Lamellar Thickness and Water–Blood Barrier mm; Wegner, 2010b) and are much less than those Distance of less-active species (Scyliorhinus, Squalus, The lamellar thickness and water–blood barrier Galeorhinus, and Raja; range: 4.85–11.27 mm; distance measured for four A. vulpinus, three A. Hughes and Wright, 1970). Finally, Alopias lamel- superciliosus, and three A. pelagicus, as well as lae are also relatively thin (12.50–14.29 mm) the mean for each species are given in Table 2. resulting in narrow vascular channels that likely Lamellar thickness did not differ significantly force red blood cells close to the water–blood between species. However, A. vulpinus has a sig- barrier. nificantly thicker water–blood barrier (2.55 6 0.27 Like lamnid sharks, the large gill surface areas mm) than both A. superciliosus (1.60 6 0.08 mm) in Alopias result from high total filament lengths and A. pelagicus (1.61 6 0.19 mm; P < 0.0001). and large lamellae. This morphometric configura- tion adheres to the model proposed by Hughes (1966) for augmenting gill surface area while mini- DISCUSSION mizing increases in gill resistance to ventilatory This study shows that all three Alopias species flow. In contrast to highly active teleosts such as possess respiratory specializations for increased tunas and billfishes that couple high total filament gas exchange, including large gill surface areas lengths with high lamellar frequencies to increase and short diffusion distances. Alopias gill surface gill area (Muir and Hughes, 1969; Wegner et al., areas are among the highest measured for any 2010a), lamellar frequencies in Alopias and lam- elasmobranch group, rivaling those of the region- nids are not greater than those of less-active elas- ally endothermic lamnid sharks (e.g., shortfin mobranchs. This likely reflects the presence of mako, Isurus oxyrinchus, and white shark, Carch- interbranchial septa in elasmobranch gills that arodon carcharias) and are greater than both inherently increase gill resistance as water is

Fig. 5. Power–law regressions of total gill surface area (cm2) to body mass (g) for the three Alopias species examined in this study alongside regressions for other elasmobranchs. Sources: Present study,a Emery and Szczepanski (1986),b Wegner et al. (2010b),c Grim et al. (2012),d Boylan and Lockwood (1962),e Hughes and Morgan (1973),f Hughes (1972),g Hughes et al. (1986),h and Hughes (1978)i. [Color ﬁgure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Journal of Morphology 8 T.P. WOOTTON ET AL. forced through septal canals (Wegner et al., 2012). musculature and result in the distinct “helmeted Because having a higher lamellar frequency would contour” which lines the dorsal surface of the head further increase gill resistance, the presence of (Fig. 6; Compagno, 2001; Smith et al., 2008). interbranchial septa in elasmobranchs, likely These nuchal grooves are commonly used to iden- causes alopiids and lamnids to augment gill sur- tify A. superciliosus and are exaggerated by the face area differently than high-performance large size of its branchial chambers relative to A. teleosts. vulpinus (Fig. 6) and other sharks. Augmentation Due to the general lack of concordance in the of gill surface area through extension of total fila- phylogentic relationship of Alopiidae and Lamni- ment length is usually limited by the volume of dae within the Lamniformes (Naylor et al., 1997, the branchial chambers. However, in A. supercilio- 2012; Shimada, 2005) and the paucity of informa- sus, it appears the branchial cavities have become tion available on the gills of other members of the enlarged to house longer filaments that ultimately group, it is difficult to assess if lamnids and alo- increase gill surface area at the cost of cranial piids separately evolved large gill surface areas as streamlining. This reduced streamlining may not a result of high activity or if the common ancestor have much impact on A. superciliosus, which to these, and some other Lamniform groups (the appears to feed heavily on the deep scattering various proposed phylogenies do not consider the layer (Preti et al., 2008), where prey are likely rel- alopiids and lamnids as sister groups) had large atively slow-moving at depth in the OMZ (Seibel gills. Given the general plasticity of gill morphol- and Drazen, 2007). ogy (Chapman, 2007; Wegner, 2011) and the less- For the more surface-oriented A. vulpinus and A. streamlined body forms (and presumably lower pelagicus, which have to catch faster and more activity levels) of the other Lamniform groups, it active prey, streamlining is likely much more is probable that the large gill areas of the two important. Correspondingly, the total filament groups are independently derived. However, addi- length in both A. vulpinus and A. pelagicus is sig- tional studies on the gills of other Lamniform spe- nificantly lower than that of A. superciliosus, and cies are needed to further address such for A. vulpinus this results in a significantly phylogenetic questions. smaller gill surface area. Despite a smaller gill area than A. superciliosus, A. vulpinus has gills that are larger than those of most elasmobranchs Alopias Interspecific Comparisons and are comparable to other regionally endother- A. superciliosus has the largest gill surface area mic sharks (Fig. 5). Although its diffusion distan- and one of the highest total filament lengths docu- ces are also short in comparison to less active mented for any elasmobranch studied to date. This elasmobranchs (Hughes and Wright, 1970), A. vul- is likely associated with its ability to tolerate pro- pinus has a thicker water–blood barrier (2.55 6 longed exposure to hypoxia. In many parts of its 0.27 vs. 1.15 6 0.22 mm) and thicker lamellae range, including the collection sites in this study, (14.29 6 0.94 vs. 11.38 6 1.61 mm) than the shortfin the daytime depth preference of A. superciliosus is mako (Wegner et al., 2010b). Bernal and Sepulveda 200–500 m (Nakano et al., 2003; Weng and Block, (2005) showed that while A. vulpinus is capable of 2004; Musyl et al., 2011), which coincides with a red muscle endothermy, it does not appear able to midwater OMZ where dissolved oxygen can be increase the temperature of the eye and brain 21 below 1.5 ml O2 l (<30% saturation; Garcia region nor the viscera, and that the elevation of et al., 2010). A. superciliosus is thus similar to red muscle temperature above ambient is less than other OMZ organisms in having a larger respira- that of the mako and other lamnids. Patterson tory surface area in comparison to closely related et al. (2011) further showed that A. vulpinus pos- species living in normoxic waters (Yang et al., sesses smaller, less complex vascular retia perfus- 1992; Childress and Seibel, 1998; Levin, 2003; ing the red muscle than lamnids. In particular, the Wegner, 2010a). In addition, the short water-blood red muscle rete of A. vulpinus has a lower arterial– barrier distance (1.60 6 0.08 mm) and thin lamellae venous contact surface area, thus indicating a (12.50 6 2.59 mm) of A. superciliosus should facili- lower ability to transfer heat from the venous to tate O2 absorption within the OMZ and are likely arterial blood and ultimately a lower capacity for required for this species to meet its metabolic heat retention. A less well-developed capacity for demands while foraging at depth. red-muscle endothermy would result in lower met- At a mass of 50 kg, A. superciliosus has a 26% abolic demands directly via a Q10 effect as well as larger gill surface area than A. vulpinus, which a reduction in the energetic costs associated with does not frequent the OMZ (Cartamil et al., 2010, the maintenance of tissue specialization needed for 2011). This results from a 28% higher total fila- high performance (e.g., large heart, high hematoc- ment length in A. superciliosus and corresponds to rits and blood volume, increased capillary den- larger branchial chambers in this species (Fig. 6). sities, and high enzyme activities; Dickson, 1995; The laterally expanded branchial chambers of A. Bernal et al., 2001). The longer diffusion distances superciliosus extend dorsally to meet the epaxial in the gills of A. vulpinus in comparison to lamnids

Journal of Morphology THRESHER SHARK GILL DIMENSIONS 9

Fig. 6. Lateral- and dorsal-view renderings of the cranial region of A. superciliosus (top) and A. vulpinus (bottom) showing the relative size of the branchial chambers. Also shown are comparative images of the gill arches (left) and scanning electron micrograph cross-sections through the lamellae (right) for the two species. Excised gill arches are from a 59.34 kg A. superciliosus and 63.93 kg A. vulpinus; lamellar cross-sections are from a 71.29 kg A. superciliosus and 14.45 kg A. vulpinus.[Colorfigurecanbeviewedinthe online issue, which is available at wileyonlinelibrary.com.] may thus correspond to lower metabolic require- exponent is closer to 0.80, which is thought to cor- ments associated with a reduced capacity for relate with the mean regression coefficient for fish regional endothermy. standard metabolic rate with body mass (50.81) Despite a generally lower capacity for regional (Palzenberger and Pohla, 1992; Wegner, 2011). endothermy, A. vulpinus has a higher scaling The high scaling exponent of A. vulpinus gill area exponent for gill surface area in relation to body with body mass suggests that its metabolic mass (1.03) than most other fishes, and this may demands may increase disproportionately with be related to an increased ability for heat reten- size, possibly associated with an increased tion and aerobic performance with size. Isometric capacity for endothermy. As these sharks grow, scaling of the gills predicts a geometric regression the body’s surface-area-to-volume ratio decreases coefficient of 0.67 for gill area to body mass (area/ and the medial red muscle becomes more insulated volume); however, for most fishes, this scaling from ambient water, likely reducing the fraction of

Journal of Morphology 10 T.P. WOOTTON ET AL. metabolic heat lost by convection across the sur- nus and A. pelagicus have relatively large gill face of the body. areas, their gill dimensions and ultimately meta- Despite the high metabolic costs presumably bolic scope appear constrained by the same associated with regional endothermy in A. vulpi- streamlining that allows for their fast and contin- nus, this species does not have a significantly uous swimming. Thus, with fewer morphological greater gill surface area than that of A. pelagicus, constraints, hypoxia-dwelling fishes may, in gen- which lacks the ability to retain body heat and eral, be able to achieve greater gill diffusion was thus hypothesized to have smaller gills. capacities than more active species that, by defi- Because A. pelagicus is a tropical species that nition, require cranial streamlining. inhabits warm waters (Compagno, 2001; Smith This study also emphasizes the effect of temper- et al., 2008), much of its body is likely warmer ature on metabolism and gill morphology. than that of A. vulpinus which is found in cooler, Although A. pelagicus was expected to have lower temperate waters (Hanan et al., 1993; Compagno, metabolic demands at a given temperature than 2001; Smith et al., 2008) and only regionally A. vulpinus, the warm temperature of its environ- warms the red muscle. Thus the aerobic require- ment (which increases its oxygen demands and ments (and performance) of A. pelagicus may decreases environmental oxygen availability) cor- equal or exceed that of A. vulpinus, resulting in relates with gill dimensions comparable to those of comparable gill areas. The shorter water–blood temperate dwelling, regionally endothermic barrier distance of A. pelagicus in comparison to sharks. Thus, in general, the diffusion capacity of A. vulpinus may be required to meet similar meta- the gills in warm-water fishes is likely to exceed bolic demands in warm tropical water which, due that of cold-water fishes of similar activity level. to a reduced solubility coefficient at warmer temperatures, has less oxygen. ACKNOWLEDGMENTS Alopias Species Relationships and The authors thank S. Aalbers, D. Bernal, D. Car- Hypotheses On Gill Evolution tamil, J.B. Graham, D. Kacev, C. McCue, E. Kis- The phylogenetic relationship of the three faludy, J. Patterson, and collaborators at the thresher shark species within the genus Alopias Pfleger Institute of Environmental Research. They remains unresolved (Naylor et al., 1997; Shimada, also thank A. Preti, J. Wraith, and two anonymous 2005; Naylor et al., 2012). The results of this study reviewers for their useful critiques of this manu- lead us to hypothesize that the Alopias ancestor script. This work was supported by NSF grant IOS- was likely an active, ectothermic, pelagic, warm- 0817774, the Pfleger Institute of Environmental water shark that had a large gill surface area and Research, and the George T. Pfleger Foundation. short diffusion distances consistent with traits exhibited by A. pelagicus. The subsequent evolu- LIETERATURE CITED tion of regional endothermy in A. vulpinus (possibly as a result of its range expansion into colder Bernal D, Sepulveda CA. 2005. 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Morpho-physiological divergence across relate with both oxygen demand and availability. aquatic oxygen gradients in fishes. In: Fernandes, MN, Ran- In this case, exploitation of a hypoxic environ- tin, FT, Mogens, LG, editors. Fish Respiration and Environ- ment. Enfield: Science Publishers. pp 13–39. ment by A. superciliosus correlates with the larg- Childress JJ, Seibel BA. 1998. Life at stable low oxygen levels: est gill surface area of any elasmobranch Adaptations of animals to oceanic oxygen minimum layers. reported to date. While the more active A. vulpi- J Exp Biol 201:1223–1232.

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