Is the Megamouth Shark Susceptible To

RECORDS OF THE WESTERN AUSTRALIAN MUSEUM 27 007–020 (2012)

Is the Megamouth Shark susceptible to mega-distortion? Investigating the effects of twenty-two years of ﬁ xation and preservation on a large specimen of Megachasma pelagios (Chondrichthyes: Megachasmidae) Brett A. Human1,3, Susan M. Morrison1 and Ian D. MacLeod2

1 Department of Aquatic Zoology, Western Australian Museum, Locked Bag 49, Welshpool DC, Western Australia 6986, Australia.

2 Western Australian Maritime Museum, Victoria Quay, Fremantle, Western Australia, 6160, Australia.

3 Deceased 2011. Corresponding author: Sue Morrison. Email: [email protected]

ABSTRACT – On 18 August 1988, the third reported Megamouth Shark (Megachasma pelagios Taylor, Compagno and Struhsaker, 1983) became stranded on a beach in Western Australia. The 5.15 m TL male shark (WAM P.29940–001) was preserved at the Western Australian Museum (WAM) and put on public display. Many deep water chondrichthyan species, including M. pelagios, possess soft and fl abby muscle tissue and a poorly calcifi ed skeleton. Such species are prone to distortion through dehydration due to the fi xation and preservation process. Such distortions may result in taxonomic and nomenclatural issues. The WAM Megamouth Shark was recently relocated, providing an opportunity to remeasure the specimen to allow a comparison with measurements taken of the specimen when it was fresh, and to assess the extent of change to these measurements after 22 years of preservation. We found that with the exception of the pelvic-fi n, that we hypothesise is the fl eshiest part of M. pelagios with the least skeletal support, the specimen has suffered little to no distortion over this time. However, there has been some degradation of teeth and denticles that is most likely due to the acidity of the formalin solution.

KEYWORDS: megamouth, long-term preservation, dimensional changes, ethanol preservation, formaldehyde ﬁ xed.

INTRODUCTION (WAM P.29940–001) was transported to Perth, then The Megamouth Shark, Megachasma pelagios frozen and put on brief display before being preserved. Taylor, Compagno and Struhsaker, 1983, is a highly Over six years later on 25 January 1995 it was placed distinctive species with a patchy circumtropical and on permanent public display at the Perth site of the warm temperate distribution. Records and sightings of Western Australian Museum (WAM), in a below-ground this enigmatic shark were few in the ﬁ rst three decades polyester resin glass-topped case, with the upper glass after the species was ﬁ rst discovered in 1976, and it was surface of the case level with the ground (Figure 1). known from only 14 specimens at the turn of the century Recent renovations at the Perth site necessitated the (Compagno 2001; Burgess 2010). However, since 2001 relocation of the Megamouth Shark specimen. The an additional 36 specimens have been recorded, with shark was removed from the below-ground case and the 50th specimen reported in June 2010 (Burgess relocated to the Western Australian Maritime Museum 2010). Many recent specimens have been retained and in Fremantle, approximately 20 km away. There it are reported as being prepared for public display and/or was placed in a custom made stainless steel tank with deposition in an ichthyological collection. viewing portholes, for continued public display. On 18 August 1988, the third recorded Megamouth The Megamouth Shark possesses soft and flabby Shark specimen was found stranded on a beach 50 km muscle tissue and its skeleton is poorly calcified south of Perth, Western Australia (Berra and Hutchins (Compagno 1988, 1990; Smale et al. 2002; Taylor et 1990, 1991; Hutchins 1992; Last and Stevens 1994, al. 1983; BAH personal observation of dissection of 2009; MacLeod 2008). The 5.15 m TL male shark Megamouth #17), and similar observations were noted 8 BRETT A. HUMAN, SUSAN M. MORRISON AND IAN D. MACLEOD

FIGURE 1 Photo of Megamouth #3 (WAM P.29940–001) on public display between 25 January 1995 to 22 September 2010, in ethanol solution in the glass-topped tank at Western Australian Museum (Perth). Image: Western Australian Museum. by Berra and Hutchins (1991) during preservation of and Kishida 1984; Stehmann and Shcherbachev 1995) and Megamouth #3. It is a well known phenomenon that all of which are currently recognised as junior synonyms such chondrichthyan species are prone to distortion of H. bickelli (Compagno et al. 2005; Eschmeyer 2010; as an artefact of fi xation and preservation (Jones and P.C. Heemstra personal communication). Geen 1977; and see below). The distortion observed Although taxonomic issues of the kind illustrated in some taxa has led to nomenclatural and taxonomic above are unlikely to apply to such a distinctive species issues, resulting in junior synonyms arising for an as the Megamouth Shark, the effects of fi xation and already available and valid species name. An example preservation on the WAM specimen are a potential issue illustrating this issue is Hexatrygon bickelli Heemstra that warrant further investigation. The preservation and and Smith, 1980, a distinctive six-gilled stingray fi xation of specimens of Megachasma pelagios, besides originally described from South Africa. The species the WAM specimen, have only been documented with uses its long, fl exible snout to probe sediments for any detail for a limited number of specimens (Taylor prey (Hennemann 2001; Last and Stevens 2009; L.J.V. et al. 1983; Takada et al. 1997), and to the best of our Compagno personal communication). The snout lacks a knowledge, there have been no reports on potential rostral cartilage and is fi lled with an acellular gelatinous morphological distortions resulting from fi xation and matrix (Heemstra and Smith 1980). Further Hexatrygon preservation of M. pelagios. At the time of this study, specimens were collected from the South China Sea the WAM Megamouth Shark specimen had been fi xed (Zhu et al. 1981, 1982), Taiwan (Shen and Lui 1984; in formalin for nearly 6½ years and subsequently in Shen, 1986a, b), Japan (Ishihara and Kishida 1984), alcohol preservative for 15¾ years, giving a total time Indonesia (Stehmann and Shcherbachev 1995), and of 22¼ years since fi rst fi xation. Berra and Hutchins Australia (Last and Stevens 1994, 2009). The highly (1990) provided a comprehensive list of morphometric variable shape of the snout is to some extent a result of measurements for this specimen prior to fi xation. The ontogeny and individual variation, but desiccation of purpose of this study is to compare those measurements the gelatinous matrix through fi xation and preservation with new ones taken by the authors during the relocation has a distorting effect that led to four new species being of the Megamouth Shark and to determine the effects, if described, all on the basis of snout morphology (Ishihara any, of more than 20 years of preservation. MEGAMOUTH DIMENSIONS AFTER 22 YEARS FIXATION 9

MATERIALS AND METHODS Forty litres of approximately 50% formalin was pumped into the body and body cavity using a 1300 mm x 10 PRESERVATION AND PUBLIC DISPLAY mm needle. The pit was drained and clean fresh water Detailed accounts of the fi xation and preservation of was added to cover the specimen by approximately the specimen are provided by Berra and Hutchins (1990, 30 cm. The water volume in the pit was estimated and 1991), Hutchins (1992) and MacLeod (2008), and are full strength formalin (40% formaldehyde in solution) summarised here. Details of the history of preservation was added. The formalin strength was tested on 21 and public display since 1992, including the recent move, October 1988 at 8.25% formalin. On 13 January 1989, are also provided to give a complete history of treatment the formalin concentration had dropped to 4.25% and additional full strength formalin was added to produce and movement of the specimen. The shark was snap a fi nal concentration of 11.75% formalin (Berra and frozen on the afternoon of its discovery on 18 August Hutchins 1990). Owing to concerns regarding residual 1988. According to bystanders it had died on the beach amounts of formaldehyde in large sharks leaching that morning. On 21 August 1988, three days after its into ethanol-based storage solutions, Megamouth #3 discovery, the frozen shark was briefl y put on public was washed in fresh water. Experiments with a small display for three hours under shade and then returned to shark (Hemigaleus australiensis White, Last and the freezer. On 15 September 1988, a month after fi rst Compagno 2005, WAM P.26190–002) had established capture, a pit lined with plastic swimming pool liner that the release of formaldehyde was linearly related was fi lled with water and the specimen was placed in it, to the logarithm of the washing time (MacLeod 2008). after being weighed at a public weighing station while Megamouth #3 was immersed in tap water for 41 frozen (690±20 kg; Berra & Hutchins 1990), and allowed days from 30 November 1994 to 3 January 1995, and to thaw over a weekend. About half of the water was removed from the temporary preservation pit on 25 removed from the pit, and Berra and Hutchins took the January 1995, at which time the formalin concentration morphometric measurements (as per Compagno 1984; was measured at 0.032% (MacLeod 2008). The liver and Table 1). intestines were also removed at this time, to minimise The body musculature was injected on 19 September the amount of liver oil leaking from the specimen and 1988 with 20 l of approximately 30% formalin using detracting from the display (MacLeod 2008). The shark 130 mm x 2 mm needles attached to 20 ml syringes. was then placed into a custom built glass-topped fi bre

FIGURE 2 Lifting Megamouth #3 from the truck in the custom stretcher outside the Western Australian Maritime Museum, Fremantle, Western Australia (22 September 2010). Image: B.A. Human. 10 BRETT A. HUMAN, SUSAN M. MORRISON AND IAN D. MACLEOD c Holotype TL)(% b M 22730) are provided for for M 22730) are provided – compared with original TL TL – compared with original d -1.2 difference d Current study Percentage a WAM P.29940.001) taken when fresh (original) and after 22 years of of 22 years taken when fresh (original) and after P.29940.001) WAM from Taylor, Compagno and Struhsaker (1983), (1983), Compagno and Struhsaker Taylor, from c Megachasma pelagios Megachasma Current study Original % current study-% original study, original study, study-% % current b a Actual measurement (mm) Proportional measurement (%TL) from Berra and Hutchins (1990), (1990), and Hutchins from Berra a n length n 2125 41.7 fi n space n 910 17.9 fi n space n 430 435 8.3 8.5 0.2 8.9 n space n 715 610 13.9 12.0 -1.9 n length n 2720 2700 52.8 53.0 0.2 56.7 fi fi fi n space n 370 360 7.2 7.1 -0.1 7.4 fi n length n 1670 1680 32.4 33.0 0.6 34.5 n–caudal- fi fi n length n 2450 2510 47.6 49.3 1.7 48.5 fi n length n 1390 1340 27.0 26.3 -0.7 24.9 n space n 640 650 12.4 12.8 0.4 13.2 preservation (current study), and percentage difference between the two studies. Proportional measurements of the holotype Proportional (BPB between the two studies. and percentagepreservation difference study), (current comparison. Morphometric and proportional measurements of Megamouth #3 ( measurement. fi fi n length n 2510 2520 48.7 49.5 0.8 50.9 fi

n–pelvic-

fi n length n 3009 59.1 63.5 fi n–caudal- n–caudal- n–anal- fi fi fi rst dorsal- rst fi Pelvic- Pelvic- Vent–caudal- Prepelvic- Prepectoral- Snout–vent length lengthTrunk Pectoral- 2645 1235 52.0 24.3 51.5 Preanal- Dorsal- Second dorsal- Morphometric length (TL)Total Original 5150 5090 100.0 98.8 Fork lengthPrecaudal lengthPre-second dorsal- Pre- Body lengthInterdorsal- 3430 3380 3890 66.6 2015 66.4 76.4 -0.2 39.6 69.3 TABLE 1 TABLE MEGAMOUTH DIMENSIONS AFTER 22 YEARS FIXATION 11 c Holotype TL)(% b difference Current study Percentage a Current study Original a Actual measurement (mm) Proportional measurement (%TL) n space n 215 150 4.2 2.9 -1.3 5.2 fi n–caudal- fi Head height 2Head width 2Interorbital spaceHead height heightTrunk widthTrunk GirthAbdomen heightAbdomen width heightTail 550 widthTail Caudal peduncle heightCaudal peduncle width 480Head length 530 630Prebranchial length 640Prespiracular length 1790 1590Preorbital length 645Eye length 280 10.7 420 350Eye height 635Spiracle lengthEye-spiracle length 250 300 1090 10.4 9.4 450 1320 935 12.4 12.4 110 230 350 34.8 31.2 1085 1290 5.4 12.7 60 8.2 -0.3 12.5 6.9 40 425 21.2 4.9 25.6 5.9 8.8 62 50 18.2 0.1 10 260 2.2 6.8 4.5 21.3 25.3 0.5 14.3 1.2 0.6 0.8 8.3 0.1 -0.3 40.4 5.3 9.9 1.2 1.0 5.1 0.2 19.1 1.5 2.4 26.5 0 5.4 10.1 0.2 1.3 1.2 3.9 0.1 MorphometricAnal- Original 12 BRETT A. HUMAN, SUSAN M. MORRISON AND IAN D. MACLEOD c Holotype TL)(% b difference Current study Percentage a Current study Original a Actual measurement (mm) Proportional measurement (%TL) ap length ap 7 10 0.1 0.2 0.1 fl n length n 535 10.5 fi n length n n anterior marginbase n height n n inner margin n posterior margin n radial length 990 330 960 870 460 260 855 230 19.2 650 690 6.4 18.9 16.9 9.0 5.1 16.8 -0.3 4.5 12.8 13.6 18.8 -1.3 -0.1 10.2 5.9 4.3 13.8 fi fi fi fi fi fi fi Morphometric lengthIntergill First gill slit heightSecond gill slit heightThird gill slit heightFourth gill slit heightFifth gill slit heightPreoral length Original Prenarial lengthMouth length 320 220 225Mouth widthNostril width 225 210Internarial space 260 215Outer internarial width 200 225Anterior nasal 230 60 220 105 215 6.2 450 4.3 4.4 580 43.5 4.4 30 155 400 4.1 5.1 570 4.2 3.9 4.4 630 4.5 32 410 1.2 4.3 455 2.0 4.2 8.7 -1.1 -0.1 11.3 0 0.9 3.0 7.8 0.6 0.1 0.2 11.2 5.9 12.4 0.3 5.8 5.9 8.1 0.6 5.7 -0.3 8.9 1 5.2 2.5 1.1 1.5 2.2 0.3 0 6.1 18.5 7.6 0.7 Pectoral- Pectoral- Pectoral- Pectoral- Pectoral- Pectoral- Pectoral- First dorsal- MEGAMOUTH DIMENSIONS AFTER 22 YEARS FIXATION 13 c Holotype TL)(% b difference Current study Percentage a Current study Original a Actual measurement (mm) Proportional measurement (%TL) n length n n anterior marginbase n height n lengthn inner margin lengthn posterior margin 240 75 155 255 200 100 285 75 150 210 100 4.7 3.0 1.5 5.0 3.9 1.9 5.6 2.9 1.5 4.1 2.0 -0.8 -0.1 0 -0.9 4.4 0.1 3.5 4.3 1.8 2.3 fi fi fi fi fi fi n anterior marginbase n height n lengthn inner margin lengthn posterior margin 280 80 295 500 435 250 70 285 460 250 5.4 5.7 1.6 9.7 4.9 8.5 5.6 1.4 9.0 4.9 3.1 -0.1 -0.2 -0.7 9.3 0 5.9 1.8 9.1 5.1 fi fi fi fi fi n length n lengthn anterior margin base n height n lengthn inner margin lengthn posterior margin 330 35 320 195 320 185 340 90 185 220 6.4 250 6.3 3.8 0.7 6.2 3.6 6.7 3.6 1.8 4.3 4.9 -0.1 5.9 -0.2 1.1 -1.9 1.3 4.1 5.5 0.9 4.6 5.7 fi fi fi fi fi fi n anterior margin 155 150 3.0 2.9 -0.1 4.4 n length n 145 140 2.8 2.8 0 fi fi Anal- MorphometricFirst dorsal- First dorsal- First dorsal- First dorsal- First dorsal- Pelvic- Original Second dorsal- Second dorsal- Second dorsal- Second dorsal- Second dorsal- Anal- Pelvic- Pelvic- Pelvic- Pelvic- Pelvic- Clasper outer lengthClasper inner lengthClasper base widthSecond dorsal- 360 560 70 370 560 60 7.0 10.9 1.4 7.3 11.0 1.2 0.3 0.1 0.2 8.0 12.3 1.1 14 BRETT A. HUMAN, SUSAN M. MORRISON AND IAN D. MACLEOD c Holotype TL)(% b difference Current study Percentage a Current study Original a Actual measurement (mm) Proportional measurement (%TL) n origin n 415 0 8.1 0 -8.1 n insertion n 270 5.3 fi fi n insertion n 230 4.5 n origin n 655 12.9 fi n insertion n 615 12.1 fi fi n origin n 430 8.4 fi n margin n 430 395 8.3 7.8 -0.5 8.5 n margin n 1210 1190 23.5 23.4 -0.1 27.4 fi fi rst dorsal- rst n length n 70 1.4 n margin n 85 85 1.7 1.7 0 1.3 fi fi fi n margin n 720 690 14.0 13.6 -0.4 14.0 fi n margin n n lobe length 105 85 85 2.0 1.7 1.7 -0.3 2.2 3.1 fi fi n insertion–anal- n n origin–anal- n n margin n 1730 1655 33.6 32.5 -1.1 32.4 fi fi fi n midpoint–pectoral- n midpoint–pelvic- n fi fi n fork width n fork length 450 480 410 460 8.7 9.3 8.1 9.0 -0.6 -0.3 10.6 6.1 fi fi n midpoint– n n midpoint–second dorsal- fi fi lengthn posterior margin 85 70 1.7 1.4 -0.3 1.8 lengthn inner margin 65 55 1.3 1.1 -0.2 1.5 n height n 80 60 1.6 1.2 -0.4 1.7 n base n 80 90 1.6 1.8 0.2 3.6 fi fi fi fi Second dorsal- Terminal caudal- Terminal caudal- Terminal First dorsal- First dorsal- Pelvic- Pelvic- Second dorsal- Subterminal caudal- Lower postventral caudal- Caudal- Caudal- Subterminal caudal- Upper postventral caudal- Dorsal caudal- Preventral caudal- Anal- Anal- Anal- MorphometricAnal- Original MEGAMOUTH DIMENSIONS AFTER 22 YEARS FIXATION 15

FIGURE 3 Megamouth #3 about to be lifted off the stretcher and placed into the custom made stainless steel tank (22 September 2010). Image: B.A. Human.

reinforced polyester tank (Figure 1), which was fi lled specimen was covered in water soaked coarse woven with carbon (to remove coloured components) and a hessian for the relocation. The journey from the Perth cotton wool pre-fi lter (to remove oil droplets). However, site to the Fremantle site took approximately 1 hour. the pump system became inoperable after four years Megamouth #3 was removed from the truck and brought (MacLeod 2008). The specimen was on display in an inside for inspection, measurements, and placement into undercover, open sided gazebo from 25 January 1995 the new tank that had been partially fi lled with a 30% until the recent move. glycerol solution (Figure 2). It was while the tank was being fi lled and the shark was on the stretcher that the On 22 September 2010, the glass top of the tank majority of morphometric measurements used in this was cut open in order to remove Megamouth #3 for study were collected (Figure 3). A few measurements relocation. The tank was drained and the shark was could not be collected (mostly of the head) because placed on a purpose built metal-framed stretcher with of the way the specimen was lying on the stretcher. A canvas slings. A crane was used to extract Megamouth number of other measurements/samples were taken #3 from the tank and place it on a fl at-bed truck, and the at this time by other researchers, including skin 16 BRETT A. HUMAN, SUSAN M. MORRISON AND IAN D. MACLEOD

FIGURE 4 Overhead view (with lids removed) of the newly constructed stainless steel tank for public viewing of Megamouth #3 at the WA Maritime Museum. This photo was taken shortly after the specimen had been lowered into the glycerol solution (22 September 2010). Image: B.A. Human.

FIGURE 5 View of the head of Megamouth #3 through one of the portholes (details as for fi gure 4). Image: B.A. Human. MEGAMOUTH DIMENSIONS AFTER 22 YEARS FIXATION 17 chromametric readings, denticle and tooth samples, and TEETH AND DENTICLES lateral line and electrosensory pore mapping (Kempster Scanning electron micrographs of the teeth and 2011; MacLeod unpublished data). The data collection denticles were taken using a back-scattered secondary and documentation took approximately one hour. electron image mode in a Phillips XL40 Controlled Megamouth #3 was then placed into a custom built Pressure cell. The samples were cut with a scalpel and 304 stainless steel (high grade) tank 560 cm L x 188 placed directly into the SEM chamber and pumped cm H x 183 cm W (maximum width in the middle of down with no coating applied to the tissues. the tank) with 50 cm diameter portholes and a fi ltration pump, containing a 30% by volume glycerol solution, MORPHOMETRICS for above ground viewing, and which also provided The morphometrics were taken according to good access for research purposes (Figures 4–5). A Compagno (1984, 2001) and Human (2006). Berra number of slings were used to position Megamouth and Hutchins (1990) acknowledged the help of A.J. #3 in the tank to prevent it fl oating due to the density Bass during the measuring process, who is well difference between the ethanol in its tissues and the acquainted with measuring sharks (Bass 1973; Bass glycerol solution, and as a result of a large amount of air et al. 1973, 1975a, b, c, d, 1976). Likewise, the senior entering the body and oral cavities. On 30 September author has extensive experience taking morphometric 2010, the specimen had settled into the glycerol solution measurements of sharks, including previous experience and personnel entered the tank to adjust the slings. At measuring Megachasma pelagios (Human 2006, 2007a, this time the remaining morphometric measurements b; Smale et al. 2002). Morphometric measurements that could not be taken previously were recorded, from Berra and Hutchins (1990) and from the current and a number of body morphometrics remeasured to study are given in Table 1, as well as percentage (of ensure accuracy because the shark had not been lying total length) differences between the two studies. The completely fl at on the stretcher. majority of measurements were taken using a standard tape measure, and the smallest dimensions measured The use of a glycerol solution as a preservative is not with digital calipers. The crane hoist was used to a standard curatorial procedure, but was chosen due to measure the weight of the shark. occupational health and safety restrictions regarding ethanol and formalin solutions, and favourable results obtained during trials with glycerol as a preservative RESULTS on chondrichthyan specimens (MacLeod and van Dam 2011). On 20 December 2010, approximately 2000 l of TEETH AND DENTICLES glycerol solution was removed from the Megamouth Scanning electron micrographs show the degradation #3 display tank and 100% glycerol was added to of a patch of denticles (Figure 6) that is most probably the remaining solution to increase the concentration due to prolonged exposure to the acidic formalin of glycerol to 45%, in accordance with a plan to solution. Figure 7 shows the upper section of a tooth incrementally increase the glycerol concentration to 65% taken from Megamouth #3. Modern elasmobranchs by volume by December 2012. This not only increased (subcohort Neoselachii) possess a triple-layered the glycerol concentration, but also reduced the levels enameloid tooth morphology, with a thin outermost of ethanol and formalin that had been leaching from the shiny layered enameloid, a thick middle layer of parallel specimen (MacLeod 2008; MacLeod and van Dam 2011). bundled enameloid, and an innermost layer of tangled In October 2010, shortly after the shark was placed bundled enameloid which surrounds a dentine core in the tank, yeast and mould contamination developed (Reif 1977, 1980; Compagno 1988, 2001; Rees and Cuny in the glycerol solution, and a fi lamentous slime and 2007). It is hypothesised that the acidic nature of the black spots of mould appeared, resulting in the glycerol formalin solution has etched away the shiny layered solution becoming opaque and the clarity of the display enameloid, leaving the parallel bundled enameloid being severely compromised. The infection probably exposed, observed as parallel striations in Figure 7 (M. occurred during the placement of the hessian over Siversson, personal communication). the specimen to avoid dehydration during the journey from Perth to Fremantle. Approximately two weeks MORPHOMETRICS after the infection was noticed, a bactericide, kathon It was not possible to weigh the specimen accurately (methylisothiazolinone), was added to the glycerol in this study. The crane used to relocate the specimen solution. The fi ltration pump was initially run only after- measured the weight of the shark at 600 kg, however hours to reduce noise pollution in the public gallery, the accuracy of the crane was ±100 kg (BAH with crane and is now operating at night with the 10 micron fi lter operator, personal communication). providing clear storage solutions and as of December Most of the measurements (actual and proportional) 2011 the infection has not returned. The suspended dead corresponded well to those of Berra and Hutchins contaminants were removed from the solution through (1990), indicating that despite 22 years of preservation, successive use of 100 μm, 50 μm and 10 μm fi lters. little distortion has occurred in the specimen. The A further benefi t of running the pump at night is that difference in measurements between the two studies periodic circulation of the glycerol solutions assists in were within 1% of each other for the majority of the the even penetration of the consolidant. morphometrics (Table 1). The measurements total 18 BRETT A. HUMAN, SUSAN M. MORRISON AND IAN D. MACLEOD

FIGURE 6 Scanning electron micrograph of a patch of FIGURE 7 Scanning electron micrograph of the upper denticles taken from Megamouth #3 during section of a tooth taken from Megamouth the recent relocation, showing the damage #3 during the recent relocation, showing likely to have been caused by prolonged the damage likely to have been caused by exposure to formalin solution. Image: M. prolonged exposure to formalin solution. Verral. Image: M. Verral. length, vent-caudal–fin length, pelvic-fin–caudal-fin proportion. There were discrepancies of greater than space, and dorsal caudal-fi n margin showed a slightly 5% for the measurements of pectoral-fi n base length and greater than 1% difference between the two studies, but anal-fi n–caudal-fi n space between the current study and these are relatively long measurements and we regard Berra and Hutchins (1990), and these most likely refl ect this as acceptable when considering the likelihood of differences in measurement technique and/or error due inconsistency in measuring a specimen as large as this. to the positioning of the shark. For instance, if a specimen of this size is lying with a slight curvature, then that will translate into a signifi cant margin of error in any long body measurements. DISCUSSION The smaller measures of anal-fi n–caudal-fi n space, Megamouth #3 has suffered little distortion despite preorbital length, prenarial length, mouth length, being in formalin for over six years and in ethanol for mouth width, pectoral-fi n base, fi rst dorsal-fi n anterior nearly 16 years. In sharks that have been otherwise margin, pelvic-fi n base, pelvic-fi n height, pelvic-fi n carefully preserved, as is the case here, one could inner margin, and pelvic-fi n midpoint–second dorsal- argue that fi ns are probably the features that are most fi n origin were more variable between the two studies. vulnerable to distortion and this could be so for the Again, the differences may refl ect discrepancies in the pelvic-fi n in this study. That the other fi ns show no method of measurement rather than distortional effects, apparent signs of distortion may refl ect the fact that despite authors in both studies being highly experienced they are relatively well supported by skeletal elements. in taking morphometric measurements of sharks. Being a lamnoid shark, Megachasma pelagios possesses An exception may be the pelvic fi n, which had many plesodic pectoral fi ns, in which the distal fi n radials different morphometric measurements between the extend to the margins of the fi n web (Compagno 1988, studies which may indicate physical distortion. 1990; Taylor et al. 1983; BAH personal observation of A number of morphometrics were collected in this dissection of Megamouth #17). With the exception of study that were not recorded by Berra and Hutchins the pectoral fi n, all other fi ns of Megamouth Sharks are (1990) and are compared to the proportions recorded for relatively small, and therefore are well supported by the holotype (Taylor et al. 1983), which is included here skeletal elements and are not fl eshy. The pelvic fi n has as a reference point for these additional measurements. the least skeletal support in Megamouth Shark (BAH All of the morphometric proportions recorded for this persernal observation of dissection of Megamouth study corresponded very closely to the proportional #17), which is the most probable explanation for the measurements of the holotype and most differed much pelvic fi n showing some distortion whereas the other less than 5% between this specimen and the holotype. fi ns do not. White et al. (2004) similarly argued that The notable exception is mouth length, which was fi n dimensions for the Megamouth Shark should be measured when Megamouth #3 was already immersed in relatively invariable. glycerol and thus diffi cult to measure, therefore probably One apparent consequence of the long-term storage refl ecting error on our part. Mouth width is also much in the formaldehyde solution is that the denticles show wider in the holotype compared with the measurements signs of signifi cant chemical attack (Figure 6) and the taken both in this study and by Berra and Hutchins teeth had become etched (Figure 7), which is consistent (1990), possibly refl ecting individual variation in this with long-term storage in acidic solutions (M. Siversson MEGAMOUTH DIMENSIONS AFTER 22 YEARS FIXATION 19 personal communication). Whilst the prolonged fi xation Siversson, curator of palaeontology WAM, examined time has apparently resulted in little damage to the body the SEM images of the denticles and teeth, provided of the shark, it seems that the acidic solution has affected an analysis, and associated literature. Barry Hutchins the teeth and the denticles. provided information on the original discovery and Another consequence, most likely due to the previous curation of the specimen. Channing Egeberg assisted with display method, is that the colouration of the skin morphometric measures. Kirsten Tullis (WAM) advised has notably faded (MacLeod 2008; MacLeod and on the photographic credit of Figure 1. We thank the van Dam 2011). In the original display, sunlight fell reviewers of the paper for helpful comments. directly onto the left side of the body of the shark, until side panels were erected at either end of the display gazebo (MacLeod 2008), resulting in the skin colour REFERENCES becoming very pale (Figures 3–5). Glycerol trials on Bass, A.J. (1973). Analysis and description of variation in the ethanol preserved shark specimens by MacLeod and proportional dimensions of scyliorhinid, carcharhinid van Dam (2011) showed that the skin of those specimens and sphyrnid sharks. Investigational Report of the appeared darker following treatment. The fading in those Oceanographic Research Institute 32: 1–28. specimens, however, resulted from ethanol preservation Bass, A.J., D'Aubrey, J.D. and Kistnasamy, N. (1973). Sharks rather than the effects of bleaching by the sun. It is likely of the East Coast of Southern Africa. I. The genus that the colour changes brought about through treatment Carcharhinus (Carcharhinidae). Investigational Report of the Oceanographic Research Institute 33: 1–168. with aqueous glycerol are due to colour saturation of the surface i.e. the glycerol solutions fully wet the shark skin Bass, A.J., D'Aubrey, J.D. and Kistnasamy, N. (1975a). Sharks of the East Coast of Southern Africa. II. The families and allow its natural colour to be viewed. This appears to Scyliorhinidae and Pseudotriakidae. Investigational Report be the underlying reason why the ‘colour’ of Megamouth of the Oceanographic Research Institute 37: 1–64. #3 has been returned. Halfway through the glycerol Bass, A.J., D'Aubrey, J.D. and Kistnasamy, N. (1975b). Sharks impregnation treatment, the colour saturation of the skin of the East Coast of Southern Africa. III. The families brought about by the removal from ethanol appears to Carcharhinidae (excluding Mustelus and Carcharhinus) and have brought back some of the darker colour that had Sphrynidae. Investigational Report of the Oceanographic been bleached out. Final colour measurements, with a Research Institute 38: 1–100. chromameter, will be done at the end of the treatment Bass, A.J., D'Aubrey, J.D. and Kistnasamy, N. (1975c). program and the effectiveness of the preservative on Sharks of the East Coast of Southern Africa. IV. The restoring colour will then be assessed. families Odontaspididae, Scapanorhynchidae, The use of glycerol as a preservative for fi shes is a new Isuridae, Cetorhinidae, Alopiidae, Orectolobidae approach that will reduce occupational health and safety and Rhiniodontidae. Investigational Report of the Oceanographic Research Institute 39: 1–102. risks and hazards. A more detailed examination of the use of glycerol on formalin preserved shark specimens Bass, A.J., D'Aubrey, J.D. and Kistnasamy, N. (1975d). Sharks of the East Coast of Southern Africa. V. The families is provided by MacLeod and van Dam (2011), and Hexanchidae, Chlamydoselachidae, Heterodontidae, continued observation of the WAM Megamouth Shark Pristiophoridae and Squatinidae. Investigational Report of will determine the long term suitability of glycerol as a the Oceanographic Research Institute 43: 1–50. preservation fl uid. Some human tissues at the University Bass, A.J., D'Aubrey, J.D. and Kistnasamy, N. (1976). Sharks of Leiden Medical Museum have been preserved in of the East Coast of Southern Africa. VI. The families glycerol solution for more than 100 years and so the Oxynotidae, Squalidae, Dalatiidae and Echinorhinidae. method can be regarded as being suitable for museum Investigational Report of the Oceanographic Research specimens. Institute 45: 1–103. Despite Megachasma pelagios being a relatively Berra, T.M. and Hutchins, J.B. (1990). A specimen of fl abby-bodied shark, the current specimen shows very megamouth shark, Megachasma pelagios (Megachasmidae) little distortion from fi xation and preservation. Although from Western Australia. Records of the Western Australian the skeleton is poorly calcifi ed (Compagno 1990; Taylor Museum 14: 651–656. et al. 1983; BAH personal observation of dissection of Berra, T. M. and Hutchins, J. B. (1991). Natural history notes on Megamouth #17), it is apparently effective in preventing the megamouth shark, Megachasma pelagios, from Western Australia. Western Australian Naturalist 18: 224–233. distortion of preserved specimens, as is demonstrated by the majority of morphometric measurements remaining Burgess, G.H. (Ed.). (2010). Florida Museum of Natural History Megamouth Shark online database, http://www.fl mnh.ufl . unchanged in Megamouth #3. The only parts of the shark edu/fish/Sharks/Megamouth/mega.htm. Accessed 16th seemingly fl eshy enough to allow morphometric distortion September 2010. from fi xatives and preservatives were the pelvic fi ns. Compagno, L.J.V. (1984). FAO Species Catalogue. Vol. 4. Sharks of the World. An Annotated and Illustrated ACKNOWLEDGEMENTS Catalogue of Shark Species Known to Date. Part 1. Hexanchiformes to Lamniformes. FAO Fisheries Synopsis Thanks to Leonie Samuelsson of the WAM library for No. 125, 4(1): 1–249. organising interlibrary loans of the Hexatrygon references Compagno, L.J.V. (1988). Sharks of the Order from China and Taiwan. Michael Verral from the ARRC Carcharhiniformes. 1st Reprint (2003). The Blackburn Centre (Technology Park, Kensington, Western Australia) Press, Caldwell, New Jersey. pp.1–572. performed the scanning electron microscopy. Mikael Compagno, L.J.V. (1990). Relationships of the megamouth 20 BRETT A. HUMAN, SUSAN M. MORRISON AND IAN D. MACLEOD

shark, Megachasma pelagios (Lamniformes: glycerol solutions. Preprints of the ICOM-CC Triennial Megachasmidae), with comments on its feeding habits. Meeting, Lisbon, Portugal, September 2011, paper 0903: In: Pratt H. L., Gruber, S. H. and Taniuchi, T. (eds). 1–8. Elasmobranchs as Living Resources: Advances in the Rees, J. and Cuny, G. (2007). On the enigmatic neoselachian Biology, Ecology, Systematics, and the Status of the Agaleus dorsetensis from the European early Jurassic. GFF Fisheries. NOAA Technical Report NMFS 90: 357–379. 129: 1–6. Compagno, L.J.V. (2001). Sharks of the World. An Annotated Reif, W.E. (1977). Tooth enameloid as a taxonomic criterion: and Illustrated Catalogue of Shark Species Known to 1. A new euselachian from the Rhaetic-Liassic boundary. Date. Volume 2. Bullhead, Mackerel and Carpet sharks Neues Jahrbuch fur Geologie und Palaontologie, (Heterodontiformes, Lamniformes and Orectolobiformes). Monatshefte 1977: 565–576. FAO Species Catalogue for Fishery Purposes. No. 1, Rome. 2: 269 pp. Reif, W.E. (1980). Tooth enameloid as a taxonomic criterion: 3. A new primitive shark from the lower Keuper. Neues Compagno, L.J.V., Last, P.R., Stevens, J D. and Alava, M.N.R. Jahrbuch fur Geologie und Palaontologie, Abhandlungen (2005). Checklist of Philippine Chondrichthyes. CSIRO Marine Laboratories Report 243: 103. 160: 61–72. Eschmeyer, W.N. (ed.). (2010). Catalog of Fishes electronic Shen, S.C. (1986a). A new species of stingray Hexatrygon version. http://research.calacademy.org/ichthyology/catalog/ taiwanensis from Taiwan Strait. Journal Taiwan Museum fi shcatmain.asp. Accessed 16th September 2010. 39: 175–179. Heemstra, P.C. and Smith, M.M. (1980). Hexatrygonidae, a Shen, S.C. (1986b). A new species Hexatrygon brevirostra and new family of stingrays (Myliobatiformes: Batoidea) from a new record Anacanthobatis borneensis (Rajiformes) from South Africa, with comments on the classifi cation of batoid Taiwan. Journal Taiwan Museum 39: 105–110. fi shes. Ichthyological Bulletin of the JLB Smith Institute of Shen, S.C. and Lui, C.S. (1984). A new stingray of the genus Ichthyology 43: pp.1–17. Hexatrygon from Taiwan. Acta Oceanographica Taiwanica Hennemann, R.M. (2001). Sharks & Rays. Elasmobranch 15: 201–206. Guide of the World. IKAN – Unterwasserarchiv, Frankfurt, Smale, M.J., Compagno, L.J.V. and Human, B.A. (2002). Germany. 1–304. First megamouth shark from the western Indian Ocean Human, B.A. (2006). Size-corrected shape variation analysis and South Africa. South African Journal of Science 98: and quantitative species discrimination in a morphologically 349–350. conservative catshark genus, Poroderma Smith, 1837 Stehmann, M. and Shcherbachev, Y.N. (1995). Second record of (Chondrichthyes: Carcharhiniformes: Scyliorhinidae). sixgill stingray of the genus Hexatrygon (Elasmobranchii, African Natural History 2: 1–15. Hexatrygonidae) from the Indian Ocean [Voprosy Human, B.A. (2007a). Size corrected shape variation analysis Ikhtiologii 35: 290–295 (1995)]. Journal of Ichthyology 35: and quantitative species discrimination in a morphologically 151–159. conserved catshark genus, Haploblepharus Garman 1913 Takada, K., Hiruda, H., Wakisaka, S., Kudo, S. and Yano, K. (Chondrichthyes: Carcharhiniformes: Scyliorhinidae). (1997). A summary of the freezing, thawing, preservation, African Natural History 3: 59–73. and display methodology used on a large megamouth Human, B.A. (2007b). Size corrected shape variation analysis shark, Megachasma pelagios. In: Yano K., Morrissey, and quantitative species discrimination in a morphologically J.F., Yabumoto, Y. and Nakaya, K. (eds). Biology of the conserved catshark genus, Holohalaelurus Fowler 1934 Megamouth Shark. Tokai University Press, Tokyo. pp.11– (Chondrichthyes: Carcharhiniformes: Scyliorhinidae). 19. African Natural History 3: 75–88. Taylor, L.R., Compagno, L.J.V. and Struhsaker, P.J. (1983). Hutchins, J.B. (1992). Megamouth: gentle giant of the deep. Australian Natural History 23 (12): 910–917. Megamouth – a new species, genus, and family of lamnoid shark (Megachasma pelagios, Family Megachasmidae) Ishihara, H. and Kishida, S. (1984). First record of the sixgill from the Hawaiian Islands. Proceedings of the California stingray Hexatrygon longirostra from Japan. Japanese Academy of Sciences 43: 87–110. Journal of Ichthyology 30: 452–454. Jones, B.C. and Geen, G.H. (1977). Morphometric changes in White, W.T., Last, P.R. and Compagno, L.J.V. (2005). an elasmobranch (Squalus acanthias) after preservation. Description of a new species of weasel shark Hemigaleus Canadian Journal of Zoology 55: 1060–1062. australiensis n. sp. (Carchariniformes: Hemigaleidae) from Kempster, R.M. and Collin, S P. (2011). Electrosensory Australian waters. Zootaxa 1077: 37–49. pore distribution and feeding in the megamouth shark White, W.T., Fahmi, M.A. and Sumadhihara, K. (2004). Megachasma pelagios (Lamniformes: Megachasmidae). A juvenile megamouth shark Megachasma pelagios Aquatic Biology 11: 225–228. (Lamniformes: Megachasmidae) from northern Sumatra, Last, P.R. and Stevens, J.D. (1994). Sharks and Rays of Indonesia. The Raffl es Bulletin of Zoology 52: 603–607. Australia. CSIRO Australia. pp.1–513. Zhu, Y.D., Meng, Q.W., Hu, A. and Li, S. (1981). Description Last, P.R. and Stevens, J.D. (2009). Sharks and Rays of four new species, a new genus and a new family of of Australia. Second Edition. CSIRO Publishing, elasmobranchiate fi shes from deep sea of the South China Collingwood, Victoria. pp. 1–644. Sea. Oceanologia et Limnologia Sinica 12: 103–116. MacLeod, I.D. (2008). Washing formaldehyde from fi xed spirit Zhu, Y., Meng, Q., Hu, A. and Li, S. (1982). New records of specimens: a mechanism for the preservation of Megamouth Chondrichthyes of Nan Hai, China. Journal of Fisheries of III. AICCM Bulletin 31: 36–43. China 6: 85–86. MacLeod, I.D. and van Dam, A.J. (2011) A migration mechanism for transfer of sharks from ethanol to aqueous MANUSCRIPT RECEIVED 19 AUGUST 2010; ACCEPTED 24 AUGUST 2011.