Traditional and digital examination of the baculum of a leopard seal (Hydrurga leptonyx)

While terrestrial breeding in polygynous species of pinnipeds allows for observations of reproductive behavior (Atkinson, 1997), similar opportunities are limited for cryptic, nonpolygynous, aquatic-breeding species. The isolated nature of solitary leopard seals (Hydrurga leptonyx; Southwell et al., 2012) restricts data collection on their reproductive behavior. Observations on reproductive behavior are limited to mating calls (Rogers, 2017; Rogers et al., 1996) and pupping (van der Linde et al., 2022). Leopard seal mating has been observed in captivity (Marlow, 1967), but not in the wild (Kooyman, 1981). Currently, the leopard seal is thought to mate aquatically, with males defending territories for mating (Atkinson, 1997). Inferences on leopard seal reproduction are further confounded by the lack of basic anatomical data for their reproductive anatomy, currently limited to the female reproductive tract (Hamilton, 1939; Harrison et al., 1952) and the ex situ baculum (Didier, 1952; Hamilton, 1939). The baculum (os penis) size and structure has been shown to be related to reproductive life history strategies in pinnipeds (Brassey et al., 2020; Dixson, 1995; Fitzpatrick et al., 2012). Any additional anatomical data for the baculum anatomy of the leopard seal, particularly its relationship with soft tissues (Harrison et al., 1952), would therefore represent a notable contribution to understanding their reproductive mating strategies. Here we provide some basic soft tissue and in situ skeletal data on the reproductive anatomy of an adult male leopard seal specimen, based on observations made during dissection and CT scanning. The leopard seal was observed hauled out on a beach at Point Ricardo prior to October 9, 2018, roughly 9 km west of Cape Conran, Victoria, Australia. It is suspected to have died on that day, and was collected by the Department of Environment, Land, Water and Planning (DEWLP) on October 10, 2018 (stranding specimen record ORB 0516). The specimen was donated to Museums Victoria and registered in the Mammalogy collections as NMV C39957. After collection, the specimen was stored at −20°C until it was thawed for dissection (the specimen had previously undergone a freeze–thaw cycle for another study, see Hocking et al., 2021). A full-body dissection of NMV C39957 was done from April 4–5, 2022. The soft tissue anatomy of the penis was observed while partitioning the hindquarters from the trunk section. After full separation, the hindquarters were CT scanned with a Siemens Somatom Go.UP at Monash Biomedical Imaging. CT scans were processed in Avizo Version 2022.1 (ThermoFisher Scientific), and measurements were taken using Meshlab (Cignoni et al., 2008). Available comparative baculum material was studied and photographed in the Mammalogy collections of Museums Victoria: a southern elephant seal (Mirounga leonina, NMV C33585), a juvenile leopard seal (NMV C39957), an immature crabeater seal (Lobodon carcinophaga, NMV C25039), and a fur seal (Arctocephalus sp., NMV C33579). A walrus specimen (Odobenus rosmarus, unregistered) from the Zoology teaching collections at Monash University was also studied. All specimens were measured with tape measure and linear calipers, except for those measured digitally (NMV C39957, unregistered walrus specimen Monash teaching collection). The walrus baculum was also CT scanned, processed, and measured on the same equipment and programs as the leopard seal. 3D scan files are available on MorphoSource (project ID 000487269). Due to the rarity of baculum specimens in pinnipeds, the comparisons are supplemented with observations in the literature (Didier, 1953; Hamilton, 1939; Miller, 2009; Miller & Burton, 2001; Miller et al., 1999, 2000; Morejohn, 2001; Oosthuizen & Miller, 2000; Scheffer, 1950; Yurkowski et al., 2011). Before dissection, we observed that the preputial opening of NMV C39957 was small, 5 cm in length (Table 1) and located just anterior to the hips. The epidermis and dermis were thick around the genital area (Figure 1b,c), although the blubber was much thinner in this region (~0.2 cm) compared to the rest of the external body cavity (~0.9 cm; Table 1, Figure 2). Phocid blubber thickness is known to vary in different parts of the body to act as thermal windows (Mellish et al., 2007), and hence this may be an adaptation for temperature regulation of the testes to avoid overheating. The testes were located between the skin/blubber and abdominal muscles. Despite testis size having a long history of study in pinnipeds (Fitzpatrick et al., 2012; Harrison et al., 1952), this is (to our knowledge) the first observation of thinning of blubber in this region. However, it is possible that the thin blubber was due to malnourishment and poor health, as the blubber thickness of the leopard seal was well below the average for phocids (~3.8 cm, Liwanag et al., 2012) and the expected variability outside of winter (>1.2 cm; Mellish et al., 2007). In a relaxed, retracted state, the penis itself was located entirely within the body cavity, in the midline ventral to the pelvis (in the scan it is displaced; Figure 3). When extended out of the preputial opening, the penis was mostly straight, although there is a slight ventral curve of the glans relative to the shaft (Figure 1c). The prepuce was elastic and stayed retracted when the penis was extended beyond the preputial opening. The CT scan revealed that the glans protrudes slightly distoventrally from the baculum (Figure 2c), with a slight projection of soft tissue at the terminal end of the glans (Figure 1b,c). The soft tissue of the glans took up most of the distal penis in cross-section (Figure 2a), although the shaft was substantially thinner relative to the baculum at mid-shaft (Figure 2b). When retracted, the preputial skin was clearly distinguishable from the penile shaft in CT images, due to the presence of radio-opaque sand particles lodged between its folds (Figure 2c). Proximally, the penis extends beyond the baculum, ending posterior to the pelvis (Figure 3). The baculum appears to be over 50% of the length of the flaccid penis (Figure 3), similar proportions to those in the walrus, Odobenus rosmarus (Fay, 1982). The data reported in this study represent the first observations of penile soft tissues in a leopard seal (Figures 1-3), and only the third observation for a monachine (southern true seal, subfamily Monachinae; Laws, 1956; Tedman, 1991). The soft tissue anatomy of the leopard seal appears to have broad similarities with harbor (Phoca vitulina), gray (Halichoerus grypus), and elephant (Harrison et al., 1952; Laws, 1956) seals described in the literature. Having soft tissue of the glans distal to the apex of the baculum appears to be a characteristic shared among phocids (Figure 2c), in contrast to otariids (Harrison et al., 1952; Laws, 1956). The prepuce appears to retract in all four of these phocid seals (Figure 1b,c; Harrison et al., 1952; Laws, 1956). One difference between the leopard seal and the two phocine seals (harbor and gray) is that the longitudinal striations noted on the glans of phocines (Harrison et al., 1952) were absent on NMV C39957; but it is possible that those striations may be artifacts caused by the formalin fixation employed in that study. We segmented out the baculum of NMV C39957 from the CT data to make observations of skeletal anatomy. The distal baculum appears laterally compressed, and much thinner compared to its shaft (Figure 4a). There is a shallow concavity on the posterior surface of the distal baculum, presumably homologous to the groove for the urethra in terrestrial carnivorans (Figure 4c) (Evans & de Lahunta, 2012). The ventral surface of the distal end of the baculum is mostly flattened (Figure 4). Immediately proximal to this flattened section, the shaft is mostly ovoid. At the midshaft, there is a ventral protuberance in the body (Figure 4b,c); this protuberance is only slightly flattened and appears to have the same thickness as the immediate dorsal surface. Proximal to this protuberance, the body is subcircular (slightly ovoid horizontally) in cross section. The base is quite short and tapers to a smaller circumference than the rest of the body (Figure 4a,b). Proximal-distally, the baculum has a slight lateral S-shaped bend when viewed dorsally or ventrally. In overall shape, the juvenile leopard seal (NMV C5603, Figure 5d) is more similar to other phocids, including juveniles of other species (Figure 5e), than it is to the adult specimen NMV C39957 (Figure 5c; Hamilton, 1939); as such, the leopard seal baculum appears to undergo substantial shape change during pubertal development (Hamilton, 1939), as it does in other pinnipeds reported in the literature such as otariids and phocines (Miller, 2009; Miller & Burton, 2001; Miller et al., 1999, 2000; Oosthuizen & Miller, 2000; Scheffer, 1950; Yurkowski et al., 2011). Overall, the baculum morphology of the adult leopard seal NMV C39957 is similar to the lobodontins described in the literature; the Weddell seal (Leptonychotes weddellii), the Ross seal (Ommatophoca rossii), and the crabeater seal are also asymmetrical and have a flattened distal apex (Didier, 1953; Morejohn, 2001). The leopard and Weddell seals both have a distinct ventral protuberance (Morejohn, 2001), although the ventral protuberance might vary in leopard seals (Hamilton, 1939). The anatomical characteristics shared amongst the lobodontins set them apart from the other pinnipeds (Figure 5b–e; Miller, 2009). The baculum of the adult leopard seal NMV C39957 is curved, similar to the hooded seal (Cystophora cristata; Miller et al., 1999), walrus (Figure 5a), and fur seal (Arctocephalus sp.; Figure 5f); however, the ventral curvature differs in the leopard seal (and the Weddell seal) by the interruption in profile from the ventral protuberance. It should be noted that this ventral protuberance has not been described in other observations of leopard seal bacula (Didier, 1952; Hamilton, 1939), and while the overall profile is consistent there appears to be some intraspecific variation in the ventral curvature. The intraspecific variation in leopard seal bacula (Didier, 1952; Hamilton, 1939) appears to be consistent with intraspecific variation observed in other phocid seals (Miller & Burton, 2001; Miller et al., 1999; Yurkowski et al., 2011). However, intraspecific variation in leopard seal bacula (and most pinnipeds) has likely not been adequately characterized and should be explored in future studies. This is important to establish, as early observations of morphological changes in leopard seal bacula in the third year of life (Hamilton, 1939) closely match when male leopard seals reach sexual maturity (Kooyman, 1981; Rogers, 2007). Noting both inter- and intraspecific variation in pinniped baculum morphology is important, as it has the potential to help infer the life history of more cryptic species (Dixson, 1995; Fitzpatrick et al., 2012; Yurkowski et al., 2011). Baculum size has been proposed to be related to intromission time (Dixson, 1995) and reproductive life history (Fitzpatrick et al., 2012). Intromission in captive leopard seals has been observed to last 10 min (Marlow, 1967). At 26 cm (Table 2), the baculum length of NMV C39957 is roughly 10% of the body length, making it the largest baculum recorded for a leopard seal, both absolutely and scaled to body size (the next largest being 24 cm; Hamilton, 1939; Scheffer & Kenyon, 1963). This places the leopard seal as having one of the larger baculum in pinnipeds (Scheffer & Kenyon, 1963). This is consistent with most phocids having larger bacula (scaled to body size) compared to most otariids and elephant seals (~6% body length), although they are still dwarfed by that of the walrus (~18% body length) (Scheffer & Kenyon, 1963). This larger relative size of the baculum in the leopard seal suggests prolonged intromission in this species, consistent with captive observations (Marlow, 1967), due to the established scaling relationship between these two variables (Dixson, 1995). In addition to size, shape complexity of bacula has also been noted to be related to reproductive strategy in carnivores, with socially monogamous taxa having more complex baculum shape, and “group-living” taxa having more simple shapes (Brassey et al., 2020). Pinnipeds, including the leopard seal, have been noted to have simple baculum shapes, on account of pinnipeds having group-living mating behavior such as land-based harems or aquatic lek-type mating (Boness et al., 2006; Brassey et al., 2020). This would imply that the leopard seal would have some form of group-living mating behavior, contrasting to their known solitary nature (Southwell et al., 2012). While the baculum of NMV C39957 is simple in shape, it should be noted that it is slightly more complex than the juvenile specimen (NMV C5603); therefore, ontogeny may need to be accounted for when making these life history inferences. When making inferences regarding leopard seal reproduction, both males and females need to be considered. The reproductive anatomy of female leopard seals has been described briefly (Hamilton, 1939; Harrison et al., 1952), based on a few individuals. The general morphology of the reproductive tract of female pinnipeds is known (Atkinson, 1997), and a study on vaginal endocasts in marine mammals found pinnipeds to have relatively simple shape complexity (Orbach et al., 2021). This seems to be the case for leopard, crabeater, Weddell, and harbor seals (Hamilton, 1939; Harrison et al., 1952). There are few descriptions of the baubellum (os clitoridis) in pinnipeds, the exception being the walrus (Fay, 1982). While some individual female pinnipeds are known to lack a baubellum completely (Lough-Stevens et al., 2018), we have no knowledge of the morphological variation in this structure. A leopard seal baubellum does not appear to have ever been reported in the literature. As such, future studies should also aim to expand on the anatomy of the baubellum. In conclusion, the soft tissue anatomy of the leopard seal penis seems to show broad similarities with other phocid seals. While the baculum of NMV C39957 largely agreed with other descriptions of leopard seal bacula, there were also some clear differences, namely the ventral protuberance, highlighting the presence of intraspecific variation. The baculum of NMV C39957 is the largest recorded for the leopard seal, and its large size is likely linked to longer intromission. Future studies should focus on recording the variation in baculum morphology of leopard seals, as well as expanding on knowledge on the reproductive anatomy of female leopard seals. James P. Rule was supported by an Australian Research Council Discovery Project (DP180101797). Thanks to Monash University's Anatomy and Developmental Biology department (in particular, Bonnie Dopheide, Stephen Thompson, Justin Adams) for providing space for the dissection. Thanks also go to Museums Victoria for access to dissection equipment and the Mammalogy collection (in particular, Karen Roberts and Steven Sparrey). The following members of the Evans EvoMorph lab and Adams Lab also assisted with the dissection: Lucy Costello, Kathleen Garland, Ruairidh Duncan, William Parker, Jake Kotevski, Jack O'Connor, Natasha Nosiara, Jonathan Edwards, and Ramon Ciccone. CT scanning was performed with the valuable assistance of Alexander McDonald and Dr. Michael De Veer at Monash Biomedical Imaging. The authors acknowledge the facilities and technical assistance of the National Imaging Facility (NIF), a National Collaborative Research Infrastructure Strategy (NCRIS) capability at Monash Biomedical Imaging (MBI), a Technology Research Platform at Monash University. Thanks to Daniel Latorre for discussions around anatomical terminology. The quality of this manuscript was improved thanks to feedback from the associate editor Frank Fish, Krista van der Linde, and two anonymous reviewers. Open access publishing facilitated by Monash University, as part of the Wiley - Monash University agreement via the Council of Australian University Librarians. James Patrick Rule: Conceptualization; data curation; formal analysis; investigation; project administration; writing – original draft; writing – review and editing. Hazel L Richards: Conceptualization; investigation; writing – review and editing. Tahlia I Pollock: Investigation; writing – review and editing. David P Hocking: Investigation; writing – review and editing. Alistair R Evans: Conceptualization; investigation; project administration; writing – review and editing.