Research methods and Comparative examination of pinniped craniofacial musculature and its role in aquatic feeding

Secondarily aquatic tetrapods have many unique morphological adaptations for life underwater compared to their terrestrial counterparts. A key innovation during the land-to-water transition was feeding. Pinnipeds, a clade of air-breathing marine carnivorans that includes seals, sea lions, and walruses, have evolved multiple strategies for aquatic feeding (e.g., biting, suction feeding). Numerous studies have examined pinniped skull and dental specializations for underwater feeding. However, data on the pinniped craniofacial musculoskeletal system and its role in aquatic feeding are rare. Therefore, the objectives of this study were to conduct a comparative analysis of pinniped craniofacial musculature and examine the function of the craniofacial musculature in facilitating different aquatic feeding strategies. We performed anatomical dissections of 35 specimens across six pinniped species. We describe 32 pinniped craniofacial muscles—including facial expression, mastication, tongue, hyoid, and soft palate muscles. Pinnipeds broadly conform to mammalian patterns of craniofacial muscle morphology. Pinnipeds also exhibit unique musculoskeletal morphologies—in muscle position, attachments, and size—that likely represent adaptations for different aquatic feeding strategies. Suction feeding specialists (bearded and northern elephant seals) have a significantly larger masseter than biters. Further, northern elephant seals have large and unique tongue and hyoid muscle morphologies compared with other pinniped species. These morphological changes likely help generate and withstand suction pressures necessary for drawing water and prey into the mouth. In contrast, biting taxa (California sea lions, harbor, ringed, and Weddell seals) do not exhibit consistent craniofacial musculoskeletal adaptations that differentiate them from suction feeders. Generally, we discover that all pinnipeds have well-developed and robust craniofacial musculature. Pinniped head musculature plays an important role in facilitating different aquatic feeding strategies. Together with behavioral and kinematic studies, our data suggest that pinnipeds' robust facial morphology allows animals to switch feeding strategies depending on the environmental context—a critical skill in a heterogeneous and rapidly changing underwater habitat.In situ measurements—maximum length (straight rostral-caudal distance from rostral tip of skull to caudal edge of occipital condyles), width (straight medio-lateral distance from lateral edges of zygomatic arch), and depth (straight dorso-ventral distance from sagittal crest to auditory bullae)—were measured for each muscle when possible. We used scaled photographs to take ex situ measurements of the maximum length and width of each muscle in two-dimensions in ImageJ. Muscle area was calculated from tracing the perimeter of the muscle in scaled photographs. We then calculated the muscle-to-skull area ratio (MSR) for each muscle, which was obtained by dividing each muscle area by the skull area to standardize for head size. We calculated mean MSR for each craniofacial muscle group within each species (e.g., facial expression, mastication, tongue, hyoid, and soft palate) for interspecific comparisons. We ran linear models to compare the relationship between relative muscle size (MSR), species, and feeding strategy and determined significance using ANOVAs (car package; Fox & Weisberg 2011; Bates et al. 2015). We examined the relationship between muscle size and species with species as the predictor variable and between muscle size and feeding strategy with feeding strategy as the predictor variable. We used least-square means to perform Tukey post-hoc pairwise contrasts between each significant predictor variable (lsmeans package; Lenth, 2016). Residual plots for each model were examined for deviations from normality and homoscedasticity. When heteroscedasticity was observed, data were log-transformed. Additionally, we examined variability in MSR for each muscle and species by quantifying the coefficient of variation [CV, standard deviation (s.d.)/mean] for each muscle. The CV measures variation in a trait. A low CV (values close to 0) indicates stereotypy, while a high CV (values close to 1) indicates high variability (Gerhardt, 1991; Wainwright et al., 2008). All statistical analyses were conducted in R v. 3.5.3 (R Core Team, 2019). Funding provided by: National Oceanic and Atmospheric AdministrationCrossref Funder Registry ID: http://dx.doi.org/10.13039/100000192Award Number: Dr. Nancy Foster Scholarship ProgramWe conducted detailed dissections of the craniofacial musculoskeletal anatomy of six pinniped species: bearded seals (Erignathus barbatus, n=6), California sea lions (Zalophus californianus, n=5), harbor seals (Phoca vitulina, n=6), northern elephant seals (Mirounga angustirostris, n=11), ringed seals (Pusa hispida, n=6), and Weddell seals (Leptonychotes weddellii, n=1; Table S1). Specimens were obtained from the Alaska Department of Fish and Game (Fairbanks, AK), the Marine Mammal Center (Sausalito, CA), Moss Landing Marine Lab (Moss Landing, CA), Ohio University (Athens, OH), the Pacific Marine Mammal Center (Laguna Beach, CA), SeaWorld San Diego (San Diego, CA), the University of Alaska (Anchorage, AK), and the University of California Santa Cruz (UCSC; Santa Cruz, CA). Specimens were obtained through NMFS permits #358-1787, #15324, #18786-04; MMHSRP #18786-04, and NMFS Southwest Region letters of authorization to A. Berta (San Diego State University) and S. Kienle (UCSC) All specimens were opportunistically collected and included different age classes and sexes. Specimens in this study consisted of either the whole head including the hyoid apparatus in situ, the whole head with the hyoid apparatus ex situ, or the whole head without an associated hyoid apparatus. All specimens showed little to no tissue decomposition and were frozen shortly after death to prevent further tissue degradation. Prior to dissection, each specimen was thawed for one to two days. We then took scaled photographs, measured, and described the external morphology of each specimen. Skull length and width measurements were collected in situ. Skull area (or lateral projected area) was measured in two-dimensions from scaled photographs in ImageJ v. 32. A dorso-caudal to dorso-rostral midline incision was made on each specimen to reveal the internal anatomical relationships. Skin, blubber, and superficial fascia were carefully peeled away to expose the underlying craniofacial muscles. The general morphology and the muscular and ligamentous connections between bony and cartilaginous elements were examined. The muscle origin, insertion, and fiber direction were documented and described for each individual muscle that could be identified. We infer the muscle action by assuming shortening along the axis of muscle fiber as they contract along the path of the whole muscle between the origin and insertion. Muscle terminology follows Evans & de Lahunta (2013), except where noted. We documented the three-dimensional (3D) arrangement between muscles and bony elements and reported inter- and intraspecific variation when observed