DOI: 10.5061/dryad.47d7wm3d3
title: Research methods and Comparative examination of pinniped craniofacial musculature and its role in aquatic feeding

description: 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.
description: We conducted detailed
 dissections of the craniofacial musculoskeletal anatomy of six pinniped
 species: bearded seals (<i>Erignathus barbatus</i>, n=6),
 California sea lions (<i>Zalophus californianus</i>, n=5),
 harbor seals (<i>Phoca vitulina</i>, n=6), northern elephant
 seals (<i>Mirounga angustirostris</i>, n=11), ringed seals
 (<i>Pusa hispida</i>, n=6), and Weddell seals
 (<i>Leptonychotes weddellii</i>, 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
 <i>in situ</i>, the whole head with the hyoid apparatus
 <i>ex situ</i>, 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
 <i>in situ</i>. 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.
description: <i>In situ</i>
 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 <i>ex situ</i> 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).