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Morphological identification in skull between spotted seal and harbor seal using geometric morphometrics

Abstract The morphology of the skull contains considerable ecological information about a species, because the skull contains sensory organs that are used to look for food, compete for mates, or to migrate. Spotted seals (Phoca largha) and harbor seals (Phoca vitulina) are similar in body size and pelage color but differ in habitat use and reproductive biology. The current study aims to clarify differences in the shapes of skulls in the spotted and harbor seals using geometric morphometrics and to discuss whether ecological differences can explain morphological differences in skulls. First, we discovered that the age at which the shape of skulls stopped changing was 7 years in both species, using the linear‐threshold model. Using a total of 75 landmarks, 54 individuals (25 spotted seals, 29 harbor seals) that were older than the age at which skulls stopped changing were correctly identified at a rate of 100%. The total of 75 landmarks was narrowed down to eight key landmarks that resulted in an identification accuracy rate of 100% using random forests. Of the eight landmarks, seven were related to feeding apparatus, indicated that the harbor seal had a broader mouth and mandible than the spotted seal. Because of both species were dietary generalists and classified as pierce feeders, we suggested that the different features in the shapes of their skulls were caused not only by differences in their feeding behavior but also other differences related to reproductive behavior.

similar. However, ecological differences in habitat and reproductive biology (McLaren, 1966;Burns, 1970) and detailed morphological and craniometric analyses (Shaughnessy & Fay, 1977;Burns et al., 1984) confirmed that substantial differences exist between these two seals (Lowry et al., 1998). Spotted seals give birth on sea ice and are monogamous.
They migrate during ice-free months, and their breeding season occurs during the months of sea ice cover. Harbor seals, on the other hand, give birth on land and use haul-out sites throughout the year, during the pupping, nursing, breeding, and molting seasons (Naito & Nishiwaki, 1972), and they are polygynous (Sullivan, 1981;Boness et al., 2002). In addition, spotted seal pups possess a white lanugo. This pelage provides insulation for the neonates of ice-breeding seals until they develop a blubber layer, and it may also serve as camouflage for protection from predators (Yochem & Stewart, 2018). Harbor seal pups, however, are normally born with a pelage similar to adults; they lose their white coat in the uterus before birth. A previous study argued that the spotted seal was, therefore, more plesiomorphic than the harbor seal (McLaren, 1966(McLaren, , 1975. A more recent study reported that the two species were closely related genetically, but their mitochondrial DNA is distinct (O'Corry-Crowe & Westlake, 1997).
An allometric comparison of the skulls showed that the harbor seal had a longer face and rostrum than the spotted seal (Shaughnessy & Fay, 1977;Burns et al., 1984;Uno, 1986). However, those features were thought to be associated with specialized types of food or the method of feeding on euphausiids in newly weaned pups (Kato, 1982;Uno, 1986).
Another study reported that the same types of food were frequently observed in the stomachs of both species located in the same area (Nakaoka et al., 1986). They are generalist pierce feeders that grip and swallow their prey whole (Adam & Berta, 2002;Kienle & Berta, 2016;Kienle et al., 2018;Kienle & Berta, 2019). We, therefore, believe that the shapes differences of these species' skulls are not necessarily a result of their diet or the way they feed. It may be possible that the differences in skull features are due to other ecological differences. Although allometric methods are convenient, it is difficult to use such methods to describe complex closed contours (Tatsuta et al., 2018). Previous studies also attempted to categorize and classify the non-metrical characteristics of skulls in these species, however they were not able to quantify them (Burns et al., 1984;Nakagawa et al., 2009).
The current study aims to analyze differences in the shapes of skulls of spotted and harbor seals using geometric morphometrics and discuss morphological differences of the skulls in the context of ecological differences.

| Age determination
The upper right canine teeth were taken from each skull, sectioned at 10 μm with a cryostat (Leica Biosystems), and stained with Delafield's hematoxylin, following fundamental methods (Hachiya & Ohtaishi, 1994;Hohn, 2002) to determine the age. Ages were estimated based on the counts of cementum annuli (Mansfield & Fisher, 1960).

| Photography of skulls
Skulls were placed on a cutting mat (57 Â 640 cm, Clover), and a digital camera (D3000, Nikon) mounted on a tripod Velbon) was used to photograph the dorsal and ventral sides, and the lateral crania and mandibles. Distance was approximately 40 cm from skull to camera. All skulls were photographed in the same place at Tokyo University of Agriculture Abashiri, Hokkaido, Japan. Adobe Photoshop CS2 was used to align the scales of the photographs.

| Landmarks on skulls
Geometric morphometrics based on two-dimensional landmarks were used to quantify the shapes of skulls. A total of 75 homologous landmarks (shown in Figure 2 and described in Table 2) were digitized from each of four images using tpsDig2 (Rohlf, 2013). Landmarks were based on Bookstein (1991). They were located at the juncture points of bones, ridge curves, and edge points, all chosen from previous studies to capture the overall shape of the skull ( , where x 0 i was positive if θ < π 2 or negative if θ > π 2 . These distance and angle (CDA) were used in the analysis. In addition, the direction of the reference landmark (dorsal cranium: No. 1, ventral cranium: No. 21, lateral cranium: No. 58, lateral mandible: No. 63) was set on the y-or x-axis, respectively. It was rotated to allow for comparison between individuals.

| Determining impact of age on shape change
In general, the growth of the body in mammals changes from rapid to gradual. Morphology of the skull also changes with growth after birth (Moore, 1981). We estimated the age at which shape changes stopped in the skull, because to capture the shape and character after the age.
We considered that, when comparing the characteristics of skulls between species, it was simpler to use the age at which shape changes stopped rather than using major shape changes in the skulls that occurred during the growth of the species. We used the linear-threshold model (Clark et al., 1985), which is presents a normal linear regression up to a boundary and a linear regression with zero angle above a boundary, to estimate the age at which the shapes of skulls in each species stopped changing. The linear-threshold model consists of one regression and threshold value. These equations are described below: where age i denotes the age of an individual, obs age i ,j is the measurement value of the CDA (j = 1 $ 150) for each individual, age θ denotes the age at which the shape of the skull stopped changing, β 0,i,j represents a constant, β 1,i,j denotes an intercept, and β 2,i,j denotes the slope of the line. The error ε i,j was assumed to be normally distributed with mean 0 and variance σ. To estimate the age θ , the AIC (Akaike, 1973) is calculated by varying value of age θ from 0 to 35 and we defined the candidate value of age θ with lowest AIC as the optimal value.

| Discrimination rate and important landmarks
We obtained the discrimination rate using random forests (Breiman, 2001) from samples of the two species that were of an age when the shape of the skull had stopped changing. Recently, some studies have used random forests for speciation by morphology (e.g., Machida et al., 2020). Random forests is an algorithm for classification developed by Breiman (2001) that uses an ensemble of classification trees (Breiman & Friedman, 1984;Ripley, 1996;Hastie et al., 2001). The algorithm for random forests in the classification  Table 2 explanatory variables were the CDA from each landmark. The discrim- We conducted the model selection by adopting appropriate explanatory variables modified by Baba and Matsuishi (2015). The model selection was based on variable importance (Breiman, 2001).
The variables were set for each landmark. We conducted a model with   We conducted a principal components analysis (PCA) using CDA to determine the major axes of variation between variables. All analyses were conducted in R version 3.5.0 (R Development Core Team, 2018). Random forest analyses were conducted using the randomForest version 4.6-14 package (Liaw & Wiener, 2002) under R.

| RESULTS
3.1 | Estimation of the age at which the shape of the skull stopped changing Using the linear-threshold model, the age at which the skull stopped changing (age θ ) was found to be 7 years old in both species (Figure 3).
AIC was lower value as the divided into species than sex in the model ( Figure 3).

| Identification rates of species and calculation of important landmarks
The 25 spotted seals and 29 harbor seals were at least 7 years old, or older. A comparison of plots at each average coordinate showed that the skull of the harbor seal was larger than that of the spotted seal ( Figure 4).
For the analysis of random forests, the overall misclassification rate for OOB data, (err oob ), showed the lowest value when the number of features (mtry) was set the number of the square roof of input variables (Shi & Horvath, 2006), and it was confirmed that the estimation was sufficiently stable when the number of decision trees (ntree) was 2000, from the change in each misclassification rate. Therefore, we  (Figures 4, 6). Landmark 56 (posterior of the external auditory meatus) of PC2 was also a common important identification landmark selected by random forests (Figures 4, 6).
Landmarks 21-27 (anterior of the rostrum) and landmarks 8-10 and 12-13 (around the squamosal) were reflected in PCs in PCA, but these were not selected by the random forests (Figures 4, 6).

| DISCUSSION
We showed that the age at which the shape of the skull stopped changing was 7 years in the spotted seal and 7 years in the harbor seal. Using all 75 landmarks, 54 individuals (25 spotted seals, 29 harbor seals) that were older than the age at which the skull stopped changing were correctly identified at an accuracy of 100%. Since each average position of landmarks on harbor seals was more on the lateral than on spotted seals (Figure 4), the skull of the harbor seal is broader and larger than that of the spotted seal. Burns (2002) and Nakagawa et al. (2009) reported that the skull of the harbor seal was more massive and robust than that of the spotted seal, a finding that was also supported by the present study.
A total of eight identification landmarks (15,28,34,35,56,70,72,73) were narrowed down while maintaining the same Of the eight landmarks, seven were related to the feeding apparatus (15,28,34,35,70,72,73). Differences in landmarks 15, 28, 34 and 35 indicated that the harbor seal has a longer rostrum and broader palate than the spotted seal. Slater et al. (2009) reported that a broader rostrum would enable to have a larger and stronger bite, and the longer, broader rostrum of the harbor seal would therefore enable it to open its mouth wider and have a stronger bite than the spotted seal. Differences in position of landmark 70 (angular process) was indicated that the harbor seal has a more developed angular process relative to the spotted seal ( Figure 4). The angular process serves for the attachment of the pterygoids medially and the masseter laterally (Evans & de Lahunta, 2013). Expansion of the angular process reflect increased size of the masseter musculature (Radinsky, 1985). Therefore, this suggests that the harbor seal may have larger masseter musculature and stronger bite force. Differences in the position of landmarks 72 and 73 indicated that the mandible of the harbor seal was broader than that of the spotted seal. Differences in position of landmark 73 indicated that the body of the mandible around landmark 73 was more angular than the spotted seal. Those positions (72,73) relate to area of attachment for the digastric muscle, which helps with opening the mouth. The broader of the body of the mandible under the row of teeth and the more ventral position of the 73 seems to support the attachment of much digastric muscle that could be developed for strong biting movement. Although bite performance is generally related to the type of prey, both species are dietary generalists and are classified as pierce feeders (Adam & Berta, 2002) and thus do not need to crush hard prey. Euphausiids are the prey of newly weaned spotted seal pups (Kato, 1982) and schooling fishes are the main prey of older seals (Kobayashi, 2015). On the other hand, harbor seals are opportunistic feeders on locally abundant and easily available prey items (Telmann & Galatius, 2018), and they have been found to prefer foraging demersal fishes in shallow waters and also cephalopod species (Andersen et al., 2004;Bromaghin et al., 2013;Nakaoka et al., 1986). Additionally, a comparative study of the feeding habitats of spotted seals and harbor seals in the same coastal area in the Nemuro district of eastern Hokkaido, Japan, for example, demonstrated that the harbor seal depends on benthic animals in shallow waters, while the spotted seal foraged for prey from the surface to middle layers (Nakaoka et al., 1986). However, a variety of common prey was also found in spotted seals and harbor seals stomachs (Nakaoka et al., 1986), suggesting that the harbor seal could not necessarily need a longer mouth and stronger bite than the spotted seal.
Also, information on the stomach contents may vary temporary, therefore it is difficult to evaluate the feeding habitat on a long-term.
However, the information of the feeding habits in the spotted seal and harbor seals along the coast of Hokkaido is insufficient (e.g., Itoo et al., 1983;Kaimoto et al., 2018;Kato, 1982;Masubuchi et al., 2017;Masubuchi et al., 2019;Nakaoka et al., 1986;Wada et al., 1992). It was also suggested that dietary constraints in the past could reflect the differences in the skull shape of the spotted seals and harbor seals.
Alternatively, differences in the skull shape related to bite force may be related to differences in reproductive behavior. In pinnipeds, polygynous species produce larger theoretical bite forces than monogamous species, they have large muscle attachment areas, and F I G U R E 6 Scatterplots of the first two axes of principal component analysis based on variables of the landmarks of spotted seals and harbor seals aged 7 or older. Circles represent spotted seals and triangles represent harbor seals. Variables that were strongly reflected in each axis are represented by the illustrations of the cranium and landmarks. Filled circles and numbers represent the landmarks. The asterisk (*) in the illustrations of cranium indicates the centroid point both males and females of combative species have more robust jaw morphology (Jones et al., 2013). In addition, highly -aggressive species have wider zygomatic arches likely to allow the emplacement of large jaw-closing muscles in the skull (Dewar et al., 2015). Male-male combat is influenced by bite force evolution in pinnipeds (Jones et al., 2013;Jones & Ruff, 2011). Relationship between combat style and skeletal morphology also reported between the intraspecific combat behaviors and morphology of the cervical vertebrae in the ruminant mammals, the morphology differed in relation to the combat style and the role of cervical vertebrae was supported to resist the forces generated in malemale combat (Vander Linden & Dumont, 2019). Scars were frequently observed around the neck of adult male harbor seals (Naito, 1973;Niizuma, 1986;Sullivan, 1981), and biting at the surface of the water between adult males for access to estrous females was observed during the breeding season (Sullivan, 1981), because the mating system of the harbor seal is polygamous and they use haul-out sites throughout the year (Burns, 2002). The spotted seal, however, gives birth and nurses pups on sea ice, and triads consisting of a female, her pup, and one attending male can be seen on the ice, or with the attending male close by, in an annually monogamous system where there is little competition between males (Burns, 2002). We conclude that a broader mouth and mandible in harbor seal had compared to spotted seal might be due to their different mating systems. Additionally, it has been reported that some spotted seals attained sexual maturity at the age of 3 years, though most reached sexual maturity at 5-6 years of age (Naito & Nishiwaki, 1972), and that most harbor seals reached sexual maturity at the age of 5 years (Suzuki & Yamashita, 1986). Mizuno and Ohtaishi (2002) also reported that skull growth ceased mostly around 5 years old and the completion ages for character development were after 5 years old in the spotted seal. Because the age at which the shape of the skull stopped changing was 7 years in both species, we found that the shape of the skull also changed after the age of sexual maturity and showed similar tendency of the previous study. Furthermore, Nakagawa et al. (2009) reported that the occipital width contributed to the discrimination between the skulls in the spotted seals and harbor seals and the feature also showed in this study as the landmarks around the squamosal contributed to the major axes ( Figure 6).
On the other hand, the landmarks around the squamosal were selected by PCA but also these landmarks were not selected in the random forests as the important identification landmarks. We estimated that the shape changes around squamosal would be great variation for individuals and occur the interaction close to landmarks, because some muscles are attachment around the area. For example, the splenius capitis, longissimus capitis, and sternocleidomastoid attach to mastoid process, furthermore, previous study reported that these muscles and the morphology of the mastoid process were associated with malemale competitions through the neck movements in some pinniped species (Boh orquez-Herrera et al., 2017). However, in the current study, landmarks around the mastoid process were not selected as the important identification landmarks. It was indicated that the important identification landmarks would represent the original features and reflect the difference of ecological behaviors in the spotted seal and harbor seal.
In conclusion, we used geometric morphometrics to show detailed differences in the shapes of skulls in the spotted and harbor seals, which are closely related species. We could discriminate between the two species with a high degree of accuracy and narrow down important landmarks for identification. We suggest that the different features in the shapes of their skulls are caused not only by differences in their feeding but also to differences in reproductive behavior.