Copyright by Lauren Taylor English 2018

The Dissertation Committee for Lauren Taylor English Certifies that this is the approved version of the following Dissertation:

Evolution of Functional Morphology of Osteoderms Across Crocodylomorpha

Committee:

Julia A. Clarke, Supervisor

Christopher J. Bell

Christopher Brochu

Matthew W. Colbert

Rowan C. Martindale

Liza J. Shapiro Evolution of Functional Morphology of Osteoderms Across Crocodylomorpha

by

Lauren Taylor English

Dissertation Presented to the Faculty of the Graduate School of The University of Texas at Austin in Partial Fulfillment of the Requirements for the Degree of

Doctor of Philosophy

The University of Texas at Austin May 2018 Dedication

To my parents, Pamela and Julian, and to my brothers, Lee and Logan, for being the first to inspire in me a love of science and continuing to do so when times were hard, for never allowing me to forget to think critically, and for always being there to commiserate over failures and celebrate over triumphs.

Acknowledgements

Many people have contributed to the research presented here, which I cannot thank enough for their assistance. I am particularly grateful to my advisor, Julia Clarke, for all the guidance and support she has gifted me from start to finish. I would also like to thank everyone who has served on my committee (Chris Bell, Chris Brochu, Matt Colbert, Brian Horton, Rowan Martindale, Tim Rowe, and Liza Shapiro) for all the feedback that has improved this manuscript. I would like to thank the following institutions and their staff for access to specimens and CT scans: the Museum of Natural History, University of Florida, the Museum of Vertebrate Zoology, University of California, Berkeley, the Saint Augustine Alligator Farm, the University of Texas at Austin – Biodiversity Collections, the Texas A&M University – Biodiversity Research and Teaching Collections, the American Museum of Natural History, the Carnegie Museum of Natural History, the Field Museum, the North Carolina Museum of Natural Sciences, the Natural History Museum in London, the Senckenberg Naturmuseum, the Science Museum of Minnesota, Stony Brook University, the University of California Museum of Paleontology, Berkeley, the National Museum of Natural History. I would also like to thank the College of Veterinary Medicine & Biomedical Sciences, Texas A&M University and the University of Texas High- Resolution X-ray Computed Tomography Facility for scanning specimens and particularly to Matt Colbert and Jessie Maisano for advice on processing CT data. Financial support was provided in part by the Ernest and Judith Lundelius Endowment in Vertebrate Paleontology, the University of Austin off-campus research funds, and by the Jackson School of Geosciences Student Travel Grant. v I would like to thank the other students in my lab (James Proffitt, Chad Eliason, Katie Browne, Nick Crouch, Sarah Davis, Zhiheng Li, Grace Musser, Chris Torres, and Xia Wang,) for helpful discussions of methodology and presentation, the collections and specimen preparation staff (Liath Appleton, Kenneth Bader, Lisa Boucher, Matt Brown, Cissy Geigerman, Ernest Lundelius, the late Ann Molineux, Tim Rowe, Chris Sagebiel, and Deborah Wagner) for showing me the ins and outs of specimen care, Philip Guerrero for his indispensable assistance in navigating campus paperwork, and the all other staff and students at the Jackson School of Geosciences for all their assistance and companionship over the years.

vi Abstract

Evolution of Functional Morphology of Osteoderms Across Crocodylomorpha

Lauren Taylor English, PhD The University of Texas at Austin, 2018

Supervisor: Julia A. Clarke

The vertebrate integument is a complex anatomical system that has evolved a wide array of specialized structures that are related to an animal’s ecology and evolutionary history, including mineralized structures found in many clades and ecological niches called osteoderms. However, the development, functional morphology, and evolutionary history of osteoderms is still poorly understood in relation to other portions of the skeleton. Osteoderms have independently evolved multiple times and can be found in the skin of many vertebrate lineages, making up a significant portion of the evolutionary history of the tetrapod skeleton. Though several hypotheses of osteoderm function have been proposed based on their overall morphology, osteoderm function has rarely been the subject of experimental analyses and only in a few taxa. The major aims of this research are to test new and previously proposed hypotheses of osteoderms function and to better understand drivers of osteoderm morphological variation using Crocodylomorpha as the study group, both because of their long and rich history of ecological diversity and because of their exemplary fossil record compared with other osteoderm bearing taxa. Quantification of within-individual variation in extant vii crocodilians suggests that relaxed selection on osteoderm morphology only occurs in taxa thought to exhibit reduced intraspecific aggression, suggesting that osteoderms evolved in Crocodylia as a defensive structure in territorial disputes. This is corroborated by an ontogenetic analysis, which demonstrates that in at least some crocodilian species, osteoderm growth coincides with the onset of territorial behavior, contradicting previously proposed hypotheses that crocodilian osteoderms function in thermoregulation, in stabilization of the vertebral column, or as a defense against predators. In contrast with previous research that had demonstrated a reduction of body flexibility when osteoderms are present, a final comparative analysis across Crocodylomorpha found no correlation between osteoderm aspect ratio and locomotor ecology, suggesting that osteoderm dimensions have no impact on flexibility. This research disputes some previously proposed hypotheses of osteoderm function and when placed in context of other research, reveals that osteoderms may have different functions in different taxa.

viii Table of Contents

Chapter 1: Variation in crocodilian dorsal scute organization and geometry with a discussion of possible functional implications ...... 1

Abstract ...... 1

Introduction ...... 2

Materials and Methods...... 4

Variation in scute dimensions ...... 5

Bilateral fluctuating asymmetry of carapace ...... 5

Scutellation pattern ...... 6

Results ...... 7

Variation in scute dimensions ...... 7

Lateral asymmetry of carapace ...... 7

Rectangular vs. pentagonal/hexagonal scute arrangement ...... 8

Discussion ...... 8

Conclusions ...... 10

Tables ...... 11

Figures ...... 12

Chapter 2: Late ontogenetic development of osteoderms and their role in social behavior...... 15

Abstract ...... 15

Introduction ...... 16

Materials and Methods...... 20

Specimens ...... 20

Assessment of maturity ...... 20

ix Results ...... 21

General developmental patterns in all species ...... 21

Life history & osteoderm development in Caiman crocodilus and Crocodylus niloticus ...... 22

Discussion and Conclusions ...... 23

Tables ...... 26

Figures ...... 28

Chapter 3: Assessment of the comparative morphology of osteoderms and locomotor ecology in Crocodylomorpha ...... 30

Abstract ...... 30

Introduction ...... 30

Materials and Methods...... 34

Specimens ...... 34

Locomotor ecology ...... 36

Statistics ...... 37

Results ...... 38

Discussion ...... 39

Conclusions ...... 40

Tables ...... 42

Figures ...... 44

Works Cited ...... 50

x Chapter 1: Variation in Crocodilian Dorsal Scute Organization and Geometry with a Discussion of Possible Functional Implications

Dermal ossifications, including osteoderms, are present in many vertebrates and are frequently interpreted as a defense against predators. Nevertheless, osteoderms remain ubiquitous in adult crocodilians while being absent in hatchlings, even though adults rarely experience predation. In other biological systems, increased variation, particularly fluctuating asymmetry, have proven useful for identifying biological structures likely to have evolved under relaxed selection, which in turn may inform their function. Therefore, using the keratinous scutes as proxies for the underlying osteoderm morphology, I investigated the average intraspecific variability of geometry and fluctuating asymmetry in dorsal scutes in five species of crocodilians (N=65). I first tested for differences in variability of scute length and width, then for differences in bilateral fluctuating asymmetry of scute number, before finally investigating scute distribution patterns for each species compared to hypothetical rectangular and hexagonal scute arrangements. The American crocodile, Crocodylus acutus, shows significantly more asymmetry than other species, which is consistent with relaxed selection on osteoderms in this species. A suspected decrease in intraspecific aggression within Crocodylus acutus, in conjunction with the inferred relaxed selection, suggests that, in general, crocodilian osteoderms function primarily as defensive armor in aggressive encounters with conspecifics. The smooth-fronted caiman, Paleosuchus trigonatus, exhibits increased variation in scute dimensions linked to the mediolateral offset of osteoderms in adjacent rows, possibly resulting in a more rigid carapace. Unfortunately, comparative data on crocodilian behavior, physiology, and development is extremely limited and restricts the ability to explore other potential explanations for the patterns observed, highlighting the need for more research on rare and cryptic crocodylians.

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INTRODUCTION

Many vertebrates of diverse anatomies, physiologies, behaviors, and habitats evolved ossified integumentary structures (Vickaryous and Sire 2009; Sire et al. 2009). In some taxa, such as turtles, these structures form a single rigid body surrounding much of the animal, but in many other taxa these structures consist of numerous small pieces of bone with flexible articulations – referred to as osteoderms in most tetrapods – which can vary considerably in their shape and articulation pattern (Ross and Mayer 1983; Hill 2004; Vickaryous and Sire 2009; Sire et al. 2009). Despite the great diversity in osteoderm morphology and in the ecology of the animals that possess them, most authors attribute the same function or functions of osteoderms and analogous structures to all taxa (e.g., Yang et al. 2012). The most commonly hypothesized function of osteoderms is as a defensive armor against predators (e.g., Ross and Mayer 1983). While ample evidence exist demonstrating that dermal plates act as predator defense in stickleback fish (e.g. Bergstrom and Reimchen 2000, 2003; Reimchen 2000), analysis of Stegosaurus osteoderms found them to be wholly inadequate for mechanical protection (Buffrénil et al. 1986), and experiments with cordylid lizards suggests that the effectiveness of osteoderms as armor is variable depending on the species of lizard and type of predator, possibly due to functional trade-offs with other biological needs (Broeckhoven et al. 2015, 2017b). Some authors proposed alternative or additional osteoderms functions including stabilization of the vertebral column during terrestrial locomotion (Frey 1988; Salisbury and Frey 2000), as intraspecific display structures (Main et al. 2005; Saitta 2015), or playing a role in physiological functions such as thermoregulation (Seidel 1979; Farlow et al. 2010) or calcium metabolism (Jackson et al. 2003; Dacke et al. 2015). One way to investigate the function of a biological structure is to find examples of a reduction or loss of function. In these cases, relaxed selection results in accumulation of mutations which would otherwise reduce fitness (Bulmer 1989; Lahti et al. 2009). This increased genetic variation then can result in increased phenotypic variation of the structure in question, particularly a kind of variation called fluctuating asymmetry, which is defined as small random developmental deviations from bilateral symmetry within individuals, but which do not change the average

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symmetry of the species as a whole (Yablokov 1966; Hallgrímsson 2003; Russell and Bauer 2005; Lahti et al. 2009). Increased total variation and increased fluctuating asymmetry are found in a variety of biological structures that are known or suspected to have reduction or loss of a particular functional compared to closely related species. Examples include the wings of recently flightless insects (Crespi and Vanderkist 1997), the vestibular system in tree sloths (Billet et al. 2012), and the scales of limbed squamates relative to limbless squamates, which use costal scales for locomotion (Kerfoot 1970). Non-pathologic variability of suspected armor has only been investigated in the stickleback, in which Bergstrom and Reimchen (2000, 2002, and 2003) demonstrated a distinct inverse relationship between predation intensity and fluctuating asymmetry of the lateral plates. They also found increased asymmetry in individual plates that were relatively less important for maintaining the total structural integrity of the stickleback armor. All extant crocodilians, and the majority of extinct crocodylomorphs, have bony carapaces, defined here as a relatively rigid dermal structure of closely articulated osteoderms, each bearing an associated keratinous scute. It has long been observed that the number, relative dimensions, positions, and articulation patterns of scutes differ among crocodilian taxa in a way that makes them useful for species identification (King and Brazaitis 1971; Brazaitis 1973; Ross and Mayer 1983). It also is known that they differ in the degree of intraspecific variability (King and Brazaitis 1971; Brazaitis 1973; Ross and Mayer 1983). Although crocodilian osteoderms as a whole garnered interest from ecologists and functional morphologists (Seidel 1979; Ross and Mayer 1983; Frey 1988; Salisbury and Frey 2000; Jackson et al. 2003; Dacke et al. 2015), no attempts have been made to propose mechanisms explaining differences in carapace morphology among different taxa. In the majority of extant species, the dorsal carapace is formed of a rectangular tessellation of osteoderms with their overlying scutes (Figure 1.1 A). Different species vary in the number of average scutes per row and although some intraspecific variation in the number and dimensions of scutes is present in all species, it was previously noted that Crocodylus acutus and to a lesser extent Crocodylus moreletii have highly irregular scute patterns and sizes (Brazaitis 1973; Ross and 3

Mayer 1983; Garcia-Grajales et al. 2009; Thorbjarnarson 2010). Paleosuchus trigonatus also was identified as having highly irregular lateral position of scute centroids, resulting in keels that are not aligned with each other cranio-caudally as they are in other species (Figure 1.1 C; Medem 1958; Brazaitis 1973; Ross and Mayer 1983). However, these patterns were only briefly mentioned for the purpose of species identification or as a curiosity, and were never systematically analyzed. Here I test for evidence of relaxed selection on dorsal osteoderms, using the associated scutes as a proxy, in five different species of crocodilian (three alligatorids and two crocodylids) and discuss the resulting implications for osteoderm function in crocodylians.

MATERIALS AND METHODS

The dorsum of sixty-five specimens from three species of alligatorid (American alligator, Alligator mississippiensis Daudin 1802; Yacare caiman, Caiman yacare Daudin 1802; smooth- fronted caiman, Paleosuchus trigonatus Schneider 1801) and two species of crocodylid (American crocodile, Crocodylus acutus Cuvier 1807; saltwater crocodile, Crocodylus porosus Schneider 1801) was photographed (Appendix A). Because the scutellation pattern in crocodilians is set very early in development (Alibardi and Thompson 2000), the maturity of the specimens was not considered an issue. All measurements were taken using ImageJ (http://imagej.nih.gov/ij/). Following the practice set by Ross and Mayer (1983), precaudal scute row 1 corresponds to the

posterior sacral osteoderm-vertebra complex and precaudal row 18, if present, corresponds with the anteriormost osteoderm-vertebra (Figure 1.1). Precaudal 17 is the anteriormost row that was present in at least some individuals of all taxa, though it was missing from some Crocodylus acutus. All other crocodilian species were qualitatively observed, but lacked sufficient sample sizes for statistical analyses. Specimens used for this study are housed in the herpetology collections of the Florida Museum of Natural History, University of Florida (UF) and the Museum of Vertebrate Zoology, University of California, Berkeley (MVZ), and were preserved as wet

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whole specimens or dry skins (Appendix A). The statistical analyses detailed below were carried out in R version 3.2.2 (R Core Team, 2015).

Variation in scute dimensions

To assess intra-individual scute variability, length and width measurements were taken from midline scutes of precaudals 7 through 11 (sensu Ross and Mayer 1983) resulting in 10 length and 10 width measurements per specimen (Appendix A). Because absolute variance scales with the mean and individuals of different body sizes were used, length and width measurements were divided by the average scute length for each specimen to assess relative variation. Variability among species was tested using a slightly modified version of Levene’s test, which is not sensitive to non-normal data (Levene 1960; Van Valen 2005), in which the absolute difference between each measurement and the mean was calculated and then used in a one-way ANOVA using the lme and anova functions in the ‘nlme’ R package (Pinheiro et al. 2017). I compared a non- phylogenetic model, a Brownian motion (BM) phylogenetic model, and an Ornstein–Uhlenbeck (OU) phylogenetic model using the phylogeny shown in Figure 1.1. The model with the lowest Akaike information criterion (AIC) value was favored as the best model. A Tukey HSD test was then performed using the glht function from the ‘multcomp’ package (Hothorn et al. 2008) to determine which particular species had significantly different variation.

Bilateral fluctuating asymmetry of carapace

Bilateral asymmetry was most evident when viewing each row individually, where there could be up to two extra or missing scutes on one side relative to the other (Figure 1.1 C). The asymmetry of any one scute row was apparently independent of the asymmetry in any other row with no systematic trend toward one side or the other as determined by a Student’s t-test on the total number of scutes on the left and right sides (p > 0.2 for all taxa and p > 0.8 in Crocodylus acutus; Appendices A and B). For example, one row might have one more scute on the right side and the adjacent row might have one more scute on the left side, and so forth, such that the total

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number of osteoderms on the left and right side are equal or nearly equal. A population of such individuals would be indistinguishable from one in which every row had exactly the same number of scutes on the left and right sides if one only considered the total number of scutes on each side. Therefore, bilateral asymmetry for each specimen was defined as the absolute difference between the number of scutes on the left and right sides of each row summed for all rows from precaudal 3 through precaudal 17 to obtain a single asymmetry count for each specimen (Appendix A). Differences in the average asymmetry values for each species was tested using a one-way ANOVA using the same R functions as described above.

Scutellation pattern

To evaluate if the different scute distribution patterns seen in each species were predominantly rectangular or hexagonal, Voronoi diagrams were generated. Voronoi diagrams are a mathematical method of partitioning a plane filled with points in such a way that all points within the resulting polygons are closer to their source point than to any other source point on the plane (Okabe et al. 2000). These diagrams have many applications, including the investigation of topological order (e.g., Kram et al. 2010). In this case, the source points were the scute centroids, of which the XY coordinates were collected from dorsal view photographs using ImageJ (http://imagej.nih.gov/ij/). There were six scutes per row for seven rows corresponding to the widest portion of the carapace by scute number. This approach was used to account for individual variation in the particular rows that had six or more scutes. It was necessary to have at least six scutes per row because the outermost Voronoi polygons were not closed (Figure 1.2) resulting in unrealistic number of sides and so were not considered in the final portion of the analysis. Crocodylus acutus was excluded in this case because this species typically has only four scutes per row, aside from non-articulated accessory scutes. I used the R package, ‘deldir’ (Turner 2015) to generate the Voronoi diagrams (Figure 1.2), from the centroids, of which only inner polygons with all sides touching another polygon were further considered. A test pattern with a perfectly rectangular point distribution resulted in a

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Voronoi diagram with six-sided polygons wherein two sides at each “corner” were considerably smaller than the other four sides due to slight variation in the location of centroids as a result of imprecise input. I found that removing sides that were less than 5% of the average polygon perimeter maximized the observed difference in average number of sides between the test rectangular and test hexagonal point distributions, so this practice was applied to the scute data. I then counted the resulting number of polygons with four, five, six, or more sides for each specimen and pooled the data for each species.

RESULTS

Variation in scute dimensions

For both length and width, Paleosuchus trigonatus was found to have the highest degree of variation, Crocodylus acutus had the second highest and all others had comparatively low variation (Figure 1.3 A). Variation in the scute length of Paleosuchus trigonatus was significantly different from all other species (Table 1.1) and Crocodylus acutus was significantly different from Caiman yacare and Crocodylus porosus. However, the scute width of both Paleosuchus trigonatus and Crocodylus acutus was significantly different from all other species including each other. Variation in both scute length and width fit a BM model (AIC = -2433 & -1503) better than the OU (AIC = -7431 & -6501) and non-phylogenetic models (AIC = -2144 & -1215).

Lateral asymmetry of carapace

All models had nearly identical AIC values (non-phylogenetic = 270, BM = 270, OU = 272), so the non-phylogenetic model was favored, because it has the fewest parameters. There was a significant difference in the average asymmetry among species (ANOVA, F = 17.3, df = 4, p < 0.0001), and the results of the pairwise-t-test indicates Crocodylus acutus has a significantly higher level of asymmetry than all other species (p < 0.001; Figure 1.3 B), while all other species were indistinguishable (p > 0.8). Also of note is that Crocodylus acutus is the only species

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observed here to have scutes from the same row of the dorsal carapace that are cranio-caudally offset from each other (Figure 1.1 C).

Rectangular vs. pentagonal/hexagonal scute arrangement

Alligator mississippiensis, Caiman yacare, and Crocodylus porosus all have a strongly rectangular scute distribution pattern (Figure 1.2 A), demonstrated by the fact that the majority (72%-81%) of the Voronoi polygons, as well as the scutes themselves, are 4-sided (Figure 1.1 A). Conversely, in Paleosuchus trigonatus, pentagonal polygons are the most abundant (44%; Figure 1.2 B) and this species has a much higher proportion of hexagonal polygons (26%) than the other species (1%-4%). Personal observation of species not analyzed here suggests that Paleosuchus trigonatus is the only extant crocodilian, including its close relative Paleosuchus palpebrosus, to have a distinctly non-rectangular arrangement of scutes. However, it should be noted that there was considerable intraspecific variation within Paleosuchus trigonatus, with some individuals possessing a more rectangular arrangement.

DISCUSSION

Scute dimensions are more variable in Crocodylus acutus and Paleosuchus trigonatus than in Alligator mississippiensis, Caiman yacare, and Crocodylus porosus. However, only Crocodylus acutus exhibits significantly greater fluctuating asymmetry relative to other species,

corroborating previous qualitative observations for this species (Brazaitis 1973; Ross and Mayer 1983; Garcia-Grajales et al. 2009; Thorbjarnarson 2010) and suggesting that Crocodylus acutus may have experienced relaxed selection on dorsal osteoderm morphology. As one of the larger species of living crocodilians (maximum length 6-7ft), Crocodylus acutus may experience reduced competition with other crocodilian species and predation on mature individuals (Thorbjarnarson 2010; Somaweera et al. 2013). Predation would still be high for hatchlings and smaller juveniles (Somaweera et al. 2013) but, an osteoderm carapace does not develop until well after hatching in

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crocodilians (Vickaryous and Hall 2008; personal observation), so it could not play a role in hatchling survival. Crocodylus porosus and Alligator mississippiensis are also among the larger species of extant crocodilians (6-7ft and 4.5ft respectively; Elsey and Woodward 2010; Webb et al. 2010) and so might be expected to experience a similar reduction in predation. However, adults and subadults of both sexes in Crocodylus porosus are known to engage frequently in intraspecific aggression (Bustard and Maharana 1983; Lang 1987; Brien et al. 2013a, 2013b), and it is well established that Alligator mississippiensis of both sexes engage in, and are victim to, cannibalism (Rootes and Chabreck 1993; Delaney et al. 2011). Crocodylus acutus, on the other hand, was suggested previously to be relatively less aggressive than other crocodilians based on anecdotal evidence (Neil 1971), and a literature search only resulted in two reported occurrences of cannibalism in this species (Richards and Wasilewski 2003). If the fluctuating asymmetry in Crocodylus acutus is the result of relaxed selection, then decreased intra- and interspecific aggression experienced by this species suggests that the osteoderms in most other species may primarily serve as a defense against other crocodilians. However, more work needs to be done on the behavior of Crocodylus acutus and other less well-studied species to establish whether the apparent difference in behavior is real or simply a reporting bias. There is also the possibility that the random gaps in the carapace left by missing osteoderms may be an adaptation for increased flexibility, particularly for lateralized behaviors (Bisazza et al. 1998; Reimchen et al. 2008). However, Crocodylus acutus is not yet known to exhibit any flexibility dependent behaviors, such as the “death roll” (Fish et al. 2007; Blanco et al. 2015), with greater frequency than other crocodilian species, further highlighting the need for more comparative research on crocodilian behavior. In contrast to Crocodylus acutus, variation in scute size in Paleosuchus trigonatus appears to be linked to the lateral offset of osteoderms in adjacent rows (Figure 1.1 C). This scutellation pattern results in relatively more osteoderms with pentagonal and hexagonal shapes as well as the absence of continuous cranio-caudal hinge-lines between osteoderms, which may make the 9

carapace more rigid than in other taxa. It is uncertain why this might have occurred in Paleosuchus trigonatus. As one of the smallest and most terrestrial species of living crocodilians, possibilities include conferring a more rigid carapace as a better defense against more abundant, larger, terrestrial predators or as a better bracing system for terrestrial locomotion (Frey 1988; Salisbury and Frey 2000). However, qualitative observations of scute morphology suggests other relatively small and terrestrial taxa lack the unique morphology of Paleosuchus trigonatus, including its close relative Paleosuchus palpebrosus. Ultimately, too little is known about the behavior and ecology of Paleosuchus (Medem 1958; Magnusson and Lima 1991; Magnusson and Campos 2010) or about the developmental causes and mechanical consequences of osteoderm morphology in Paleosuchus trigonatus to draw causal conclusions. Clearly, the unusual morphology of the dorsal carapace warrants further investigation. Hypotheses of a role for osteoderms in physiological functions or terrestrial locomotion are more difficult to evaluate in this context because there are few comparative data published. Rather, the majority of information about crocodilian physiology and behavior are generalizations stemming from a few well-studied taxa (e.g., Smith 1979, Somaweera et al. 2013). Qualitative observation of other species not analyzed in this study suggests that Crocodylus moreletii, Crocodylus acutus, and Paleosuchus trigonatus are the only extant crocodilians to exhibit the morphologies described here (Medem 1958; Brazaitis 1973; Ross and Mayer 1983; Garcia- Grajales et al. 2009; Thorbjarnarson 2010; pers. obs.). Hopefully future studies of rare and cryptic taxa will reveal whether these three species are unique in other respects as well.

CONCLUSIONS

Variation in the dimensions, bilateral fluctuating asymmetry, and centroid positions of dorsal scutes were examined for five species of crocodilians. Alligator mississippiensis, Caiman yacare, and Crocodylus porosus have regularly spaced rectangular scutes with comparatively low variation. In contrast, Crocodylus acutus has more variably sized scutes with an asymmetrical

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scute distribution. Because relaxed selection can result in an increase in fluctuating asymmetry, the pattern seen in Crocodylus acutus is consistent with a reduction or loss of function of osteoderms in this species. The greater fluctuating asymmetry in conjunction with behavioral differences between Crocodylus acutus and the other species studied here suggests that in most species the dorsal osteoderms primarily function as defensive structures against conspecifics and other crocodilians. It is clear, however, that more comparative research on crocodilian behavior and physiology is necessary to evaluate other hypotheses of osteoderm function. Paleosuchus trigonatus, among the smallest and most terrestrial crocodilians, shows novel scute morphology as a result of the lateral offset of osteoderms in adjacent rows. The resulting carapace morphology, which lacks continuous cranio-caudal hinges between osteoderms, may be more structurally rigid than in other species, but the cause of this shift in morphology is still unclear. As deviants from the ‘average’ crocodilian scute morphology, both Crocodylus acutus and Paleosuchus trigonatus provide new insights into the function of osteoderms and these species are potentially interesting new avenues of research on crocodilian behavior.

W Am Pt Cy Ca Cp L Am - < 0.001* 0.992 < 0.001* 0.952

Pt < 0.001* - < 0.001* <0.001* < 0.001*

Cy 0.870 < 0.001* - < 0.001* 0.997

Ca 0.082 0.348* 0.004* - 0.015*

Cp 0.964 < 0.001* 1.000 0.043* -

Table 1.1: Corrected P-values for between-species comparisons of variation in length (L: bottom left) and width (W: top right). Am = Alligator mississippiensis, Pt = Paleosuchus trigonatus, Cy = Caiman yacare, Ca = Crocodylus acutus, Cp = Crocodylus porosus.

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Figure 1.1: Representative dorsal carapace morphologies including (A) a symmetrical rectangular distribution of scutes seen in Caiman yacare, UF 120665, (B) a symmetrical carapace with laterally offset scutes in Paleosuchus trigonatus, UF 55873, and (C) an asymmetrical carapace seen in Crocodylus acutus, UF 87575, with an example of craniocaudal offset of scutes from the same row visible near the anterior region of the carapace. In all views anterior is toward the top of the page and the scale bar equals 1 cm. The phylogenetic tree represents the consensus relationships of all taxa included in this study from multiple sources (Brochu 1997; Gatesy et al. 2003; Roos et al. 2007; Oaks 2011). Precaudal (PC) rows 3, 7, 11, and 17 are labeled on A and B. The arrow in C indicates a scute on the left side that has expanded anteriorly so that it is in contact with scutes from both precaudal rows 13 and 14 on the right side.

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Figure 1.2: A and B show example Voronoi diagrams represented by (A) Caiman yacare, UF 120665 with a well-ordered, rectangular arrangement and (B) Paleosuchus trigonatus, UF 55873 with a more disordered arrangement of largely pentagonal and hexagonal polygons. The silhouette shows the region examined for this portion of the study in grey and the orientation corresponding to the Voronoi diagrams. C shows the frequency with which polygons with different numbers of sides occur in Voronoi diagrams generated from each species. 13

Figure 1.3: Boxplots showing the (A) size corrected relative variability of scute width and (B) the degree of bilateral fluctuating asymmetry as measured by the sum of the difference in the number of scutes on the right and left sides of the carapace. Asterisks indicate species which are significantly different from all others.

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Chapter 2: Late Ontogenetic Development of Osteoderms and Their Role in Social Behavior

All extant and most extinct crocodylomorphs possess bony carapaces made up of articulating osteoderms, which are most commonly described as defensive structures against predators. Yet, qualitative observations in the literature suggest that the carapace of extant crocodilians does not fully mature into a completely imbricated structure until well after predation is greatest during the hatchling stage. If osteoderms primarily serve a defensive function, strong selection pressure should yield an earlier appearance in ontogeny. Therefore the absence of osteoderms in hatchlings requires an alternative explanation. Many aspects of crocodilian locomotor, social, and feeding behavior are known to change markedly throughout ontogeny and would be expected to exert different kinds of selective pressures on crocodilians as they grow. Alternative hypotheses of osteoderm function can be tested in part by tracking the completion of growth in the osteoderm carapace in relation to the aforementioned shifts in ecology and inferred selective regime. I examined X-ray computed tomography (CT) data from juveniles and adults of eight species of extant crocodilians including two ontogenetic series of Crocodylus niloticus (N=6) and Caiman crocodilus (N=4). Specimens were coded for total body length, as well as osteoderm shape and the presence or absence of contact among osteoderms. Osteoderms were considered to have mature morphology when all nuchal and dorsal osteoderms were rectangular and fully articulated mediolaterally and overlapping craniocaudally. The timing of osteoderm initial growth and maturation was evaluated with respect to five hypotheses of function and development: (1) osteoderms assist in thermoregulation in larger individuals; (2) osteoderms aid in terrestrial locomotion in larger individuals; (3) osteoderm growth is constrained by diet; (4) hatchlings are protected by adults and predator self-defense is only necessary later in life; (5) osteoderms serve as armor during intraspecific conflicts over resources. I found that the absolute body length at the time of carapace maturation varies by species as a consequence of differences in maximum body length, but generally corresponds to a stage when individuals begin competing with adults for territories and resources, shortly before sexual maturity. The observed ontogenetic pattern across

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sampled species is more consistent with osteoderms acting as defensive structures against conspecifics than any other proposed hypothesis.

INTRODUCTION

Osteoderm function has fascinated many biologists in part because of the ubiquity of osteoderms and analogous structures among some taxonomic groups in spite of documented and suspected physiological costs. Studies of crocodilians and lizards demonstrates that osteoderms can severely reduce flexibility, which can hinder attempts to catch prey or escape predators capable of penetrating the osteoderm covering (Frey 1988; Salisbury and Frey 2000; Losos et al. 2002). Dermal ossifications are also suspected to have high nutrient costs (Giles 1983; Spence et al. 2012) and to significantly alter body density in aquatic taxa, which can be a detriment under some circumstances (Houssaye 2009; Myhre and Klepaker 2009). This raises the question of why so many taxa develop dermal ossifications and maintain them for long periods of geologic history. The most popular interpretation of osteoderm function is as armor for defense against predators, to the extent that the terms ‘osteoderms’ and ‘armor’ are sometimes treated as synonyms (e.g., Huxley 1859; Ross and Mayer 1983; Yang et al. 2012). Research on analogous structures in some fish confirms the original presumption that bony dermal plates act as a predator defense mechanism in those taxa (e.g., Bergstrom and Reimchen 2003). Yet, in a few taxa dermal ossifications originally presumed to act as armor have been found to be structurally inadequate to protect against most predators (de Buffrénil et al. 1986; Main et al. 2005; Marinho 2007; Butler & Galton 2008; Broeckhoven et al. 2015). Furthermore, several additional or alternative hypotheses of osteoderm function have been proposed for various taxa, including thermoregulation (Seidel 1979; de Buffrénil et al. 1986; Farlow et al. 2010; Hayashi et al. 2010; Broeckhoven et al 2015; Owerkowicz 2016; Clarac et al. 2017), structural support of the vertebral column (Frey 1988; Schwarz-Wings et al. 2009; Buchwitz et al. 2012), social display (Main et al. 2005; Hayashi et al. 2010), metabolic regulation (Jackson et al. 2003; Marinho 2007; Dacke et al. 2015), and defense

16 in intraspecific aggression (Broeckhoven 2017a; English 2018). Therefore, osteoderms should not automatically be assumed to act as defensive structures in all taxa. All adult crocodilians have osteoderms, and those cover an extensive portion of the body in some species (Fig. 1; Ross and Mayer 1983; King and Brazaitis 1971). Yet, osteoderms develop relatively late in crocodilian ontogeny compared to the rest of the skeleton, with ossification beginning after crocodilians hatch (King and Brazaitis 1971; Vickaryous and Hall 2008). This pattern of osteoderm development does not fit with a predator defense function. Hatchlings have a fairly high mortality rate due to predation, but adult crocodilians are rarely killed by predators other than humans (Somaweera et al. 2013). Several other aspects of crocodilian biology change through ontogeny and may explanation the delayed ontogenetic development of osteoderms as well as provide insight into osteoderm function. First, some aspects of crocodilian biology change through ontogeny as a consequence of allometric scaling, because certain mechanically relevant dimensions change at different rates. For any organisms of a similar shape, larger individuals have less surface area of any given surface relative to their body volume than small individuals, altering any physiologic or mechanical process that depends on surface to volume ratios (McGowan 1983; Schmidt-Nielsen 1984; Alexander 1989; Vogel 2009). These principles may apply if osteoderms assisted with thermoregulation (Seidel 1979; Clarac et al. 2017, 2018) or if they helped to brace the vertebral column during terrestrial locomotion (Frey 1988; Schwarz-Wings et al. 2009; Buchwitz et al. 2012). Because crocodilian species vary little in their postcranial anatomy (Meers 2003; Allen et al. 2014) and analysis of Alligator mississippiensis suggests crocodilian postcranial dimensions tend to scale isometrically or nearly isometrically through ontogeny (Livingston et al. 2009; Allen et al. 2010), allometric changes in biology should be expected to occur at approximately the same absolute body size in all taxa regardless of differences in life history. The rate at which body temperature changes is dependent on the surface area of the skin relative to body volume, such that small crocodilians warm and cool much faster than large crocodilians. Though, the rate of heat transfer is also dependent on the thermal conductivity of the material the heat is passing 17

through – in this case, the dermis. Seidel (1979) was the first to propose that osteoderms might be an adaptation for thermoregulation by increasing the rate at which heat is absorbed while basking. Skin and body temperature measurements of alligators of different sizes with and without osteoderms suggests that osteoderms do change the thermal conductivity of the dermis (Owerkowicz 2016). Conversely, finite element analysis failed to find a significant effect of osteoderms on heat transfer, though the authors still suggested that osteoderms might play a role in thermoregulation through their vascularization (Clarac et al. 2017). If osteoderms are an adaptation for thermoregulation via changing the thermal conductivity of the skin and thus changing the rate of heat transfer, then the timing of osteoderm development should be more dependent on absolute body size than on life history events, such as reaching sexual maturity. In that case, different crocodilians species that reach maturity at different body sizes should still complete their osteoderm development at roughly the same body size. A similar line of reasoning might be applied to the hypothesis originally proposed by Frey (1988) that osteoderms help to brace the trunk region during terrestrial locomotion. The strength of muscle and bone is proportional to their cross-sectional area, whereas the force exerted on the body due to gravity is proportional to the body volume (McGowan 1983; Schmidt-Nielsen 1984; Alexander 1989; Vogel 2009). Therefore, in the absence of shape changes, larger crocodilians should have greater difficulty supporting their body weight on land than smaller crocodilians. If osteoderms help to support the vertebral column and alleviate some of the stress on muscle and joints in larger individuals, then, once again, osteoderm development should be more closely tied to absolute body size rather than life history events. In addition to body size, crocodilians also undergo marked changes in diet and social behavior as they age. Young crocodilians have diets dominated by invertebrates, but vertebrates become a major food source in larger crocodilians (Hutton 1987; Thorbjarnarson 1993; Tucker et al. 1996; Horna et al. 2001; Platt et al. 2006, 2013; Wallace and Leslie 2008; Saalfeld et al. 2011). It is possible that the late development of osteoderms is a dietary constraint, in that hatchlings lack

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sufficient mineral resources from their diet to devote to osteoderm growth. If that is the case, then osteoderm growth should begin after the shift towards a vertebrate-based diet. Newly hatched crocodilians tend to stay hidden in vegetation close to the nesting area where their mothers, and other adults in some taxa, will respond to their distress calls by attacking predators (Hunt 1977; Staton 1978; Rodda 1984; Lang 1987; Cintra 1989; Thorbjarnarson and Hernández 1993; Whitaker 2007; Antelo et al. 2008; Campos et al. 2012; Somaweera and Shine 2012). After the hatchling phase, crocodilians disperse and must defend themselves against predators (Staton 1978; Rodda 1984; Lang 1987; Antelo et al. 2008; Campos et al. 2012; Somaweera et al. 2013). It is possible that osteoderms are essential for predator defense in juveniles after they lose parental protection, but would be of no benefit before that point. In this case, the completion of osteoderm development should coincide with dispersal from the nesting grounds. On the other hand, it is also possible that osteoderms act as a defensive armor during aggressive intraspecific encounters in adults competing for territories, nesting sites, and mates. Depending on the species, sub-adult crocodilians may begin competing with conspecifics for territory at the same time as dispersal or at a later point shortly before sexual maturity (Gorzula 1978; Hutton 1989; Tucker et al. 1997; Campos et al. 2006; Subalusky et al. 2009; Hanson et al. 2015; Balaguera-Reina et al. 2016). Brien et al. (2013a) also found that, in captivity, frequency and intensity of aggressive interactions tended to increase with age for several species and that bites tended to occur on the head, body, or tail. If osteoderms are a defense against other crocodilians, then osteoderm development should finish when competition for territories begins, well after dispersal from the nesting grounds in those species which separate those two events. A final social role for osteoderms is for visual display, as has been proposed for some non-crocodilian taxa (e.g., Stegosaurus; Main et al. 2005). However, as crocodilians mature, the growth of osteoderms does not make any distinct visible changes to their external appearance (pers. obs.), so a display function is considered unlikely. I used the timing of osteoderm development in crocodiles to test five hypotheses of primary osteoderm function. These are: (1) osteoderms are necessary for larger crocodilians to 19

thermoregulate as effectively as small individuals (Seidel 1979); (2) osteoderms are necessary for bracing the trunk of larger crocodilians during terrestrial locomotion (Frey 1988); (3) osteoderm growth is constrained by diet; (4) crocodilians change their predator defense strategy from crypsis and maternal protection to a passive armor defense during the juvenile stage; and (5) osteoderms are defensive armor for intraspecific conflicts.

MATERIALS AND METHODS

Specimens

Twenty-four X-ray computed tomography (CT) scans representing multiple developmental stages of eight species of extant crocodilians were obtained for this study (Table 2.1 & Appendix C). Sixteen scans were downloaded from CrocBase, an online repository for crocodilian CT scans (Hutchinson and Sumner-Rooney 2016). Five other scans were obtained from live animals for the purpose of medical diagnosis by St. Augustine Alligator Farm, and were subsequently donated upon request for this study. One specimen is housed in the herpetology collections of the Texas Memorial Museum and was scanned at The University of Texas High-Resolution X-ray CT Facility. The final two specimens came from the Texas A&M Biodiversity Research and Teaching Collections and were scanned at the Texas A&M College of Veterinary Medicine and Biomedical Sciences. Of the species included in the study, Crocodylus niloticus and Caiman crocodilus are represented by comparatively well-sampled ontogenetic series which were used for further hypothesis testing. The remaining species were compared with the expectations derived from the well-sampled species. Scanning details and specimen numbers are reported in Table 2.1 and Appendix C. All osteoderms were segmented in Avizo 8.1.

Assessment of maturity

Body size in relation to size at sexual maturity was used as a proxy for overall maturity of specimens in part because size data were available for all specimens and for all taxa in the literature, and because research of growth patterns shows size to be a more appropriate measure of

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maturity in crocodilians than age (Rootes et al. 1991; Lance 2003; Woodward 2010). Total body length was used even though it is one of the less reliable measures of overall size, because total body length was more readily available for the specimens and in the literature, and because rough approximations of body size were adequate for this study. In some cases snout-vent-length of Crocodylus niloticus was converted to total length using the equations provided by Hutton (1987). Carapace maturity was determined in part by the presence of osteoderms in all osteoderm-bearing regions of the body: nuchal/cervical, dorsal, caudal, ventral, and appendicular (Figure 2.1). Maturity was also ascertained (1) when osteoderms clearly overlapped anteroposteriorly, taking into account any flexing of the specimen, and (2) when osteoderms were closely articulated along the mediolateral edges resulting in approximately rectangular osteoderms with morphology corresponding with the morphology of the overlying keratinous scutes (Figure 2.2). The term ‘juvenile’ is applied to all specimens smaller than those assessed to be mature. I established the relative timing of carapace maturation in relation to major life history events and ontogenetic shifts in ecology reported in the literature (Staton & Dixon 1977; Gorzula 1978; Hutton 1987, 1989; Thorbjarnarson 1993, 1994; Magnusson and Sanaiotti 1995; Wallace and Leslie 2008). These results were then compared to the previously described predictions based on each hypothesis of osteoderm function and development.

RESULTS

General developmental patterns in all species

The least mature specimens had small, widely spaced, oval osteoderms in the nuchal shield, the medial columns in the dorsal shield, and at the base of the tail, but lacked osteoderms in the limbs, ventral surfaces, the lateralmost column of the dorsal shield, and the more distal portion of the tail, although a small amount of individual variation was observed (Figure 2.1A). In juveniles, nuchal osteoderms were more morphologically mature than the dorsal osteoderms and, within the dorsal carapace, medial osteoderms were more mature than lateral ones. In all species except

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Crocodylus moreletii, individuals large enough to be sexually mature possessed osteoderms in the neck, the dorsal and ventral surfaces of the trunk, the tail, and the limbs (Table 2.2). These specimens also had tightly articulated/overlapping, roughly square-shaped osteoderms in the nuchal shield, dorsal shield, and at the base of the tail. Other aspects of mature osteoderm morphology and articulation were more variable among species. In Crocodylus moreletii, appendicular and ventral osteoderms were only present in a single individual much larger than the minimum reproductive size (Table 2.2). Furthermore, osteoderms of Crocodylus moreletii were generally more irregularly shaped and widely spaced than other species.

Life history & osteoderm development in Caiman crocodilus and Crocodylus niloticus

Life history timelines of Crocodylus niloticus and Caiman crocodilus compiled from data reported in the literature are summarized in Figure 2.3. Most of the reports of body size at sexual maturity were based on females, though when males were included in a study they were reported to mature at the same size as females (e.g., Wilkinson and Rhodes 1997; van Weerd 2010). As with other crocodilians, Crocodylus niloticus and Caiman crocodilus spend their hatchling period in the general vicinity of the nesting grounds, before dispersing at total body lengths of 1.2 m and 0.7 m respectively (Gorzula 1978; Hutton 1989). Dietary shifts from primarily invertebrates (variable taxa depending on local ecology) to primarily vertebrates (largely fish) occurs at total lengths of approximately 0.6-0.9 m for Caiman crocodilus and 1.2 m for Crocodylus niloticus (Hutton 1987; Thorbjarnarson 1993; Wallace and Leslie 2008). Note that the shift in diet is likely more gradual than is depicted here and that there is another dietary shift later in ontogeny when invertebrate consumption is greatly reduced again, which is not marked in Figure 2.3. After leaving the nesting area, Caiman crocodilus begin competing with conspecifics for territory well before the occurrence of reproductive maturity at 1.2 m (Staton & Dixon 1977; Gorzula 1978; Thorbjarnarson 1994; Magnusson and Sanaiotti 1995). Crocodylus niloticus, on the other hand, enter a temporary ‘dispersal phase’ when they reach a total length of about 1.2 m, during which juveniles avoid contact with adults and lack distinct territories, before they begin

22 competing with adults for resources at total length of 2.2 m, followed shortly after by sexual maturity at 2.5 m (Hutton 1989). In both species, osteoderms began ossification during the nestling phase (Figure 2.3), though osteoderm growth in Caiman crocodilus appears to begin earlier in development than in Crocodylus niloticus, with osteoderms appearing larger and more developed in a 30 cm caiman hatchling (~25% length at sexual maturity) than in an 80 cm crocodile hatchling (~32% length at sexual maturity). Final osteoderm maturation into a fully articulated carapace occurs at ~0.6-0.7 m in Caiman crocodilus and ~1.5-2 m in Crocodylus niloticus, shortly before individuals begin competing with adult conspecifics for territories (Figure 2.3).

DISCUSSION AND CONCLUSIONS

What limited data is available suggests crocodilian postcranial anatomy varies little among species (Meers 2003; Allen et al. 2014) and through ontogeny (Livingston et al. 2009; Allen et al. 2010). Consequently, allometric principles of mechanics and heat transfer predict that if osteoderms assist in thermoregulation or in structural support of the vertebral column, then osteoderms of both crocodilian species evaluated here should finish maturation at roughly the same body size. Yet, osteoderm maturation in Crocodylus niloticus occurs at more than twice the body length as in Caiman crocodilus (Fig. 3 and Table 3). This is not a definitive refutation of a thermoregulatory or trunk bracing function for crocodilian osteoderms, because some as-yet- undocumented differences in the anatomy or ecology of both species might lead to different functional requirements which could then lead to different developmental patterns. But, the dramatic difference in body size when osteoderms mature does weaken the strength of these hypotheses as plausible explanations of osteoderm primary function. More species and more lines of evidence need to be evaluated. If osteoderm development is delayed because of availability of nutrients in the early post-hatching diet, then ossification should begin after juveniles change their diet. Although many of the specimens in this study were raised in captivity where a primarily vertebrate diet is common

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(Huchzermeyer 2003), three of the juvenile caimans were wild-caught in their native range. Of those caimans two would have been young enough to still have a primarily invertebrate diet, yet still demonstrated the beginning of carapace development (Fig. 1A). Furthermore, osteoderm development in wild-caught and captive-raised specimens were not appreciably different from each when comparing similar relative ontogenetic stages. It is possible that osteoderm mineralization is merely slow at first before accelerating when juveniles start consuming vertebrates, but, in such a case, there should still be a difference between wild and captive individuals. Therefore the dietary constraint hypothesis is not supported. The maturation of the osteoderm carapace occurs well after juvenile Crocodylus niloticus lose maternal protection (Fig. 3). This suggests that armor does not replace maternal protection as predator defense in juveniles. Instead, osteoderm maturation coincides well with the period of time when juveniles start exhibiting territorial behavior. In Caiman crocodilus, Gorzula (1978) noted a marked increase in the occurrence of injuries consistent with intraspecific conflicts at a total length of about 70 cm, which is approximately the same size at which osteoderms fully mature (Fig. 3, Table 3). Smaller caimans lacked any such scarring (Gorzula 1978). The relative timing of carapace formation supports a primary function as defensive armor in intraspecifi¬c conflicts in the sampled Crocodylia, with the exception of Crocodylus moreletii. This conclusion is further supported by research on patterns of intraspecific variation, in which Crocodylus acutus, a species that is purportedly less aggressive towards conspecifics, demonstrated evidence of relaxed selection on the osteoderm carapace (English 2018). Research on the cordylid lizard, Hemicordylus capensis, similarly found that osteoderms develop in this species at sexual maturity (Broeckhoven et al. 2017a). Broeckhoven et al. (2017a) also found that osteoderm volume was much greater in males than in females, consistent with the observation that intraspecific aggression is greater among males than among females in cordylids as opposed to crocodilians in which both sexes fight for resources (Brien et al. 2013b; Brien 2015). The irregularly shaped and widely spaced osteoderms of Crocodylus moreletii may be another case of relaxed selection on the dorsal

24 carapace, though not enough is known about their behavior to assess if there could be a similar cause. Bite marks on crocodyliform fossils dating as far back as the Cretaceous suggests that intraspecific aggressive encounters among crocodilians and their close relatives has been an important selective force for millions of years (Buffetaut 1983; Mackness and Sutton 2000; Avilla et al. 2004; Katsura 2004; Martin 2013). Nevertheless, osteoderms should not be assumed to act as armor against conspecifics in all crocodilian relatives. Many of the earliest-known crocodylomorphs only had two rows of osteoderms along their spinal column (Irmis et al. 2013), and some purported herbivorous taxa had extensive osteoderm covering in spite of a lack of any obvious means of inflicting injury on each other (e.g., Simosuchus; Hill 2010; Kley et al. 2010; Sertich and Groenke 2010). It is hypothesized here that osteoderms originated in the ancestors of extant crocodilians in response to an as-yet undetermined selective force, and that in subsequent lineages, such as the crown clade, osteoderms were exapted for defense against conspecifics in territorial aggression. The results of this research adds to a growing body of literature demonstrating the importance of intraspecific interactions in shaping anatomy and emphasizes the importance of testing hypotheses of biological function.

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Sexual Taxon N Size Range Maturity Maximum Size Alligator sinensis 1 150 cm 100 cm 200 cm Caiman crocodilus 4 30-90 cm 150 cm 270 cm Caiman latirostris 1 170 cm 150 cm 300 cm Crocodylus mindorensis 2 90 cm 150 cm 250 cm Crocodylus moreletii 5 145-205 cm 150 cm 450 cm Crocodylus niloticus 8 80-230 cm 250 cm 600 cm Crocodylus suchus 1 230 cm 250 cm 600 cm Osteolaemus tetraspis 3 120-150 cm 100 cm 200 cm

Table 2.1: Species, number of specimens (N), and range of total body lengths of specimens used in this study compared with average size metrics for each species. Approximate sizes at sexual maturity and maximum body sizes were taken from the following sources: Moulton et al. (1999), Thorbjarnarson et al. (2001), Eaton (2009, 2010), Fergusson (2010), Jiang (2010), Platt et al. (2010), van Weerd (2010), Velasco and Ayarzagüena (2010), Verdade et al. (2010).

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Specimen Nuchal Dorsal Caudal Ventral Limbs Taxon Length Number LA DVO LA DVO LA DVO P/A LA P/A Alligator sinensis A07012 150 cm 1 1 1 1 1 1 1 0 1 Caiman crocodilus TCWC 23569 30 cm 1 1 0 0 0 0 1 0 0 Caiman crocodilus TMM 28671 50 cm 1 1 0 0 0 0 0 NA 0 Caiman crocodilus RVC-JRH-FCC1 85 cm 1 1 1 1 1 1 1 1 1 Caiman crocodilus TCWC 19285 90 cm 1 1 1 1 1 1 1 0 0 Caiman latirostris A07056 170 cm 1 1 1 1 1 1 1 1 1 Crocodylus mindorensis A03033 ~90 cm 0 0 0 1 0 0 0 NA 1 Crocodylus mindorensis A03036 ~90 cm 0 0 0 1 0 0 0 NA 0 Crocodylus moreletii RVC-JRH-FMC1 145 cm 1 ? § 1 0 0 0 0 NA 1 Crocodylus moreletii RVC-JRH-FMC3 165 cm 1 0 0 0 0 0 0 NA 0 Crocodylus moreletii RVC-JRH-FMC5 170 cm 1 0 0 0 0 0 0 NA 0 Crocodylus moreletii RVC-JRH-FMC2 205 cm 1 0 1 1 0 0 1 0 1 Crocodylus moreletii RVC-JRH-FMC4 ? 1 0 0 0 0 0 0 NA 0 Crocodylus niloticus RVC-JRH-NNC2 80 cm 0 0 0 0 0 0 0 NA 0 Crocodylus niloticus RVC-JRH-FNC1 80 cm 1 0 1? * 0 0 0 0 NA 0 Crocodylus niloticus RVC-JRH-NNC1 100 cm 1 1? ǂ 0 0 0 0 0 NA 0 Crocodylus niloticus RVC-JRH-FNC4 120 cm 1 ? ǂ 0 0 0 0 0 NA 0 Crocodylus niloticus RVC-JRH-FNC2 130 cm 1 0 0 0 0 0 0 NA 0 Crocodylus niloticus RVC-JRH-FNC6 140 cm 1 1 1 1 1 0 0 NA 1 Crocodylus niloticus RVC-JRH-FNC0 230 cm 1 1 1 1 1 1 1 0 1 Crocodylus suchus 14006196 230 cm 1 1 1 1 1 1 1 0 1 Osteolaemus tetraspis RVC-JRH-FDC2 120 cm ? § ? § 1 1 1 1 1 0 1 Osteolaemus tetraspis RVC-JRH-FDC3 135 cm 1 1 1 1 1 1 1 0 1 Osteolaemus tetraspis RVC-JRH-FDC1 150 cm ? § ? § 1 1 1 1 1 0 1

Table 2.2: Maturity scoring of osteoderms by region. Only the base of the tail was considered for the caudal region. LA = laterally articulated; DVO = dorsoventrally overlapping; P/A = presence/absence; 1 = present; 0 = absent; NA = not applicable; ? = uncertain/indeterminable due to [*] low scan resolution, [ǂ] excessive flexion, or [§] absence or partial absence of skin.

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Figure 2.1: Osteoderm anatomy in dorsal view in a (A) yearling (TCWC 23569), a (B) slightly older juvenile (TMM 28671), and a (C) subadult (RVC-JRH-FCC1) Caiman crocodilus. The ventral shield (visible along the margins of the specimen) and appendicular osteoderms are only present in C. Osteoderms along the back and tail are relatively narrower and more widely spaced in A and B, whereas the dorsal osteoderms in C are so closely articulated that they appear continuous in the scan. On the other hand, osteoderms in the nuchal shield are relatively closely articulating and rectangular in outline even in the smallest individual.

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Figure 2.2: Schematic showing the differences in morphology between immature (top) and mature (bottom) osteoderms. Osteoderms are seen in lateral view on the left and dorsal view on the right.

Figure 2.3: The timing of osteoderm maturation and the sizes of the specimens used in this study mapped onto the life histories of Crocodylus niloticus and Caiman crocodilus including primary source of food and social behavior.

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Chapter 3: Assessment of the comparative morphology of osteoderms and locomotor ecology in Crocodylomorpha

The relative degree of vertebral flexibility is an important aspect of vertebrate locomotion, and osteoderms have been demonstrated to reduce, but not eliminate, this flexibility. Crocodylomorphs span a wide range of locomotor ecologies, from marine taxa dominated by lateral bending to terrestrial taxa presumed to exhibit more dorsoventral bending of the spinal column. Theoretically, these different locomotor styles should result in morphological adaptations that allow flexibility in one direction and stability in another. Cursory observation suggests that terrestrial taxa had relatively narrow osteoderms whereas marine taxa had wider osteoderms, with amphibious taxa inhabiting an intermediate morphospace. Therefore, to investigate the influence of osteoderm aspect ratio on vertebral flexibility, I tested for a correlation between osteoderm aspect ratio and locomotor ecology across Crocodylomorpha, including sensitivity analyses to account for ambiguity in ecological classification. I also calculated estimates of phylogenetic signal, tested for allometry in a regression of length versus width, and qualitatively investigated within-individual variation to assess the impact of these factors on variation in osteoderm aspect ratio across taxa. The results show that there is no significant correlation between osteoderm aspect ratio and locomotor ecology and that the previously observed pattern is, in fact, an artifact of osteoderm allometry and phylogeny. Because osteoderms have been demonstrated to influence body flexibility, but flexibility is not a product of osteoderm aspect ratio, other aspects of osteoderm anatomy must be the determining factors.

INTRODUCTION

The axial skeleton is an important component of animal locomotion, in which the relative flexibility and rigidity of the skeleton can have substantial effects on the energy required to flex or maintain posture as well as determining limits on the magnitude and direction of bending (Rockwell et al., 1938; Frey, 1988; Gal, 1993; Salisbury and Frey, 2000; Molnar et al., 2014).

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Some taxa have relatively compact, rigid vertebral columns well suited for withstanding strong forces during locomotion, such as hopping in frogs and flight in birds (Norberg, 1990; Handrigan and Wassersug, 2007). On the other hand, pronounced vertebral bending can provide direct propulsion in swimming or limbless animals, or allow for greater stride length in running animals (Rockwell et al., 1938; Hildebrand, 1961; Fish, 1984; Bramble, 1989; Long et al., 1997). The direction of bending dependent on phylogenetic history in swimming taxa (e.g., cetacean vs. fish) and on posture in terrestrial animals, in which taxa running with a more parasagittal gait (e.g., most mammals) use more dorsoventral bending and taxa with a sprawling gait (e.g., most lizards) use lateral bending (Rockwell et al., 1938; Hildebrand, 1961; Fish, 1984; Bramble, 1989; Long et al., 1997). It should be noted, however, that both dorsoventral and lateral bending may occur in the same animal when walking at slower speeds (Shapiro et al., 2001; Schilling and Hackert, 2006). Axial flexibility is determined not only by the vertebral column, but also by the various tissues surrounding it, including dermal ossifications such as osteoderms (Frey, 1988; Salisbury and Frey, 2000; Losos et al., 2002; Schwarz-Wings et al., 2009). Osteoderms independently evolved multiple times and can be found in the skin of many vertebrate lineages, making up a significant yet understudied portion of the evolutionary history of the tetrapod skeleton (Hill, 2004; Vickaryous and Sire, 2009). They exhibit a wide range of morphological diversity and may vary in abundance, shape (e.g., rectangular, hexagonal, or rounded), relative and absolute size, aspect ratio, the presence or absence of keels or pitted ornamentation, as well as location on the body and articulation pattern (Hill, 2004; Vickaryous and Sire, 2009). The drivers of this diversity are not yet understood, but differing selective pressures for relative flexibility in animals with different locomotor ecologies may be a major selective force. Recent comparative studies (Broeckhoven, 2017a; English, 2018 and unpublished data) suggests that within some taxa, osteoderms are primarily an adaptive response to within species conflicts, but while this may explain the presence of osteoderms it does not account for variation in morphology among taxa. Biomechanical models as well as experimental removal of osteoderms from freshly killed crocodilians demonstrated that the presence of osteoderms 31 noticeably decreases body flexibility (Frey, 1988; Salisbury and Frey, 2000). Additionally, a comparative analysis of escape behavior in several species of armored lizards (Cordylidae) found that more heavily armored taxa had reduced capacity for running and maneuvering than less armored taxa (Losos et al., 2002). What has not yet been investigated is whether different osteoderm shapes and articulation patterns may influence the directionality of axial bending. Crocodylomorphs are an ideal group to study the potential relationship between osteoderm morphology and locomotor ecology, because osteoderms are present in nearly every species and they have a long evolutionary history in which they evolved diverse morphologies and ecologies, while leaving a relatively decent fossil record of articulated specimens (Naish, 2001; Hill, 2004; Vickaryous and Sire, 2009; Irmis et al., 2013). Their earliest fossils are from the Late Triassic and comprise remains of small, terrestrial predators that are frequently interpreted to be cursorial with a parasagittal gait (Clark, 1994; Nesbitt, 2011; Irmis et al., 2013). After surviving the end-Triassic extinction, crocodylomorphs diversified to inhabit many different ecological niches including the adoption of herbivory and the invasion of aquatic and marine habitats, with ecological diversity peaking during the Cretaceous (Clark, 1994; Naish, 2001; Irmis et al., 2013). The smallest known crocodylomorphs include the approximately 30cm long, terrestrial Gracilisuchus (Lecuona and Desojo, 2012), while the largest were estimated to be just over 10m long and included amphibious, ambush predators such as Deinosuchus (Farlow et al., 2005) and marine predators like Machimosaurus (Fanti et al., 2016). In addition to body size and habitat, crocodylomorphs are frequently characterized by their snout proportions which range from ‘short/blunt’ to ‘long and narrow’ to ‘long and broad/flat’ to ‘deep/laterally compressed’, with intermediate/generalized morphologies existing in between these extremes, each of which has independently evolved multiple times (Brochu, 2001, 2003; Naish, 2001; Wilberg, 2012). Several different modes of locomotion have been observed in living crocodilians – undulatory swimming, the sprawling gait, the high walk, and galloping/bounding (Manter 1940; Cott, 1961; Zug, 1974; Webb and Gans, 1982; Frey, 1984, 1988; Parrish, 1987; Grigg and Gans, 1993; Meers, 1999). The bounding gait is a relatively high speed terrestrial gait during which the 32

vertebral column experiences a large degree of dorsoventral bending (Cott, 1961; Zug, 1974; Singh and Bustard, 1976; Webb and Gans, 1982; Frey, 1984, 1988; Meers, 1999; Allen et al., 2014). It is restricted to some, and potentially all, taxa within Crocodylidae along with Gavialis, while seemingly absent in Alligatoridae, but even amongst those taxa in which it has been observed, bounding is apparently a rare behavior and is often restricted to juveniles (Cott, 1961; Zug, 1974; Singh and Bustard, 1976; Webb and Gans, 1982; Meers, 1999; Allen et al., 2014). All other locomotor categories have been observed in all of the well-studied taxa across Crocodylia and are presumed to occur in the poorly-studied taxa as well, with the caveat that the high walk is restricted to juveniles in Gavialis, though species likely differ in how frequently each locomotor mode is used (Bustard and Singh, 1977; Meers, 1999; Milàn and Hedegaard, 2010; Allen et al., 2014). Undulatory swimming and the terrestrial sprawling slide both involve small (low speed) to large (high speed) amounts of lateral bending of the vertebral column, particularly in the tail during swimming and in the trunk during the sprawling gait (Manter 1940; Fish, 1984; Frey, 1984, 1988; Grigg and Gans, 1993; Meers, 1999; Carpenter, 2009; Milán and Hedegaard, 2010). The high walk is a relatively low speed gait which involves raising the body off the ground and adopting a nearly erect posture that subjects the vertebral column to a relatively small degree of torsional and lateral bending forces (Frey, 1984, 1988; Gatesy, 1991; Meers, 1999; Salisbury and Frey, 2000; Carpenter, 2009). Dorsal osteoderms within Crocodylomorpha are typically rectangular with aspect ratios that vary greatly among taxa, and they are arranged in roughly parallel rows and columns with each row associated with a single vertebra (King and Brazaitis, 1971; Ross and Mayer, 1983; Vickaryous and Hall 2008; pers. obs.). In mature individuals, osteoderms overlap each other in the anterior-posterior direction facilitated by a smooth glide plane on the anterior edge of the dorsal surface, while the rest of the osteoderm exhibits some manner of pitted ornamentation and, in some taxa, a keel (Figure 1; Buffrénil, 1982; Sun and Chen, 2013; Buffrénil et al., 2015; Clarac et al., 2018; pers. obs.). Osteoderms of different taxa may vary dramatically in aspect ratio (pers. obs., e.g., Figure 4 in Sues et al., 2003 and Figure 5 in Puértolas-Pascual et al., 2015). The shape of 33

osteoderms (e.g., wide vs. narrow) may restrict bending in different directions, in which case crocodylomorphs using the same modes of locomotion should have similar osteoderm morphologies. Therefore I hypothesized that among extant and extinct crocodilians, lateral bending was more important for taxa that would have spent little time on land and dorsoventral bending would have been more important for upright, terrestrial taxa, while amphibious taxa most likely employed multiple gaits or a single rolling gait that incorporated bending of the spine in all directions and that these physical factors will manifest in the osteoderm morphologies. Based on preliminary observations, I expected marine taxa to have relatively wide osteoderms, terrestrial taxa to have relatively narrow osteoderms, and amphibious taxa to have equidimensional osteoderms. To test this hypothesis, I gathered length and width ratios of dorsal osteoderms from across Crocodylomorpha, representing the full range of known locomotor ecologies and I then tested for a correlation between aspect ratio and locomotor ecology. Additionally, I evaluated non- functional sources of morphological variation by testing for allometric patterns and for phylogenetic signal, and I estimated the minimum within-individual variation of osteoderm aspect ratio.

MATERIALS AND METHODS

Specimens

Photographs were taken of 38 specimens representing 10 extant and 26 extinct taxa housed in the following institutions: AMNH, the American Museum of Natural History, New York, U.S.A.; CM, the Carnegie Museum of Natural History, Pittsburgh, U.S.A.; CUP, the former FuJen Catholic University of Peking collection now housed at the Field Museum; MVZ, the Museum of Vertebrate Zoology at Berkeley, U.S.A.; NCSM, the North Carolina Museum of Natural Sciences, Raleigh, U.S.A.; NHMUK, the Natural History Museum, London, U.K.; SMF, the Senckenberg Naturmuseum, Frankfurt, Germany; SMM, the Science Museum of Minnesota, St. Paul, U.S.A.;

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UA, the Université d'Antananarivo, Antananarivo, Madagascar currently on loan to Stony Brook University, U.S.A.; UCMP, the University of California Museum of Paleontology, Berkeley, U.S.A.; UF, the Florida Museum of Natural History, Gainesville, U.S.A.; USNM, the National Museum of Natural History, Washington D.C., U.S.A. (Table 1). Because all living crocodylians are amphibious to a degree (Grigg and Gans, 1993) and have osteoderms of comparable aspect ratio (personal observation), only a subset of modern taxa were sampled determined by the availability of relatively larger (i.e., more mature), and well-preserved individuals. This, unfortunately, excluded the relatively unique Gavialis from the dataset, however this was offset by inclusion of the closely related and morphologically similar extinct Eogavialis (Buffetaut, 1982). When multiple specimens were available for a taxon, the largest individuals exhibiting the most mature morphology were selected for the analyses. Relatively flat specimens and isolated osteoderms were photographed dorsally and the resulting images were subsequently processed in ImageJ 1.50e (Schneider et al., 2012) to obtain measurements (see below). All other specimens were photographed a minimum of 40 times from different angles to produce photogrammetric models using AgiSoft PhotoScan Standard version 1.3.4 (2017). Osteoderm measurements were obtained from the 3D surface models in Meshlab version 2016.12 (Cignoni et al., 2008). Length and width measurements were obtained from up to seven randomly selected osteoderms along the dorsal midline in articulated specimens. In disarticulated specimens, osteoderms were measured if their morphology was consistent with dorsal midline osteoderms in articulated specimens of closely related taxa. In this study, width corresponds with the mediolateral direction and length corresponds with the anteroposterior direction (Figure 1). Measurements excluded the anterior process when present, and because the glide plane was usually obscured by the anteriorly imbricated osteoderm, the glide plane was excluded from length measurements as well (Figure 1). Extant taxa consisted of skins and wet preparations of whole specimens. Observations of the skins suggest that the keratinous sheath covering each osteoderm closely correlates with osteoderm dimensions excluding the glide plane in mature individuals. Therefore, the keratinous sheath was measured in place of measuring osteoderms directly in 35

modern taxa. Because of differences in preservation, it was not possible to obtain the same number of measurements in all specimens or to guarantee that osteoderm measurements came from the same anterior-posterior position within the dorsal shield. To test sensitivity to within-individual and within-taxa variation, statistical analyses were performed with all data, taxon-averages, and a dataset composed of the widest measurement from each taxon.

Locomotor ecology

Following Stubbs et al. (2013), locomotor ecology classifications for extinct taxa were classified as marine, amphibious, terrestrial, or as ambiguous (Table 1) based on the following criteria: the depositional environments specimens have been recovered from, ecological assessments reported in the literature, key aspects of their postcranial anatomy, and the ostensible ecologies of closely related taxa (see Table 1 for taxon specific references). Taxa were classified as amphibious if they were found primarily in lacustrine and fluvial deposits, with postcranial morphology similar to modern crocodilians. Taxa were categorized as marine or terrestrial if they were primarily found in marine and terrestrial depositional environments, respectively, and are noted as marine or terrestrial taxa in the literature. Additionally, marine taxa typically had relatively small forelimbs and highly curved and laterally compressed femora, whereas terrestrial taxa frequently possessed relatively strait femora and humeri (Parrish 1986; Stubbs et al. 2013). A fossorial category was also recognized by Stubbs et al. (2013), but only one taxon from my dataset (Simosuchus) would potentially fit in that category (Buckley et al., 2000; Stubbs et al., 2013). Because of the uncertainty of the identification of Simosuchus as fossorial and because the mechanics of burrowing vary among taxa and frequently include more lateral bending (Dorgon, 2015), Simosuchus was classified as ambiguous. Extant taxa were classified entirely as amphibious with the exception of Paleosuchus, which has been reported to be among the more terrestrial of living crocodilians, though they still spend a considerable amount of time in rivers (Magnusson and Lima, 1991), so this taxon was classified as ambiguously terrestrial/amphibious. Taxa with ambiguous ecological classifications were alternately removed from the dataset and

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iteratively reclassified as either marine, amphibious, or terrestrial, in order to assess the sensitivity of this study to classification.

Statistics

All statistical analyses were carried out in R version 3.2.2 (R Core Team, 2015). To evaluate phylogenetic signal, I used osteoderm aspect ratio in the form of width divided by length, and used the functions ‘abouheif.moran’ and ‘phylosig’ from the ‘adephylo’ and ‘phytools’ packages respectively, to calculate Moran's I (Moran, 1950), Abouheif's Cmean (Abouheif, 1999), Pagel's λ (Pagel, 1999), and Blomberg's K (Blomberg et al., 2003), while also testing for statistical significance of the signal (Jombart and Dray, 2010; Revell, 2012). To assess allometry and factor out its effects on subsequent analyses, I first performed a regression of length versus width using the ‘gls’ function from the ‘nlme’ package (Pinheiro et al., 2017). The residuals from the regression were then used in a one-way analysis of variance (ANOVA) testing for a correlation between osteoderm aspect ratio and locomotor ecology using the ‘lme’ and ‘anova’ functions also from the ‘nlme’ package (Pinheiro et al., 2017). I compared a nonphylogenetic model, a Brownian motion phylogenetic model, and an Ornstein–Uhlenbeck (OU) phylogenetic model (Martins and Hansen, 1997) using the phylogeny shown in Figure 2 to assess the effects of phylogenetic signal on the results. Because no single published phylogenetic tree encompassed all of the taxa analyzed for this study, the tree used here draws on multiple non- conflicting sources (Bronzati et al., 2012; Brochu, 2013; Puértolas-Pascual et al., 2015; Drumheller and Brochu, 2016; Drymala and Zanno, 2016; Buscalioni, 2017; Johnson et al., 2017) and was built by hand in Mesquite version 3.31 (Maddison and Maddison, 2017). Sample sizes were insufficient to test for significant differences in within-individual variation, so basic quantification of observed variation was used to explore minimum within- individual variation in osteoderm length and width. When evaluating variation, only taxa with at least five measurements were included and the length and width measurements were standardized across species by dividing by the average osteoderm length for each specimen for ease of

37 comparison. All R-generated plots were made with the ‘ggplot’ function from the ‘ggplot2’ package (Wickham, 2016).

RESULTS

Osteoderm aspect ratio, expressed as width:length, varied from 0.53:1 in Terrestrisuchus gracilis to 3.8:1 in Pholidosaurus sp. (Table 1). The observed within-individual variation was generally greater for width than for length measurements (Figure 3). The greatest amount of variation of osteoderm length was observed in Goniopholis sp. (medium sized, amphibious, goniopholid), Araripesuchus gomesii (small, terrestrial, notosuchian), and Protosuchus richardsoni (small, terrestrial, “protosuchian”), whereas the greatest variation in osteoderm width was observed in Paleosuchus trigonatus (small, terrestrial/amphibious, extant alligatoroid), Pholidosaurus sp. (medium, marine/amphibious, pholidosaurid), Pelagosaurus typus (large, marine, thalattosuchian), Araripesuchus gomesii, and Protosuchus richardsoni. There was no apparent relationship between within-individual variation and locomotor ecology, body size, or phylogeny (Figure 3). Estimates for various metrics of phylogenetic signal are listed in Table 2, and all are statistically significant and indicate similarity to a Brownian motion model of evolution. Across all taxa osteoderms exhibited significant positive allometry with the largest osteoderms proportionately wider than small osteoderms (slope ~1.5, residual standard error = 18.5, adjusted R2 from ‘lm’ function = 0.7363, p-value < 1e-10; Figure 4). When using a Bonferroni correction for multiple comparisons, there was no significant difference in osteoderm shape between different ecologies in any variation of the analysis (Figure 5). The presence of phylogenetic data and different methods of summarizing osteoderm shape in an individual had little impact on the analyses. The residuals of Paleosuchus trigonatus, Simosuchus clarki (small, possibly fossorial, notosuchian), Theriosuchus pusillus (small, possibly terrestrial, atoposaurid) and Gavialosuchus americana (large, marine/amphibious, crocodyloid) fell well within the ranges observed for all ecological classifications and had no impact on the analyses. Pholidosaurus sp. was the main

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outlier, with osteoderms much wider than expected given their size, however its classification as either a marine or amphibious taxon was insufficient on its own to result in a significant difference between ecological categories.

DISCUSSION

The narrowest osteoderms are found among the terrestrial taxa (i.e., Terrestrisuchus, Dromicosuchus, and NCSM 21722) and marine taxa were consistently wider than long, while a large number of amphibious taxa had roughly equidimensional osteoderms (Table 1), which would suggest at first glance that osteoderm morphology is related to ecology. The close mechanical and developmental association between osteoderms and vertebrae in crocodilians (Frey, 1988; Salisbury and Frey, 2000), and comparative performance studies of lizards (Losos et al., 2002), further suggests that osteoderm morphology should evolve in response to different locomotor regimes. In contrast with these expectations, however, the lack of statistical correlation between osteoderm aspect ratio and locomotor ecology suggests that relative osteoderm width is most likely not a determining factor of the directionality of torso flexibility. Nevertheless, there remains the possibility that the assumptions made here regarding vertebral bending during locomotion in extinct taxa were erroneous and that locomotor ecology is not closely correlated with the main axis of vertebral bending across distantly related taxa. Alternatively, non-locomotor behaviors, such as the death roll (Fish et al., 2007), could have been more important factors than rapid locomotion with regards to selection on vertebral flexibility, although these behaviors remain poorly studied. More data on the range of crocodilian behaviors, their prevalence amongst different taxa, their mechanics, and osteological correlates need to be assessed before their impact on trunk flexibility or osteoderm morphology can be studied. In the absence of additional evidence, the null hypothesis that osteoderm aspect ratio does not impact vertebral flexibility must be accepted for the time being. Instead, overall osteoderm shape appears to be primarily driven by allometry (Figure 4) and phylogeny (Figure 2).

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Across Crocodylomorpha, osteoderm width increases with positive allometry with respect to osteoderm length, such that small osteoderms from smaller individuals are much narrower than large osteoderms with a roughly 1.5 unit increase in width for every unit increase in length (Figure 4). Because most terrestrial taxa are relatively small, most marine taxa are relatively large, and many amphibious taxa are intermediate in size, this created the illusion of an association between locomotor ecology and osteoderm aspect ratio. In addition to size, phylogeny is also a statistically significant influence on osteoderm aspect ratio, the evolution of which appears to follow a Brownian motion pattern. Goniopholididae and Pholidosaurus notably deviate by having unusually wide osteoderms for their size (Figures 2, 4, 5, and Table 1). The final source of variation of aspect ratio is random variation within an individual, which is usually the result of variability in osteoderm width rather than length, though within-individual variation appears to be minimal overall for most taxa (Figure 3). The few taxa that do exhibit increased variability of osteoderm aspect ratio do not have any other traits in common (i.e., size, locomotor ecology, phylogenetic relationships), and if real may well be the result of different evolutionary processes.

CONCLUSIONS

Flexibility of the osteoderm carapace is not influenced by the aspect ratio of individual osteoderms; instead, variation in aspect ratio is the result of allometry, phylogenetic history, and

individual variation. Instead of aspect ratio, flexibility may be determined by morphology of the glide plane and mediolateral articulating surfaces. It is also possible that osteoderm morphology is largely irrelevant to flexibility, and it is the mass of osteoderms in a given region of the body, along with the mechanical properties of the connective tissues, which are most important to movement. If this is the case, then osteoderm morphology within Crocodylomorpha, and likely many other taxa as well, may primarily be the result of evolutionary relationships and, consequently, they may make good phylogenetic characters, provided effects attributable to size are removed prior to consideration. Osteoderm aspect ratio may also be influenced by how they

40 distribute impact forces, such as from a bite from a predator or competitor, or by their thermal properties (Broeckhoven et al., 2015; 2017). In any case, mechanical modeling of both osteoderms and the vertebral column and additional studies of other taxa may prove useful for addressing many of the questions raised here.

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Table 1

Taxon Specimen Length (mm) Width (mm) 18.2, 16.6, 18.1, 21, 19.9, 18.4, 15.3, 17, 16.7, 20.7, Alligator mississippiensis MVZ 129958 17.6, 16.3 17.7, 14.4 12.3, 17, 16, 17, 16.2, 15.4, 8.3, 18.4, 17.6, 18.2, 18, Alligator sinensis MVZ 67884 15.5 18.7, 16.3 Amphicotylus lucasii AMNH FR 5766 30.8, 29.7 75.1, 68.1 Anteophthalmosuchus hooleyi NHMUK R 3876 58.8, 51.8 124.5, 143.8 4.6, 5.3, 6.5, 7.2, 7.2, 7.6, 11, 15, 16.3, 16.2, 15.8, Araripesuchus gomesii AMNH FR 24450 7.5 14.1, 12 Borealosuchus wilsoni USNM V 12990 45.8, 44.4, 41.2 67.8, 64.6, 62.3 40.1, 42.5, 45, 47.8, 48.5, 43.4, 50.9, 50.4, 52.3, 52.9, Brachychampsa sp. UCMP 133901 47.8, 45.9 53.1, 49.7 16.4, 19.1, 19.3, 18.8, 17, 13.1, 15.6, 16.9, 16.5, 17, Caiman latirostris UF 120077 17.1, 19 14.6, 14.4 14.6, 15.9, 15.4, 15.1, 16.1, 17.7, 17.8, 19.5, 17.7, 17.2, Crocodylus acutus UF 52749 15.7, 16.8 20.4, 18.8 25.3, 17.4, 17, 18.2, 16.9, 13.4, 16.7, 16.7, 17.7, 17.6, Crocodylus niloticus MVZ 74884 17.5, 16.8 17.1, 20.3 11, 10.8, 10.4, 10.8, 11.4, 8.6, 11.7, 10.6, 11.1, 11.7, Crocodylus rhombifer UF 68614 12.6, 12.8 13.6, 13.2 15.9, 16.4, 18.5, 17.6, 17.3, 17.2, 18, 18.2, 20.7, 19.6, Crocodylus siamensis UF 119455 17.1, 16.6 20.4, 18.8 Diplocynodon darwini SMF ME 1158a 21.6, 21.5, 21.5, 22.4, 20.9 32.1, 32.8, 34.9, 33.8, 33 Dromicosuchus grallator NCSM 13733 18.1, 18.1, 18.3, 19.6, 19.9 10.7, 11.2, 12.4, 12.4, 13.4 Eogavialis africanum NHMUK R 3335 78.9, 74.2 118.2, 101.7 Eogavialis gavialoides NHMUK R 3343 69.1 92.1 Eogavialis gavialoides NHMUK R 3432 74, 63.9 101.1, 91.3 Eutretauranosuchus sp. USNM V 5836 9.7, 10 21.9, 22.6 Gavialosuchus americana USNM V 25243 57.7, 60.4, 53.9 90.5, 86.4, 85.9 Goniopholis sp. SMM P 2003.20.1 31.3, 31.8, 40.5, 40.1, 46.2 90.3, 91.6, 96.3, 101.2, 96.8 Hesperosuchus agilis CM 29894 21.8, 28.2, 29.9 25, 28.8, 29.6 Hoplosuchus kayi CM 11361 3.73, 3.74 6.22, 8.81 Lemmysuchus obtusidens NHMUK R 3169 63.3, 56.5, 72.8 124.5, 112.1, 98.8 16.3, 22.7, 21.5, 22.4, 20.2, 18.2, 22.9, 25.7, 26.6, 27.5, Mecistops cataphractus UF 88891 20.4, 20.1 22, 26.1 13.6, 13.6, 14.5, 11.8, 14.5, 13.8, 16.2, 24.5, 9.85, 22.8, Paleosuchus trigonatus UF 55873 12, 15.5 9.56, 15.6 20, 20.7, 22.8, 23.6, 23.5, 22.5, 30.4, 35.9, 39.3, 36.9, Pelagosaurus typus NHMUK 32598 24.5, 24.2 31.6, 28.9 Pholidosaurus sp. NHMUK R 3494 32.1, 21.5, 30.1, 32.4 106.4, 107.8, 106, 101.8 105.4, 80.9, 89.7, 73.7, Pholidosaurus sp. NHMUK R 3956 29.1, 28.9, 28.2, 21.1, 30 113.3 8.61, 9.45, 13.7, 12.5, 14.1, 19.2, 18, 24.4, 23.2, 20.5, Protosuchus richardsoni AMNH FR 3024 13.7 17.9 Simosuchus clarki UA 8679 14.4, 14.6, 17.2 14.8, 15.5, 17.3 67.6, 68.8, 76.6, 74.3, 63.8, 85.7, 90.3, 92.7, 91.1, 92.7, Steneosaurus durobrivensis NHMUK R 2865 71.9, 74.8 95.7, 81.4 Steneosaurus edwardsi NHMUK R 2074 39.6, 36.6, 42.2, 40.8, 35 61.4, 62.7, 57.3, 59.7, 55.5 52.7, 44.7, 56.5, 49.2, 51.6, 70.2, 66.7, 68.8, 67.6, 69, Steneosaurus leedsi NHMUK R 3806 59.9, 50.5 68.5, 66.2 Teleosaurus cadomensis NHMUK R 4207 33.8, 35 70.1, 69.7 Terrestrisuchus gracilis NHMUK R 10002 8.25, 8.11, 8.16 4.63, 4.68, 4.3

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Theriosuchus pusillus NHMUK 48216 7.97 12.9 26.3, 25.4, 25.9, 25.6, 24.8, 17.9, 18.7, 19.8, 20.1, 20.7, Tomistoma schlegelii UF 87168 26.8, 25.5 20.7, 19.1 Unnamed taxon NCSM 21722 17.6, 17.8, 19.5, 18.4, 19.5 11.6, 15.7, 17.2, 14.7, 14.6

Table 1, Continued.

Taxon Width/Length Ecology Primary Reference Alligator mississippiensis 0.941113373 amphibious Elsey and Woodward, 2010 Alligator sinensis 1.079630883 amphibious Jiang, 2010 Amphicotylus lucasii 2.363263721 amphibious Foster and McMullen, 2017 Anteophthalmosuchus hooleyi 2.426613877 amphibious Puértolas-Pascual et al., 2015 Araripesuchus gomesii 2.189443089 terrestrial Sereno and Larsson, 2009 Borealosuchus wilsoni 1.482825052 amphibious Erickson, 1976 Brachychampsa sp. 1.110295808 amphibious Norell et al., 1994 Caiman latirostris 0.854189697 amphibious Verdade et al., 2010 Crocodylus acutus 1.178933338 amphibious Thorbjarnarson, 2010 Mecistops cataphractus 1.176424701 amphibious Shirley, 2010 Crocodylus niloticus 0.92681679 amphibious Fergusson, 2010 Crocodylus rhombifer 1.008702948 amphibious Ramos Targarona et al., 2010 Crocodylus siamensis 1.112656712 amphibious Simpson and Bezuijen, 2010 Diplocynodon darwini 1.544930917 amphibious Delfino and Smith, 2012 Dromicosuchus grallator 0.639623916 amphibious Irmis et al., 2013 Eogavialis africanum 1.436416128 amphibious Buffetaut, 1982 Eogavialis gavialoides 1.333396082 amphibious Buffetaut, 1982 Eogavialis gavialoides 1.395213923 amphibious Buffetaut, 1982 Eutretauranosuchus sp. 2.257927116 amphibious Pritchard et al., 2013 Gavialosuchus americana 1.528196002 marine? Buffetaut et al., 1984 Goniopholis sp. 2.508001694 amphibious Andrade et al., 2011 Hesperosuchus agilis 1.044247776 terrestrial Clark et al., 2000 Hoplosuchus kayi 2.012371259 terrestrial Foster, 2003 Lemmysuchus obtusidens 1.740795731 marine Johnson et al., 2017 Paleosuchus trigonatus 1.176072707 terrestrial? Magnusson and Campos, 2010 Pelagosaurus typus 1.416398815 marine Pierce and Benton, 2006 Pholidosaurus sp. 3.634797588 marine? Martin et al., 2016 Pholidosaurus sp. 3.372177713 marine? Martin et al., 2016 Protosuchus richardsoni 1.710306275 terrestrial Irmis et al., 2013 Simosuchus clarki 1.030021714 terrestrial? Buckley et al., 2000 Steneosaurus durobrivensis 1.263686826 marine Mueller-Töwe, 2006 Steneosaurus edwardsi 1.526571839 marine Mueller-Töwe, 2006 Steneosaurus leedsi 1.343462478 marine Mueller-Töwe, 2006 Teleosaurus cadomensis 2.032650134 marine Mueller-Töwe, 2006 Terrestrisuchus gracilis 0.555459734 terrestrial Irmis et al., 2013 Theriosuchus pusillus 1.621455401 terrestrial? Schwarz and Salisbury, 2005 Tomistoma schlegelii 1.315722609 amphibious Bezuijen et al., 2010 Unnamed taxon 0.795571027 terrestrial Lecuona et al., 2016

Table 1: Specimens included in this study, with ecological categorizations, osteoderm length and width measurements, width/length ratios, and primary references.

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p-value of phylogenetic Estimated Value signal Moran's I 0.620 0.001 Abouheif's Cmean 0.624 0.001 Pagel's λ 0.975 3.15e-83 Blomberg's K 5.43e-05 0.001

Table 2: Estimates for Moran's I, Abouheif's Cmean, Pagel's λ, and Blomberg's K along with p-values for significance of the phylogenetic signal.

Figure 1: Schematic showing general dorsal osteoderm morphology in Crocodylomorpha when viewed dorsally and the regions used to obtain length and width measurements. The black spotted region indicates where pitting occurs and the dark grey region indicates the location of the keel.

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Figure 2: Phylogenetic tree used for the phylogenetic ANOVAs (see methods). Colors indicate locomotor ecology and the aspect ratio of the squares is proportional to the average osteoderm aspect ratio for each taxon. Extinct taxa marked with †.

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Figure3: Violin plots of within-individual variation. 46

Figure 3: Violin plots showing within-individual variation of standardized length (length of osteoderm divided by average length of osteoderms for that individual) and standardized width (width divided by average length). Only specimens with at least five measurements were included. Colors indicate locomotor ecology, with ambiguous taxa categorized as terrestrial or marine. Taxa are arranged phylogenetically as in Figure 2: Ca = Crocodylus acutus, Cn = Crocodylus niloticus, Cr = Crocodylus rhombifer, Cs = Crocodylus siamensis, Mc = Mecistops cataphractus, Ts = Tomistoma schlegelii, Am = Alligator mississippiensis, As = Alligator sinensis, Pat = Paleosuchus trigonatus, Cl = Caiman latirostris, Bs = Brachychampsa sp., Dd = Diplocynodon darwini, Gs = Goniopholis sp., Ps = Pholidosaurus sp., Sl = Steneosaurus leedsi, Se = Steneosaurus edwardsi, Sd = Steneosaurus durobrivensis, Pet = Pelagosaurus typus, Ag = Araripesuchus gomesii, Pr = Protosuchus richardsoni, Dg = Dromicosuchus grallator, Ut = unnamed taxon (NCSM 21722).

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Figure 4: Example scatterplot showing allometric relationship of osteoderm length versus width (mm) using all measurements. Taxa in which the ecology is ambiguous are indicated by unique symbols, as shown in the key. The shaded region indicates the 95% confidence interval. Width = 1.545 * Length – 1.632, adjusted R2 = 0.7363 (R2 calculated using ‘lm’ function in R).

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Figure 5: Example plot of residuals from the osteoderm average length versus average width regression analysis against ecological locomotor category using all datapoints and assigning ambiguous taxa as marine (Gavialosuchus americana and Pholidosaurus sp.) or terrestrial (Paleosuchus trigonatus, Simosuchus clarki, and Theriosuchus pusillus). The black dot and line indicate the mean and standard deviation respectively. The spread of points in the horizontal direction is to aid in vizualizing the density of points in the vertical direction.

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