THE FUNCTIONAL MORPHOLOGY OF CONTROL SURFACES:

A COMPARATIVE ANALYSIS

by

Sarah Louise Hoffmann

A Dissertation Submitted to the Faculty of

The Charles E. Schmidt College of Science

In Partial Fulfillment of the Requirements for the Degree of

Doctor of Philosophy

Florida Atlantic University

Boca Raton, FL

March, 2019

Copyright 2019 by Sarah Louise Hoffmann

ii THE FUNCTIONAL MORPHOLOGY OF SHARK CONTROL SURFACES:

A COMPARATIVE ANALYSIS

by

Sarah Louise Hoffmann

This dissertation was prepared under the direction of the candidate's dissertation advisor, Dr. Mariani)e E. Porter, Department of Biological Sciences, and has been approved by all members of the supervisory coinmittee. It was submitted to the faculty of the Charles E. Schmidt College of Science and was accepted in partial fulfillment of the requirements for the degree of Doctor of Philosophy.

SUPERVISORY COMMITTEE:

/~ *V' Marianne E. Porter, Ph.D. Dissertation Advisor

A!A;;Saraj:edini, Ph.D. I1e8n, Charles E. Schmidt College of Science ~_s~ Khaled Sobhan, Ph.D. Interim Dean, Graduate College

iii ACKNOWLEDGEMENTS

I am in humble gratitude for the thoughtful encouragement, patience, and guidance of my committee chair, Marianne E. Porter, and my dissertation committee,

Stephen M. Kajiura, Philip J. Motta, and Oscar M. Curet.

This research would not have been possible without the help of countless volunteers, undergraduate researchers, and honor’s thesis students. Remy Sanders,

Andrea Hernandez, Matthew Warren, Valerie Tovar, and Kala Hall all aided in the digitization of video. Conrad Testagrose and Wilmer Lopez spent countless hours dissecting shark pectoral fins and digitizing images. Matthew Warren, Conrad

Testagrose, and Wilmer Lopez further contributed to the analysis of data and authoring of various presentations and manuscripts. Matthew Warren, Christina Coppenrath, Tori Erb,

Samantha Leigh, Caitlin Shea-Vantine, Wilmer Lopez, Matthew Warren, and Valerie

Tovar aided in husbandry and live animal experiments. The Keys Marine Lab,

Gumbo Limbo Nature Center, University of Washington Friday Harbor Labs, New

England Aquarium, Carolyn Wheeler, Keith Hermann, the 2016 Friday Harbor Fish

Class, and the FAU IACUC staff aided in the procurement and health of live .

Countless volunteers and fishermen, including Beth Bowers, Doug Adams, Bryan

Frazier, Katie Viducic, and Kelsey James, sampled fins for me, primarily through the organization and labor of Lisa Natanson, who bears the scars of removing pectoral fins for the rest of her life.

iv Cassandra Donatelli, Elizabeth Brainerd, Benjamin Knorlein, Jeremy Lomax, and

Thaddaeus Buser helped me wade my through 3D point tracking, flow visualization modeling, and phylogenetic analyses. Geri Mayer and Jeannette Wyneken supported my teaching fellowships and I am extremely grateful for the life lessons and many laughs they provided. I am grateful to my labmates in the Florida Atlantic Biomechanics lab and the FAU Elasmobranch research lab for their many thoughtful contributions over the years.

Generous support from the following made this research possible: FAU startup funding to Marianne Porter, FAU Graduate College, FAU Office of Undergraduate

Research and Inquiry, National Save the Sea Turtle Foundation, FAU Graduate and

Professional Society, FAU Kelly Foundation Scholarship, Florida SeaGrant Guy Harvey

Ocean Foundation, Blinks Family Scholarship, Delores A. Auzenne Fellowship, Newell

Doctoral Fellowship, Marine Technology Society, Society for Integrative and

Comparative Biology, Ruth Wainright Fellowship, Friends of Gumbo Limbo, and the

American Elasmobranch Society. I am also indebted to the FAU Biological Sciences staff who helped me navigate graduate school: Sharon Ellis, Stacee Caplan, Michelle Cavallo,

Rebecca Dixon, and Corey Jasper.

Thank you to Marianne Porter, Stephen Kajiura, Adam Summers, Jessica Pate, and Lisa Natanson, who served as role models in my academic career and made my forays into the field possible. You allowed me to experience the animals that fueled my passion live and in person, and for that I am ever grateful. The practical and personal skills I learned from you have shaped the researcher and person that I am today.

Finally, I am forever indebted to those who stuck by me in these turbulent years.

First to my parents, Lynn and Brian Hoffmann for their endless encouragement and v random shark facts. Christina Coppenrath, Boris Tezak, Tori Erb, Daniel Alempijevic,

Lisa Natanson, Justin Perrault, Bethany Augliere, Danielle Ingle, Cassie Volker, and

Jessica Pate took me in as family and are now stuck for life. And Boone, who sacrificed five years of his best life away from the snowy mountains for reasons he will never comprehend. I would not have made it through without you all, thank you.

vi ABSTRACT

Author: Sarah L. Hoffmann

Title: The functional morphology of shark control surfaces: a comparative analysis

Institution: Florida Atlantic University

Dissertation Advisor: Marianne E. Porter

Degree: Doctor of Philosophy

Year: 2019

Sharks are an objectively diverse group of animals; ranging in maximum size from 2,000cm () to 17cm (dwarf lantern shark); occupying habitats that are periodically terrestrial (epaulette shark) to the deepest parts of the ocean (); relying on a diversity of diets from plankton to marine mammals; with vast amounts of morphology diversity such as the laterally expanded heads of hammerhead species, the elongate caudal fins of thresher species, and the tooth embedded rostrum of saw shark species representing some of the anatomical extremes. Yet despite these obvious differences in morphology, physiology, and ecology, the challenges associated with studying hard to access, large bodied, pelagic animals have limited our comparative understanding of form and function as it relates to swimming within this group. The majority of shark swimming studies examine species that succeed in captivity, which are usually benthic associated that spend time resting on the substrate. These studies have also been limited by the use of flumes, in which the unidirectional flow and small working area precludes the analysis of larger animals, volitional swimming, and vii maneuvering. The few existing volitional kinematics studies on sharks quantify two- dimensional kinematics which are unable to capture movements not observable in the plane of reference. With this study, we quantified the volitional swimming kinematics of sharks in relation to morphological, physiological, and ecological variation among species. We developed a technique to analyze three-dimensional (3D) kinematics in a semi-natural, large volume environment, which, to our knowledge, provides the first 3D analysis of volitional maneuvering in sharks. We demonstrated that Pacific and bonnethead sharks rotate the pectoral fins substantially during yaw (horizontal) maneuvering and is correlated with turning performance. We proposed that ecomorphological differences correlate with the varied maneuvering strategies we observed between the two species. We also found that there is some mechanical constraint on shark pectoral fin shape that is explained by phylogenetic relationships but describe a continuum of morphological variables within that range. We propose standardized terminology and methodology for the future assessment of shark pectoral fin morphology and function. As with previous studies, the ease of access to species was a challenge in this study and future studies should continue to assess the functional ecomorphology of shark pectoral fins among species.

viii THE FUNCTIONAL MORPHOLOGY OF SHARK CONTROL SURFACES:

A COMPARATIVE ANALYSIS

LIST OF TABLES ...... xiv

LIST OF FIGURES ...... xv

LIST OF EQUATIONS ...... xxiv

CHAPTER 1: INTRODUCTION ...... 1

Control surfaces ...... 2

Locomotor modes in fishes ...... 3

Shark pectoral fin functional ecomorphology ...... 5

Volitional swimming kinematics ...... 7

Summary...... 8

Chapter 1 Figures ...... 10

CHAPTER 2: REGIONAL VARIATION IN UNDULATORY KINEMATICS OF

TWO HAMMERHEAD SPECIES: THE BONNETHEAD (SPHYRNA TIBURO)

AND THE SCALLOPED HAMMERHEAD (SPHYRNA LEWINI) ...... 14

Abstract ...... 14

Introduction ...... 15

Materials and methods ...... 18

Study animals ...... 18

Morphological measurements ...... 19

Kinematic analysis...... 20

ix Statistical analysis ...... 22

Results ...... 23

Morphology ...... 23

Kinematics ...... 24

Discussion ...... 25

Morphological Variation ...... 26

Undulatory Variation ...... 27

Conclusions ...... 30

Chapter 2 Figures ...... 32

CHAPTER 3: THREE-DIMENSIONAL MOVEMENTS OF THE PECTORAL FIN

DURING YAW TURNS IN THE PACIFIC SPINY DOGFISH, SQUALUS

SUCKLEYI ...... 38

Abstract ...... 38

Introduction ...... 39

Methods ...... 43

Volitional swimming trials ...... 43

Muscle stimulation trials ...... 45

3D marker tracking ...... 45

VROMM animation...... 47

Data analysis ...... 47

Results ...... 49

Pectoral fin kinematics during routine turns ...... 49

Pectoral fin muscle stimulation ...... 50

x Discussion ...... 51

Pectoral fin rotation during volitional turning ...... 51

Pectoral fin musculature ...... 54

3D volitional kinematics...... 56

Conclusion ...... 57

Chapter 3 Figures and Tables ...... 59

CHAPTER 4: BODY AND PECTORAL FIN KINEMATICS DURING YAW

TURNING IN BONNETHEAD SHARKS (SPHYRNA TIBURO) ...... 68

Abstract ...... 68

Introduction ...... 69

Methods ...... 72

Marker placement ...... 72

Volitional swimming trials ...... 73

Muscle stimulation trials ...... 74

3D marker tracking ...... 74

2D whole body kinematics ...... 75

Data analysis ...... 76

Results ...... 77

Pectoral fin kinematics ...... 77

Whole body kinematics ...... 78

Turning performance ...... 78

Muscle stimulation ...... 79

Comparison to Pacific spiny dogfish ...... 80

xi Discussion ...... 80

Pectoral fin kinematics during volitional turning ...... 81

Whole body kinematics ...... 83

Turning performance ...... 84

Muscle stimulation ...... 85

Ecomorphological differences in maneuvering strategy ...... 87

Conclusions ...... 88

Chapter 4 Figures and Tables ...... 90

CHAPTER 5: COMPARATIVE MORPHOLOGY OF SHARK PECTORAL FINS ....100

Abstract ...... 100

Introduction ...... 101

Methods ...... 104

External morphology ...... 104

Geometric morphometrics of fin shape ...... 105

Skeletal morphology ...... 105

Cross sectional morphology ...... 106

Data analysis ...... 107

Results ...... 108

Geometric morphometrics and phylogenetic comparative methods ...... 109

Gross morphology ...... 110

Cross-sectional morphology ...... 112

Relationships among morphological variables ...... 113

Discussion ...... 114

xii Geometric morphometrics and phylogenetic comparative methods ...... 115

Aspect ratio ...... 116

Skeletal extent ...... 117

Cross sectional morphology ...... 119

Conclusion ...... 121

Chapter 5 Figures and Tables ...... 122

CHAPTER 6: SYNTHESIS, FUTURE DIRECTIONS, AND SIGNIFICANCE ...... 140

Synthesis ...... 140

Function and ecomorphology ...... 141

Volitional swimming kinematics ...... 144

Future directions ...... 144

Significance and broader impacts ...... 146

APPENDICES ...... 148

Appendix A: Permission To Reproduce Hoffmann Et Al., 2017 ...... 149

Appendix B: Permission To Reproduce Hoffmann Et Al., 2019 ...... 150

REFERENCES ...... 151

xiii

LIST OF TABLES

Table 3.1: Pectoral fin muscle terminology as previously described in the literature...... 66

Table 3.2: Precision of point tracking and mean rigid body error for three volitional

trials per individual. Precision is measured by the standard deviation of the

distances between markers within a rigid body and was on average 0.684 mm.

Rigid body error, calculated as the error between the optimized marker

constellation from all frames and the reconstructed location of the markers,

averaged 0.24 mm over all trials and rigid bodies (Knorlein et al., 2016)...... 67

Table 4.1: Top five best fit models predicting turning angular velocity. All top five

models included total fin rotation as a significant variable and the top two also

included one of the body bending variables (BC or MBA)...... 98

Table 4.2: Fin rotation and turning performance variables compared between the

bonnethead and Pacific spiny dogfish (Hoffmann et al., 2019). Values represent

the mean ± standard error and significance is denoted by *. The absolute values

of X, Y, Z axis rotation and the change in velocity were used for statistical tests.

Only the absolute value of change in velocity during turning was significantly

different between the two species ...... 99

Table 5.1: Sample size, habitat use, ecological designation, and biology of species

encompassed in the present study (Compagno, 1984)...... 139

xiv

LIST OF FIGURES

Figure 1.1: Six axes of movement in a fully aquatic environment. Translation occurs

medio-laterally (slip), dorso-ventrally (heave), and cranio-caudally (surge).

Rotation about the medio-lateral axis (pitch) adjusts vertical orientation, about

the dorso-ventral axis (yaw) adjusts horizontal position, and about the cranio-

caudal axis (roll) changes the longitudinal orientation of the body axis. Adapted

from Parson et al., 2011...... 10

Figure 1.2: Range of undulatory swimming modes (Adapted from: Breder, 1926;

Lindsey, 1978). Black bars shade the amount of body that is being laterally

displaced and the black line underneath the fishes represent the relative

amplitude of lateral displacement during undulation. Lateral displacement of the

body axis ranges from movement of the majority of the body (anguilliform) to

solely caudal oscillation (thunniform). This is accompanied with a shift from

undulation, where there is more than one wave along the body, to oscillation,

which is a simplified flapping with less than one wave along the structure

(Breder, 1926)...... 11

Figure 1.3: General pectoral fin skeleton of aplesodic and plesodic fins (Marinelli

and Strenger, 1959; Liem and Summers, 1999; Compagno, 1984; Wilga and

Lauder, 2000, 2001; Maia et al., 2012). At the proximal fin base, three basal

elements (propterygium, metapterygium, mesopterygium) articulate with the

scapulo-coracoid. Extending distally from the fin web are three series of radial

xv elements. Thin, flexible certaotrichia are embedded in the connective tissue that

anchors the skin to the fin. Fins with less than 50% skeletal support are

considered aplesodic (A) while fins with greater than 50% skeletal support are

plesodic (B)...... 12

Figure 2.1: Eight anatomical landmarks were digitized on each individual for both

species. For regional flexion frequency and amplitude analyses, four isolated

regions are outlined in brackets as the anterior body (angle ABC), mid-body

(angle BCD), posterior-body (angle CDE), and caudal peduncle (angle DEF)...... 32

Figure 2.2: Morphological differences between bonnethead (black) and scalloped

hammerhead (white) bodies. (A) The cephalofoil (standardized by dorsal body

area) of the scalloped was 39% larger than the bonnethead

though the standardized pectoral fin area (B) of the bonnethead was 38% larger.

(C) Standardized planning surface area (cephalofoil area + pectoral fin area) of

the scalloped hammerhead was 5% greater than the bonnethead. (D) Anterior

body fineness ratio was 30% greater in the scalloped hammerhead. Error bars

represent standard error. Stars denote significant statistical differences...... 33

Figure 2.3: Regional variation in second moment of area of the body between

bonnethead (black) and scalloped hammerhead sharks (white). (A) Second

moment of area (Ix) was greater in the bonnethead compared to the scalloped

hammerhead at all five regions. (B) Ix:Iy was greater in the scalloped

hammerhead at four of the five regions, demonstrating that the scalloped

hammerhead has a more laterally compressed trunk. The biggest differences in

shape between the two species was observed in the anterior trunk region...... 34

xvi Figure 2.4: Variations in anterior body cross-sectional morphology, quantified as

Ix:Iy at third gill slit. (A) There was no difference in cross sectional shape across

body sizes for either species. (B) Cross sectional shape was approximately

circular (Ix:Iy = 1) in the bonnethead in comparison to the scalloped

hammerhead in which the body was laterally compressed (Ix:Iy > 1). Error bars

represent standard error. Stars denote significance...... 35

Figure 2.6: Flexion variables differed between bonnethead (black) and scalloped

hammerhead (white) sharks. (A) Midline amplitude increased significantly along

the length of the body with the largest amplitudes produced in the caudal region.

Midline amplitude differed between species at MB and PB regions where

bonnethead amplitude was on average 28% greater than the scalloped

hammerhead. (B) AB had significantly higher frequencies than the three

posterior regions (MB, PB, CF) in both species and was not different between

species. The frequency of flexion was consistent across the three posterior

regions (MB, PB, CF) for both species. Error bars represent standard

error. Lower case letters denote significant statistical differences...... 37

Figure 3.1: Video reconstruction of moving morphology (VROMM) experimental

design. (A) Pacific spiny dogfish were outfitted with white bead markers along

the anterior body and leading edge of the pectoral fin. (B) Two fully submerged

Go-Pro cameras were angled approximately 45o to one another and focused on

the same 1m3 volume outlined with bricks. (C) Cameras were time synchronized

with a flashing light and calibrated for 3D analyses with a 7 x 9 checkerboard

calibration object...... 59

xvii Figure 3.2: Pectoral fin rotation relative to the body axes. (A) The 3D shapes

represent the body and inside fin and the white dots represent tracked points. A

joint coordinate system (FB-JCS) was placed at the proximal fin base to measure

relative fin rotation about the dorso-ventral (X; red), medio-lateral (Y; green),

and cranio-caudal (Z; blue) axes. (B-D) Sample rotation trace of one turn

highlighted in the gray box. The pectoral fin was (B) protracted, (C) supinated,

and (D) depressed during the turn. This pectoral fin rotation pattern was observed

in all nine trials. Protraction rotates the fin cranially, supination causes the

trailing edge of the fin to translate ventrally, increasing the angle of ...... 60

Figure 3.3: We found significantly greater depression of pectoral fins during turning

(F2,24=20.6537, P < 0.0001). For all nine trials, the inside pectoral fin was

protracted, supinated, and depressed. The absolute values of rotation were used

in an ANOVA to compare the range of rotation in each axis and lower-case

letters denote significant differences. The box represents the mean (middle line)

± standard error of the mean. Whiskers represent the upper and lower extremes...... 62

Figure 3.4: Relation among pectoral fin movement and turning kinematics. (A) Both

depression and protraction were positively related to turning angular velocity

while supination was not (P=0.253, P=0.0008). (B) When considering the sum of

rotations about all three axes, there was a significant positive relation with

turning angular velocity (P=0.0010). (C) The area of fin to flow was significantly

positively related to turning angular velocity (P=0.0474). (D) Drag was also

significantly positively correlated with turning angular velocity (P=0.0098)...... 63

xviii Figure 4.1: Camera set-up and bead placement for VROMM. (A) Black

hemispherical beads were placed along the body and pectoral fins of four

bonnethead sharks. (B) Three GoPro Hero 5 Black cameras (outlined in yellow)

were mounted to cement blocks and angled at a common volume of interest,

marked with white coral fragments. (C) The trial arena was calibrated for 3D

analyses by taking images of a 7 square x 9 square checkerboard at various

regions throughout the volume...... 90

Figure 4.3: Range of pectoral fin rotation about the body axes during turns, in which

the fin did not contact the substrate. The magnitude of rotation did not differ

among axes. Boxes represent the mean (middle line) ± the standard error of the

mean, and whiskers represent the minimum and maximum values. During

turning, the fin was protracted (X), pronated (Y), and depressed (Z)...... 93

Figure 4.4: Whole body flexion kinematics. Individuals demonstrated a range of

body bending coefficients (BC) and maximum bending angles (MBA), which

were significantly related to each other. Significance (P < 0.05) is denoted by the

corresponding trendline and R2 value...... 94

Figure 4.5: Fin rotation relative to turning performance. (A) Fin depression was the

only axis of rotation significantly related to turning angular velocity. Total fin

rotation was significantly positively related to turning angular velocity (B) and

the change in velocity (C). Significance (P < 0.05) is denoted by the

corresponding trendline and R2 value...... 95

Figure 4.6: Whole body kinematics relative to turning performance. Body bending

coefficient (BC; A) and maximum body angle (MBA; B) were significantly

xix positively related to turning angular velocity. (C) MBA was also significantly

related to the change in velocity. Significance (P < 0.05) is denoted by the

corresponding trendline and R2 value...... 96

Figure 4.7: Fin rotation relative to the body from stimulation of three target muscles

in the pectoral fin. dorsal pterygoideus (A; orange), ventral pterygoideus (B;

purple), and cranial pterygoideus (C; yellow). Lead placement targeted the

middle of the muscle body, represented by black dots. (D-F): Fin rotation relative

to the X (D), Y (E), and Z (F) axes described in Fig. 4.2. The CP was the only

muscle to produce fin protraction (D) whereas the DP and VP both resulted in

retraction. The DP and CP both pronated the fin (E) and the VP supinated the fin.

The DP was the only fin elevator (F) and stimulation of the VP and CP both

resulted in fin depression. Error bars represent standard error of the mean...... 97

Figure 5.1: Gross anatomy of cartilaginous skeletal elements in shark pectoral fins.

Fins generally have a longer, tapered leading edge (anterior) and a trailing edge

(posterior) fork that terminates in a lobe. Internally, three basal cartilages (the

propterygium, mesopterygium, and metapterygium) articulate at the proximal fin

base with the scapulo-coracoid. Three sets of radials (proximal, intermediate, and

distal) extend distally from the basals to support the fin web. Thin, flexible

ceratotrichia are embedded in the connective tissue that overlays the skeleton and

attaches it to the fin. Fins have been categorized based on the extent to which the

skeletal elements (radials) extend into the fin web. If less than 50% of the fin

web is supported by the skeletal elements, the fin is termed “aplesodic” (A),

whereas greater than 50% skeletal support is considered “plesodic” (B). For both

xx fin types, the leading edge lobe is generally more supported by the radials, and

thus more rigid, than the trailing edge lobe...... 122

Figure 5.2: Shark fin classification by family (Phylogeny: Velez-Zuazo and

Agnarsson, 2011; Classifications: Compagno, 1977; Maisey, 1985; Wilga and

Lauder, 2001; Maia et. al., 2012; Sakai, 2011; Crawford, 2014)...... 123

Figure 5.3: Meristics and morphometrics used to quantify pectoral fin differences

among species. A) External fin morphology was measured as the fin area, length,

and width at the base and fork. Skin and connective tissue were removed to

reveal the skeletal anatomy, comprised of three sets of radials that extend distally

into the fin. The leading edge radial, longest radial, and trailing edge radials were

further dissected to examine cross sectional morphology (B). In general, radials

were more dorso-ventrally compressed on the leading and trailing edges than at

the longest radial. C) The radii along the dorso-ventral (NAx; rx) and lateral

(NAy; ry) neutral axes were measured to calculate the second moment of area (I)

in each axis. Total and calcified areas were measured for each cross section as

well ...... 124

Figure 5.4: Landmarks used for shape analysis. Outline from lateral photograph of

the left pectoral fin of limbatus (fork length 67 cm, fin length 13.8

cm) used in this study. Five putatively homologous landmarks are indicated on

the outline of the pectoral fin: 1) proximal insertion of the trailing lobe, 2) distal

tip of trailing lobe, 3) inflection of the trailing edge fork, 4) distal fin tip, 5)

proximal base of leading edge as described in Fig. 5.3...... 125

xxi Figure 5.7: Exemplar fin overlays for five shark families exemplifying fin shape and

the extent of skeletal support among families. Squatinidae (A) and Alopiidae (B)

had the greatest amount of radial support, (C) was intermediate, and

Carcharhinidae (D) and Sphyrnidae (E) had the least amount of radial support...... 130

Figure 5.8: Fin classification and shape by species. (A) Aspect ratio varied greatly

among orders and there was no real trend among the groups. (B) Five species, all

Carcharhiniformes, had aplesodic fins. All Lamniformes and the one

Squatiniformes species had plesodic fins. In the observed species, skeletal extent

ranged from 40% - 86%. The majority of the unsupported fin web, particularly in

the plesodic fins, was along the trailing edge (see Fig. 5.7). Error bars represent

the standard error of the mean...... 131

Figure 5.9: External and skeletal morphology by family and order. (A) Alopiidae had

on average the greatest aspect ratio and (B) skeletal support. On average, all

families observed in this study can broadly be classified as having plesodic fins,

despite the differences observed by species (see Fig. 5.1). (C) The number of

radials was substantially greatest in Squatinidae and least in Carcharhinidae,

Sphyrnidae, and Cetorhinidae. Lamnidae and Alopiidae were intermediate. Error

bars represent the standard error of the mean...... 133

Figure 5.10: Cross-sectional morphology of the fin and radials. (A) For all species,

both the leading and trailing edge proximal radials were substantially more

dorso-ventrally compressed than the longest proximal radial, but only in the

Lamniformes was the longest radial laterally compressed. (B) The amount of

calcification was highly varied among radials and families, but in general the

xxii Lamniformes had less radial calcification than other groups. (C) For all species

except Sphyrnidae, taper angle was greater along the trailing edge. When

considering the means of all species, trailing edge taper angle was significantly

greater than leading edge taper angle by a factor of two (P = 0.0032)...... 135

Figure 5.12: Relationship between skeletal extent and morphology in order

Carcharhiniformes, separated by migratory behavior. Both aspect ratio (A) and

trailing edge taper (B) were positively related to skeletal extent (P = 0.0201, R2 =

0.4325; P = 0.0061, R2 = 0.6307)...... 138

xxiii

LIST OF EQUATIONS

ab3 I = π (Eq. 1.1) ...... 20 y 4

푎3푏 퐼 = 휋 (Eq. 1.2) ...... 20 푥 4

퐴푓 푆푡 = (Eq. 2.3) ...... 21 푉

퐻 2+퐻 2−퐻2 cos(훽) = 푖 푓 (Eq. 3.1) ...... 48 2퐻푖퐻푓

θ 푤 = (Eq. 3.2) ...... 48 푡 푡

1 퐹 = 퐶 퐴휌푉2 (Eq. 3.3) ...... 49 푑 2 푑

3 푑 ⁄2 푑3 퐶 = 1 + 1.5 + 7 (Eq. 3.4) ...... 49 푑 푙 푙

퐻 2+퐻 2−퐻2 cos(훽) = 푖 푓 (Eq. 4. 1) ...... 75 2퐻푖퐻푓

θ 푤 = (Eq. 4.2) ...... 75 푡 푡

퐿 퐵퐶 = 1 − (Eq. 4.3)...... 76 푇퐹

퐿 2 퐴푅 = 푓 (Eq. 5.1) ...... 105 퐴푓

퐴푠푘 푆퐸푎 = (Eq. 5.2) ...... 105 퐴푓

퐿푠푘 푆퐸푙 = (Eq. 5.3) ...... 106 퐿푓

퐴푐 % 퐴푟 = × 100 (Eq. 5.4) ...... 106 퐴푡

Π 푟 푟 3 퐼 = 푦 푥 (Eq. 5.5) ...... 106 푦 4 xxiv

Π 푟 푟 3 퐼 = 푥 푦 (Eq. 5.6) ...... 106 푥 4

퐼푦 푆푟 = (Eq. 5.7) ...... 106 퐼푥

퐷푙표푛푔−퐷푙푒푎푑푖푛푔 푇퐿퐸 = (Eq. 5.8) ...... 107 푊푠

푇 훼 = tan−1( ) (Eq. 5.9) ...... 107 2

xxv

CHAPTER 1: INTRODUCTION

Aquatic employ a variety of strategies to move around and interact with their environments, which is often reflected in their morphological differences.

Despite major differences in swimming styles, aquatic vertebrates share similar generalized body plans: a central body axis that tapers to a caudal peduncle with a variety of fins or flippers inserting along the body. Fins and flippers are often characterized as control surfaces (structures that adjust position in space) that balance (or imbalance) forces for swimming and maneuvering (Fish and Lauder, 2017). Among swimmers, the major thrust producing structure and the type of movement it undergoes varies substantially. For example, sea turtles and mobulid rays “flap” extensive flippers/fins in underwater flight, delphinids rapidly oscillate semi-lunate caudal fins in the dorso-ventral plane in one type of axial undulation, sharks also displace caudal fins but in the lateral plane instead, and elongate fishes, such as eels, have a whole-body foil which is undulated for thrust production along the central axis (Rosenberger, 2001; Vogel, 2003;

Fish et al., 2008; Fish and Lauder, 2017). All of these strategies rely on the movement of water in some form to produce a forward reactionary force, which can be accomplished in many ways as is reflected in the diversity of locomotor strategies documented in these groups.

1 Control surfaces

Specific control surface function and design varies greatly throughout aquatic vertebrates but maintains one general role: to generate forces for stability or maneuverability. Control surface length, width, surface area, and thickness all affect the movement, hydrodynamic

efficiency, and thus role of control surfaces (Fish and Lauder, 2017). Tapered control surfaces with a spindle cross-sectional shape promote lift generation but have limited flexibility, due to chord thickness (Felts, 1966; Lang, 1966; Magnuson, 1970;

Fish, 2002). Thinner control surfaces, on the other hand, are more flexible and may undergo more complex movements for a finer degree of control over locomotion (Wilga and Lauder, 2000, 2001; Lauder and Drucker, 2004). Aspect ratio (AR: fin length/surface area2) impacts the amount of lift produced relative to drag such that longer, thinner fins

(high aspect ratio) are more hydrodynamically efficient (Lighthill and Blake, 1990).

Comparative studies on these control surface characteristics of delphinids and actinopterygian fishes demonstrate that morphology correlates with habitat use as ecomorphological adaptations within groups (Wainwright et al., 2002; Lauder and

Drucker, 2004; Fulton et al., 2005; Fish et al., 2008; Weber and Howle, 2013; Fish and

Lauder, 2017).

The position of control surfaces also greatly affects an organism’s intrinsic stability. Fins positioned at a dihedral angle to the body are situated for inherent resting stability, while those at a negative dihedral angle to the body are destabilizing and promote maneuverability (Aleyev, 1977; Webb et al., 1996; Wilga and Lauder, 2000;

Fish, 2002; Webb and Weihs, 2015). In addition to maintaining stability, control surfaces

2 are used to move about the six axes within in aquatic environment: three translations

(surge, heave, slip) and three rotations (pitch, roll, yaw) (Fig. I.1; Fish, 2002; Walker,

2004; Webb, 2006; Parson et al., 2011). Most aquatic vertebrates primarily translate in the surge axis (forward/backward) and maneuver about the rotational axes. Maneuvering occurs when an imbalance of forces causes the body to reorient about one of the three axes (Fish and Nicastro, 2003). There are two general maneuvering strategies based on the force that is used to reorient the body: (1) control surfaces that reorient lift to initiate whole body rolling (also called banking) for turning or (2) control surfaces that generate asymmetrical drag, creating a pivot about which the body rotates (Vogel, 2003; Fish and

Lauder, 2017). At high speeds, swimmers use lift-based maneuvering to minimize the turning radius and avoid deceleration throughout the turn (Fish, 1997). However, the ability of a control surface to generate lift decreases with velocity, and low speed, precision maneuvering may instead be drag based (Fish and Nicastro, 2003; Fish and

Lauder, 2017). The stability and maneuverability of an aquatic organism is inherently tied to control surface morphology, which differs predictably among varied ecosystems.

Locomotor modes in fishes

Ecomorphological adaptations have been well studied in fishes in particular, likely because there is a wide range of body design, habitat use, and locomotor strategy.

They are frequently categorized in two overarching categories based on the thrust producing mechanisms they use: (1) swimming with the median paired fins (MPF) and

(2) thrust production via the body and caudal fin (BCF) (Breder, 1926; Lindsey, 1978;

Webb, 1998; Blake, 2004). MPF swimmers use complex fin movements to generate thrust by moving water primarily with their pectoral fins. In contrast, BCF swimmers

3 undulate some amount of the body axis with increasing amplitude towards the caudal fin, which sheds vortices from the trailing edge resulting in a forward reaction force (Webb,

1998). Unsurprisingly, morphological studies on MPF swimmers tend to focus on differences in paired fins while BCF studies describe differences in the body axis and caudal fin (Thomson and Simanek, 1977; Webb and Keyes, 1982; Webb, 1984;

Wainwright et al., 2002; Lauder and Drucker, 2004; Blake, 2004; Fish and Lauder,

2017). Yet, differences in the non-primary control surfaces of fishes likely have implications on locomotor behavior too, whether it be in intrinsic stability (i.e. paired fins in BCF swimmers) or hydrodynamic efficiency (i.e. body axis shape in MPF swimmers) that vary with morphology. Further, body shape also correlates with differences in habitat use where selective pressures drive the adaptation of morphological features to a particular ecological setting (Norton et al., 1995).

The relationship between form and function of control surfaces in fishes is well described for actinopterygians, but is less well understood in sharks. The size range, availability of specimens, and logistics of captivity makes this group less accessible for swimming kinematics studies. Generally, sharks are BCF swimmers that laterally displacement (undulate) differential amounts of the body axis during swimming depending on species (Webb and Keyes, 1982; Donley and Shadwick, 2003; Donley et al., 2004; Blake, 2004). Undulation originates anywhere from the first dorsal fin

(subcarangiform), second dorsal fin (carangiform), or even at the caudal peduncle

(thunniform) and increases in amplitude rostro-caudally (Fig. I.2; Breder, 1926; Lindsey,

1978; Webb and Keyes, 1982; Blake, 2004; Donley and Shadwick, 2003; Donley et al.,

2004). Differences in the amount of axial displacement are hypothesized to correlate with

4 body shape, such that species with thinner, flexible bodies fall towards the subcarangiform end of the spectrum and fusiform, stiffer bodied species only oscillate the caudal fin (thunniform) (Webb and Keyes, 1982; Donley and Shadwick, 2003; Donley et al., 2004). These variations in axial morphology and locomotor strategy are also hypothesized to correlate with habitat use. Subcarangiform sharks tend to be smaller, reef associated species while thunniform species are high performance pelagic predators

(Webb and Keyes, 1982; Donley and Shadwick, 2003; Donley et al., 2004). Individuals may display a number of BCF strategies, particularly throughout unsteady swimming behaviors, but these categories serve as a generalized overview of most frequent locomotor strategy observed within a group (Long and Nipper, 1996; Webb, 1998; Blake,

2004). A comprehensive understanding of volitional swimming behavior among shark species would greatly improve our understanding of ecomorphology as it relates to swimming.

Shark pectoral fin functional ecomorphology

Even less well described than whole body functional ecomorphology in sharks is the diversity of control surfaces. Throughout the literature, the extent of the pectoral fin skeleton into the fin is used as a morphological characteristic to assess the level of relatedness among species (Compagno, 1977; Maisey, 1984; McEachran, 1989;

Compagno, 1990; Shirai, 1996). Historically, fins are classified into two distinct groups based on the skeletal extent: (1) aplesodic fins have less than 50% of the fin supported by the skeleton and (2) plesodic fins have greater than 50% skeletal support (Fig. I.3;

Compagno, 1984; Wilga and Lauder, 2000, 2001; Maia et al., 2012). Difference in the shape of the basals (the three cartilaginous elements that articulate with the scapulo-

5 coracoid at the proximal body axis) and the amount of muscle associated with the fin are hypothesized to vary along with skeletal extent, but a broad comparative analysis of these parameters is lacking (Fig. I.3; Marinelli and Strenger, 1959; Compagno, 1984; Liem and

Summers, 1999; Maia et al., 2012). Additionally, there is some disagreement about the function of shark pectoral fins. Based on their morphology and position, it is long hypothesized that shark pectoral fins generate lift at the mid body to counteract the resultant lift produced by the caudal fin and balance forces acting on the body during steady swimming (Fig. I.4; Harris, 1936; Alexander, 1965; Ferry and Lauder, 1996;

Wilga and Lauder, 2000,2002; Fish and Shannahan, 2000). Volitional swimming studies document changes in fin angle of attack, further supporting the lift generating hypothesis

(Fish and Shannahan, 2000). For at least one species though, particle image velocimetry reveals that negligible lift is generated by the pectoral fins during steady swimming, and that the body acts as the primary lift producer (Wilga and Lauder, 2000). This study further demonstrates that shark pectoral fins undergo complex conformational changes when moving, suggesting that they may have more dynamic roles than solely functioning as rigid, lift generating mechanisms (Wilga and Lauder 2000, 2001). Differences in these functional descriptions may also result from ecomorphological variations in study species. Given the objective diversity of fin morphology, it is likely that these structures have variable roles among species.

The hypothesis that shark pectoral fins are rigid, immobile control surfaces is further challenged by descriptions of fin movements throughout the literature. Epaulette sharks ( ocellatum) use sequential “swinging” (protraction and retraction) of the pelvic and pectoral fins to walk along the benthos (Pridmore, 1994), Common

6 thresher sharks (Alopias vulpinus) “laterally rotate” the pectoral fins during braking

(Oliver et al., 2013), and both bonnethead sharks (Sphyrna tiburo) and Pacific spiny dogfish (Squalus suckleyi) differentially move pectoral fins during yaw turning (Kajiura et al., 2003; Domenici et al., 2004). Further, the use of abduction and adduction have been used interchangeably throughout the literature, carried over from the description of actinopterygian pectoral fin movement, to describe a fin motion that is less applicable to sharks (Marinelli and Strenger, 1959; Pridmore, 1994; Wilga and Lauder, 2001; Kajiura et al., 2003; Domenici et al., 2004; Oliver et al., 2013). These descriptions demonstrate that fins are dynamic control surfaces moving relative to the body, though the actual movement of the fin remains unclear due to a lack of standardized terminology and 3D analyses.

Volitional swimming kinematics

Our ability to evaluate the ecomorphology of an organism depends on our capacity to observe them interacting with their environment. There are critical gaps in our understanding of the interaction between morphology, function, and ecology in shark species which may be a result of the logistical issues that accompany the study of unsteady swimming and large, aquatic organisms. For this reason, the majority of fish swimming studies occur in flumes and the behaviors documented are likely not indicative of natural swimming (Lowe, 1996). Further, the size restrictions and unidirectional flow of flumes precludes the study of horizontal maneuvering. There are a number of volitional shark swimming studies, but they are limited by the use of 2D video (Lowe,

1996; Kajiura et al., 2003; Domenici et al., 2004; Porter et al., 2009; Porter et al., 2011;

Hoffmann et al., 2017). Recent advances in 3D kinematic analyses now make the

7 comprehensive study of volitional kinematics possible (Ros et al., 2011; Sellers and

Hirasaki, 2014; Jackson et al., 2016; Fish et al., 2018; Jimenez et al., 2018; Hoffmann et al., 2019). An array of cameras with overlapping fields of view are time-synchronized and calibrated for 3D space, through which trials are conducted. The use of 3D video in conjunction with volitional swimming provides an avenue for the study of a broader variety of behaviors (i.e. maneuvering) and animals that were previously infeasible in traditional flume studies. These techniques also provide data that are likely more representative of truly natural behavior (Lowe, 1996).

Summary

The overarching goal of this study was to document the relationship between morphology and function in free swimming sharks. First, we quantified differences in axial undulation that correlated to morphological variations between two hammerhead species (Chapter 2; Hoffmann et al., 2017). For a comprehensive understanding of movement in all planes, we adapted existing 3D kinematic analyses for use with fully submerged low cost cameras in a relatively large environment. This allowed for the first ever quantification of pectoral fin rotation relative to the body axis during volitional yaw maneuvering in the Pacific spiny dogfish, Squalus suckleyi (Chapter 3; Hoffmann et al.,

2019). Next, we applied similar techniques to a morphologically, physiologically, and ecologically different species (the bonnethead, Sphyrna tiburo) to compare turning performance (Chapter 4; Hoffmann and Porter, in review). Finally, we assessed the comparative external and skeletal morphology of pectoral fins from 18 shark species from various orders, families, and ecological categories (Chapter 5). These studies

8 provide a baseline understanding of the relationship between morphology and function among ecologically diverse species during volitional swimming and maneuvering.

9 Chapter 1 Figures

Figure 1.1: Six axes of movement in a fully aquatic environment. Translation occurs medio-laterally (slip), dorso-ventrally (heave), and cranio-caudally (surge). Rotation about the medio-lateral axis (pitch) adjusts vertical orientation, about the dorso-ventral axis (yaw) adjusts horizontal position, and about the cranio-caudal axis (roll) changes the longitudinal orientation of the body axis. Adapted from Parson et al., 2011.

10

Figure 1.2: Range of undulatory swimming modes (Adapted from: Breder, 1926;

Lindsey, 1978). Black bars shade the amount of body that is being laterally displaced and the black line underneath the fishes represent the relative amplitude of lateral displacement during undulation. Lateral displacement of the body axis ranges from movement of the majority of the body (anguilliform) to solely caudal oscillation

(thunniform). This is accompanied with a shift from undulation, where there is more than one wave along the body, to oscillation, which is a simplified flapping with less than one wave along the structure (Breder, 1926).

11

Figure 1.3: General pectoral fin skeleton of aplesodic and plesodic fins (Marinelli and

Strenger, 1959; Liem and Summers, 1999; Compagno, 1984; Wilga and Lauder, 2000,

2001; Maia et al., 2012). At the proximal fin base, three basal elements (propterygium, metapterygium, mesopterygium) articulate with the scapulo-coracoid. Extending distally from the fin web are three series of radial elements. Thin, flexible certaotrichia are embedded in the connective tissue that anchors the skin to the fin. Fins with less than

50% skeletal support are considered aplesodic (A) while fins with greater than 50% skeletal support are plesodic (B).

12

Figure 1.4: Hypothesized force balance in swimming sharks (Harris, 1936; Alexander,

1965; Thomson and Simanek, 1977; Nakaya, 1995; Ferry and Lauder, 1996; Wilga and

Lauder, 2000, 2002). Undulation of the body axis results in vortex shedding at the caudal fin (Fvortex jet), causing and upward reactive force at the tail (Fcaudal fin), which is the primary thrust production mechanism (Fcaudal fin x). In general, the weight of the body

(Fweight) exceeds the buoyant force (Fbuouyant), meaning that some other must produce lift to balance forces on the body. It is hypothesized that the body (Fbody) and/or pectoral fins

(Fpec fin) generate lift in the anterior body to counteract the negative buoyancy and upward lifting force generated at the tail (Fcaudal fin y).

13 CHAPTER 2: REGIONAL VARIATION IN UNDULATORY KINEMATICS OF TWO

HAMMERHEAD SPECIES: THE BONNETHEAD (SPHYRNA TIBURO) AND THE

SCALLOPED HAMMERHEAD (SPHYRNA LEWINI)

Reproduced with permission: Hoffmann, S. L., Warren S. M., and Porter, M. E.

(2017). Regional variation in undulatory kinematics of two hammerhead species, the bonnethead (Sphyrna tiburo) and the scalloped hammerhead (Sphyrna lewini). J. Exp.

Bio. 220(18). 3336-3343.

Abstract

Hammerhead sharks (Sphyrnidae) have a large amount of morphological variation within the family, making them the focus of many studies. The size of the laterally expanded head, or cephalofoil, is inversely correlated with pectoral fin area. The inverse relation in cephalofoil and pectoral fin size in this family suggests that they might serve a complimentary role in lift generation. The cephalofoil is also hypothesized to increase olfaction, electroreception, and vision; however, little is known about how morphological variation impacts post-cranial swimming kinematics. Previous studies demonstrate that the bonnethead and scalloped hammerhead have significantly different yaw amplitude and we hypothesized that these species utilize varied frequency and amplitude of undulation along the body. We analyzed video of free swimming sharks to examine kinematics and 2D morphological variables of the bonnethead and scalloped hammerhead. We also examined the second moment of area along the length of the body and over a size range of animals to determine if there were shape differences along the

14

body of these species and if those changed over ontogeny. We found that both species swim with the same standardized velocity and Strouhal number but there was no correlation between two-dimensional morphology and swimming kinematics. However, the bonnethead has a dorso-ventrally compressed anterior trunk and undulates with greater amplitude whereas the scalloped hammerhead has a laterally compressed anterior

trunk and undulates with lower amplitude. We propose that differences in cross- sectional trunk morphology account for interspecific differences in undulatory amplitude.

We also found that for both species, undulatory frequency is significantly greater in the anterior body compared to all other body regions. We hypothesize that the bonnethead and scalloped hammerhead swim with a double oscillation system.

Introduction

Hammerhead sharks, (Sphyrnidae) are characterized by a laterally expanded head

(cephalofoil) that varies greatly among species (Lim et al., 2010; Thomson and Simanek,

1977). The most basal hammerhead lineage, represented by the

(Eusphyra blochii), possesses a cephalofoil that is proportionally the largest and measures up to 50% of their total body length (Lim et al., 2010). In comparison, the bonnethead shark (Sphyrna tiburo) is the most recently derived species and their cephalofoil width is

18% of total body length. Generally, as cephalofoil width increases among species, pectoral fin area decreases (Thomson and Simanek, 1977). Previous studies on hammerhead sharks focus primarily on cephalofoil morphology and its effects on hydrodynamics and sensory efficiency; however, little is known about the morphology and function of the post-cranial body. The significant morphological variation and the close phylogenetic relationship among hammerheads make them an ideal study system to examine the effects of shape on swimming performance. 15 Morphological differences in the body axis and caudal fin are known to affect swimming style in sharks (Flammang, 2014; Lindsey, 1979; Long and Nipper, 1996; Webb and

Keyes, 1982). Stiff bodied species tend to displace their nearly homocercal caudal fin whereas flexible bodied sharks originate undulation anterior to their heterocercal tails

(Donley et al., 2005; Lindsey, 1979; Long and Nipper, 1996; Webb and Keyes, 1982).

Stiff bodied swimmers also undulate using smaller lateral displacements, encounter less incurred drag, and tend to be less maneuverable than their flexible bodied counterparts

(Alexander, 2003; Webb and Keyes, 1982). Previous studies show that scalloped hammerheads have significantly smaller body stiffness and are more flexible than a non- hammerhead species; however, these comparisons have not been made among hammerheads (Kajiura et al., 2003). Given the influence of body shape on swimming, our goal was to examine the effects of morphology on undulatory kinematics in two closely related hammerhead species.

Morphological variables such as body flexural stiffness, body profile, and caudal fin shape are known to affect fluid movement around the body and the shape of the vortex wakes produced during swimming (Long and Nipper, 1996; Webb and Keyes,

1982). To produce forward movement, sharks must generate an undulatory wave that reaches maximum amplitude at the caudal fin, shedding a wake of water that results in forward thrust (Alexander, 2003; Ferry and Lauder, 1996; Flammang et. al., 2011;

Lindsey, 1979; Webb and Keyes, 1982; Wilga and Lauder, 2002). Fishes modulate this undulatory wave by changing the frequency and amplitude of lateral displacement

(Hunter and Zweifel, 1971; Long et al., 2010). Increasing frequency, specifically tail beat frequency, is correlated with increasing swimming velocity, but the relation between amplitude and velocity is less well understood (Lauder et al., 2016; Sfakiotakis et al., 16 1999). Changes in frequency and amplitude also affect Strouhal number, which describes the cyclical motion of undulatory swimming (Alexander, 2003).

Undulatory reconfiguration, or changes in body shape during a tailbeat, can be observed when frequency and amplitude are determined along the length of the body, where the traveling wave is present during forward swimming (Long et al., 2010).

Previous studies on hammerheads show that the amplitude of head yaw increases with cephalofoil size, but body frequencies and amplitudes have not been documented

(McComb et al., 2009). The goals of the present study were to: (1) quantify variations in anterior trunk morphology (fineness ratio and second moment of area) between two species of hammerhead shark, the bonnethead (Sphyrna tiburo) and scalloped hammerhead (Sphyrna lewini); (2) compare the undulatory kinematics during volitional swimming; and (3) examine the relationship among morphological variables and swimming kinematics in these two species. Firstly, we hypothesized that anterior body morphology varied such that species with larger heads have smaller pectoral fins

(Thomson and Simanek, 1977). We also predicted that bonnethead anterior trunk stiffness, as measured by second moment of area and the anterior trunk fineness ratio, is intermediate to the values previously reported for scalloped hammerheads and sandbar sharks (Carcharhinus plumbeus), and the anterior body fineness ratio is positively correlated with head width (Kajiura et al., 2003). Secondly, we hypothesized that the scalloped hammerhead undulates at higher amplitude than the bonnethead based on previous studies demonstrating this trend in head yaw (McComb et al., 2009). We also predicted that both species have a higher undulatory frequency in the anterior body compared to the rest of the body. Finally, we hypothesized that within each species, morphological variables correlate with swimming kinematics. We predicted that 17 cephalofoil area is negatively correlated with anterior body amplitude, anterior body fineness ratio is positively correlated with anterior body amplitude, and pectoral fin area is negatively correlated with mid-body amplitude.

Materials and methods

Study animals

Volitional swimming of scalloped hammerhead sharks, Sphyrna lewini, was filmed at the Hawaii Institute of Marine Biology as part of an approved IACUC protocol to T. C. Tricas at the University of Hawaii at Manoa. Young of the year scalloped hammerhead sharks (mean total length: 60.0 cm; n=4) were caught using hook and line from Kaneohe Bay, Oahu, HI, USA. Animals were housed and filmed together in a twelve-foot diameter tank at the Hawaii Institute of Marine Biology after a 24hr acclimation period. Individuals of the same species were filmed together and distinguished by anatomical differences such as total length. Bonnethead sharks,

Sphyrna tiburo, were collected in Long Key, FL, USA, and cared for under an approved

IACUC protocol granted to the authors. Sub-adult to adult bonnethead sharks (mean total length: 89.2 cm; n=4) were caught in shallow waters using a gill net near Long Key, FL.

Animals were housed and filmed in a sixteen-foot diameter tank at the Florida Atlantic

University Marine Research Facility, FL, USA. Bonnetheads acclimated for 24hrs prior to swimming trials and were filmed one individual at a time.

In this study, bonnethead and scalloped hammerhead sharks are in different life history stages, but they are approximately matched for body size. Adult scalloped hammerheads mature between 140cm TL (males) and 212 cm TL (females) and pose many captivity and husbandry challenges in a lab setting (Compagno, 1984). As a result of varying life history, there may be ontogenetic differences not captured in these data. 18 We attempted to mitigate these differences by using data standardized by total length for swimming velocity and tail beat amplitudes, and size independent variables like flexion frequency (Hz) and flexion amplitude (º).

Morphological measurements

For each shark, we captured still images from video footage and measured the whole dorsal body area (cm2) using ImageJ (1.38X available from the National Institutes of Health). We partitioned out the cephalofoil area and total pectoral fin area, which were standardized by dorsal body area. Total planning area was calculated as the sum of the cephalofoil area and pectoral fin area and was standardized by dorsal body area. To make direct comparisons with published data, we also quantified cephalofoil width (cm) as measured from eye to eye (McComb et al, 2009; Thomson and Simanek, 1977). We measured the anterior body fineness ratio defined as the maximum width divided by the length of the anterior trunk region from the base of the cephalofoil to the origin of the pectoral fins.

To examine cross-sectional morphology of the anterior trunk, we CT scanned a bonnethead (n=1, TL = 68.83cm) and scalloped hammerhead (n=1, TL = 51.94cm) shark on a GE Medical Systems LightSpeed 16 CT scanner with 0.625mm slice thickness at

South Florida Radiation Oncology. Single slice images (512x512p) from the scans were used to calculate the second moment of area (I), a structural predictor of stiffness, at multiple points along the body as determined by individual slices at those landmarks.

Second moment of area (I) was measured at five anatomical landmarks along the total length: (A) base of the cephalofoil, (B) third gill slit, (C) anterior pelvic fin origin, (D) anterior origin of the second dorsal fin, (E) and caudal peduncle. I was calculated in both the dorso-ventral 19 풂풃ퟑ 푰 = 흅 (Eq. 1.1) 풚 ퟒ and lateral

풂ퟑ풃 푰 = 흅 (Eq. 1.2) 풙 ퟒ orientations. The ratio of these measurements was used to compare cross-sectional shape between the two specimens to remove the effect of body size (Mulvany and Motta, 2013).

To investigate the effect of ontogeny on anterior body shape, a cross-section at the third gill slit (region B) was dissected from fresh frozen individuals of varied body length for the scalloped hammerhead (n=4, TL=51.9cm - 250cm) and the bonnethead (n=10,

TL=14.6cm - 96.5cm). Cross-sections were scaled with a 15cm ruler and photographed with a Nikon D3300 for ImageJ analysis and I was calculated as outlined above. A simple linear regression of Ix:Iy vs. total length (cm) was used to determine if there were differences in shape through ontogeny.

Kinematic analysis

Video was filmed from a dorsal view with a GoPro Hero3 at 30 fps and

1080x1920p as the sharks swam volitionally. A flat port housing was used and the cameras were set to narrow field of view to eliminate the barrel distortion of GoPro cameras. Sharks occupied less than 5% of the frame and we selected trials in which individuals were centered in the frame to avoid potential distortions at the edges of the field of view. Cameras were mounted 1m above the water surface and water depth was approximately 1m. Variables were standardized to animal total length or variables were size independent (units of Hz or º) to minimize effects of varying sizes among individuals and depth of each swim in the tank. A 30cm ruler was placed at the bottom of the tank to provide scale for the camera. To ensure that the same trials were not analyzed twice,

20 video was analyzed sequentially to select clips in which sharks completed at least three full tail beat cycles of straight, steady swimming.

We analyzed eight anatomical landmarks using LoggerPro 3.10.1 point tracking software (Vernier Software & Technology, Beaverton, OR, USA; Fig. 2.1). Anatomical landmarks included: (A) the tip of the rostrum, (B) midline at the gills, (C) anterior origin of the first dorsal fin, (D) anterior origin of the second dorsal fin, (E) caudal peduncle, (F) tip of the caudal fin, (G) left posterior margin of the cephalofoil, and (H) right posterior margin of the cephalofoil (Fig. 2.1). To account for noise, point tracking data were filtered using a low-pass, fourth order butterworth filter at 5Hz (Erer, 2007).

We measured variables related to the overall swimming performance. We calculated velocity (V; cm∙s-1) by measuring the displacement of point C from frame to frame over the time between frames. This was averaged over the duration of the clip (at least three tail beats). A tail beat cycle is defined as the excursion of the tail from peak lateral flexion on one side back to peak lateral flexion on the same side. We measured tail beat frequency (f; Hz) for each clip as cycles per second. Tail beat amplitude (A; cm) was measured as the peak-to-peak distance covered by the tail from full lateral flexion on one side to the full lateral flexion on the other. We then calculated Strouhal number (St) as

푨풇 푺풕 = (Eq. 2.3) 푽 where A is peak-to-peak tail beat amplitude (cm), f is tail beat frequency (Hz), and V is velocity (cm∙s-1) (Rohr and Fish, 2004). To remove the effect of body size when comparing between species, both tail beat amplitude (St. A) and velocity (U; body lengths·s-1) were standardized by total length.

21 Regional flexion and amplitude were measured for the anterior body (AB) as angular displacement of the anterior trunk (point B); for the mid-body (MB) as angular displacement at the dorsal fin origin (point C); for the posterior body (PB) as angular displacement at the second dorsal fin origin (point D); and for the caudal fin (CF) as angular displacement at the caudal peduncle (point E) (Fig. 1.1). Flexion amplitude was measured as the maximum displaced angle from the straightened midline (180o) and flexion frequency was calculated as the maximum displacement per time averaged over at least three tail beats. The anterior body (AB) flexion frequency is synonymous with head yaw quantified in previous studies (McComb et al., 2009).

Statistical analysis

For both species, video was obtained for four individuals. We selected a maximum of three clips per individual to maintain a balanced design. For each variable, we calculated the mean for each shark and we used those mean values for statistical analyses. Each variable was evaluated to ensure normality and homoscedasticity using a

Shapiro-wilk test, and variances were analyzed using ANOVA with JMP v.5.0.1.a (SAS

Institute Inc., Cary, NC, USA). The flexion frequency and flexion amplitude were analyzed using mixed-effects ANOVA with species, region, and the interaction term as fixed effects and individual was coded as a random effect. Post-hoc t-tests were used to compare the means of each species at each region, using a standard Bonferroni corrected alpha value of P = 0.0125. Linear regressions analyses were used to assess the relations of kinematic variables with the morphological variables outline above.

22 Results

Morphology

Total length (TL) and dorsal body area were significantly different between species (F1,6 = 67.7894, P = 0.0002; F1,6 = 53.0927, P = 0.0003; respectively;

Supplemental Table 2.1). To account for size and body area differences when comparing between species, we standardized cephalofoil and pectoral fin area by dorsal body area.

As predicted, standardized cephalofoil area was significantly greater in the scalloped hammerhead whereas standardized pectoral fin area was significantly higher in the bonnethead (Fig. 2.2A,B; F1,6 = 51.4146, P = 0.0004; F1,6 = 76.3849, P < 0.0001). When considering the total area of the planing surface (cephalofoil area + pectoral fin area), we found that the standardized planing area was significantly greater in the scalloped hammerhead (Fig 2.2C; F1,6 = 18.4857, P = 0.0051). Additionally, the anterior body fineness ratio was significantly higher in the scalloped hammerhead (Fig. 2.2D; F1,6 =

63.2371, P = 0.0002).

For all five regions of the body axis, second moment of area (I) was greater in the bonnethead (Fig. 2.3A). To remove the effect of body size, we calculated the ratio of second moment of area (I) measured in the dorsal-ventral direction (Iy) to second moment of area measured in the lateral direction (Ix) as a quantification of shape (Mulvany and

Motta, 2013). A ratio of one indicates a perfect circle, greater than one indicates lateral body compression, and less than one indicates dorso-ventral body compression. For four of the five cross sectional body regions, the scalloped hammerhead had a greater Ix:Iy ratio (Fig. 2.3B). Additionally, the greatest differences in cross-sectional shape between species were in the first two cross-sections at the base of the cephalofoil and third gill slit

(the anterior trunk region). 23 To ensure the variations in anterior body cross-sectional morphology reported in

Fig. 2.2 were not an effect of total body length (a proxy for ontogeny in each species), we examined the Ix:Iy ratio at the third gill slit among animals of varying size in the scalloped hammerhead (n=4, TL 51.9cm – 250cm) and the bonnethead (n=10, TL 18.5cm –

111.5cm) (Fig. 2.4A). There was no significant difference in the cross-sectional shape for either species across total length; however, Ix:Iy was significantly greater in the scalloped hammerhead (Fig. 2.4B: F1,12 = 13.4256, P= 0.0032).

Kinematics

Neither velocity (m∙s-1) nor standardized velocity (body lengths∙s-1) was different between species (Fig. 2.5A; Supplemental Table 2.2; P = 0.1712 and P = 0.2334, respectively). Strouhal number was not different between species (Fig. 2.5B;

Supplemental Table 2.2; P = 0.2180). Neither tail beat frequency nor standardized tail beat amplitude differed between species (Fig. 2.5C and D; P = 0.2072 and P = 0.8199; respectively).

A mixed effects ANOVA showed that flexion amplitude was significant with species and region as fixed effects and individual as a random effect (Fig. 2.6A; R2 =

0.9496, P < 0.0001). Flexion amplitude was significantly greater in the bonnethead with species as a fixed effect (F1,6 = 9.7661 P=0.0205). Region was also a significant fixed effect (F3,18 = 114.356, P < 0.0001). Post-hoc comparisons show that flexion amplitude in both species was lowest in the anterior body (AB), greatest at the caudal fin (CF), and the mid-body (MB) and posterior body (PB) were intermediate and not significantly different from one another. Between species, there was no difference in flexion amplitude in the

CF; however, AB, MB and PB amplitude were significantly greater in the bonnethead (P

= 0.0141, P = 0.0159, P = 0.0057; respectively). 24 Flexion frequency was also significant as a mixed effects ANOVA with species, and region as fixed effects and individual as a random effect (Fig 2.6B; R2 = 0.8899, P

<0.0001). Region was the only significant effect (F3,18 = 2.2007, P < 0.0001). Post-hoc analyses show that for both species, flexion frequency was greatest in the AB and there was no significant difference among the MB, PB, and CF (P < 0.0001).

We used simple linear regressions to examine the effects of morphology on flexion amplitude, which was the only kinematic variable different between species. For both species, there was no relation between anterior body fineness ratio and anterior body amplitude, contrary to our predictions. There was also no relation between standardized cephalofoil area and AB flexion amplitude. Finally, MB flexion amplitude was not correlated with pectoral fin area.

Discussion

Previous studies have demonstrated that closely related bonnethead (Sphyrna tiburo) and scalloped hammerhead (Sphyrna lewini) sharks differ in morphology, sensory physiology, body flexibility, and head yaw amplitude; however, there are limited data on the effect of morphology on swimming kinematics in these two species (Kajiura et al.,

2003; Lim et al., 2010; Mara et al., 2015; Mccomb et al., 2009; Thomson and Simanek,

1977). Our goal was to link morphological variation between species with differences in swimming kinematics. In addition to differences noted in previous studies (cephalofoil width, pectoral fin area), we showed whole body variation in morphological variables between these species (cephalofoil area, pectoral fin area, dorsal body area, anterior fineness ratio, second moment of area; Figs 2.2-4). Despite having different morphologies, the swimming kinematics of these species was similar (Fig. 2.5). We were not able to show any correlations among these morphological variables and kinematics 25 within each species. However, we found that these species employ different undulatory strategies to produce similar standardized velocity (body lengths∙s-1) and Strouhal number (Fig. 2.6). The scalloped hammerhead has a larger cephalofoil, narrower anterior trunk, and swims at a low undulatory amplitude whereas the bonnethead has a smaller cephalofoil, wider anterior trunk, and swims at a higher amplitude.

Morphological Variation

Bonnethead and scalloped hammerhead sharks show large variations in external morphology. We supported our hypothesis that the bonnethead has a proportionally smaller cephalofoil and larger pectoral fin area whereas the scalloped hammerhead had a proportionally larger cephalofoil and smaller pectoral fin area (Fig. 2.2). A similar inverse relation between the cephalofoil and pectoral fin area has also been noted in the smooth hammerhead (Sphyrna zygaena) (Thomson and Simanek, 1977). Previous data show the inverse relation between pectoral fin area and cephalofoil area results in the same total area of planning surfaces in the bonnethead, scalloped hammerhead, and smooth hammerhead; however, we found that the scalloped hammerhead has a significantly larger planning surface area than the bonnethead (Thomson and Simanek,

1977). These data also support our hypothesis that the anterior body fineness ratio of the scalloped hammerhead shark was significantly greater than the bonnethead, demonstrating that the scalloped hammerhead has a narrower anterior trunk (Fig. 2.2D).

We predicted that the bonnethead would have an anterior trunk that is stiffer than the scalloped hammerhead but not as stiff as the sandbar shark. We found that the anterior body (AB) second moment of area (Ix) of the bonnethead is greater than the scalloped hammerhead, suggesting that the bonnethead anterior body is structurally stiffer (Fig. 2.3A; Kajiura et al., 2003). However, the second moment of area 26 measurements we found are greater than values previously reported for the scalloped hammerhead due to overall differences in body size, and we are unable to compare our Ix results to those reported for a non-hammerhead species (Kajiura et al., 2003).

To account for the differences in body size between the bonnetheads and scalloped hammerheads in this study, we compared the cross-sectional trunk shape quantified as the ratio Ix:Iy, where values greater than one indicate lateral compression and less than one indicate dorso-ventral compression. The greater Ix:Iy ratios at four of the five cross-sectional regions further suggest that the trunk of the scalloped hammerheads is laterally compressed and likely more flexible than the bonnethead (Fig.

2.3B). The bonnethead has significantly more dorso-ventrally compressed anterior trunk and is probably stiffer than the scalloped hammerhead (Fig. 2.3B). We also demonstrated that across ontogeny, there is no change in anterior trunk shape in both species, and the bonnethead has a significantly more dorso-ventrally compressed anterior trunk and is likely stiffer than the scalloped hammerhead (Fig. 2.4)

Undulatory Variation

In addition to differences in trunk cross-sectional morphology and stiffness, we found that bonnethead and scalloped hammerhead sharks modulate the frequency and amplitude of the undulatory wave to produce overall similar kinematic outputs.

Standardized velocity (U), Strouhal number (St), tailbeat frequency (f), and standardized tail beat amplitude (St. A) were statistically similar between species (Fig. 2.5). Based on previous studies showing that the scalloped hammerhead has a greater amplitude of head yaw, we hypothesized that the scalloped hammerhead would have a greater undulatory amplitude at all body regions compared to the bonnethead. We found differences in regional flexion amplitude between species; however, the mid-body and posterior body 27 were significantly more displaced in the bonnethead rather than the scalloped hammerhead as predicted (Fig. 2.6A). We suggest that differences in mid-body amplitude may be a result of differing cross-sectional trunk morphology; however, we were not able to directly test this relation with our data, as we were unable to obtain cross-sectional data from the sharks used in the kinematic analyses.

We supported our hypothesis that the anterior body undulates at a higher frequency than the rest of the body in both species (Fig. 2.6B). The white sturgeon,

Acipenser transmontanus, swims using a double oscillating system, where undulatory frequency in the anterior body differs from the rest of the body (Fig. 2.6B; Long, 1995).

These complexities in undulatory wave propagation have also been observed in lamprey and eel, and they may stand apart from the traditional ridged bodied teleost model where undulatory amplitude increases rostro-caudally (Long, 1995; Root et al., 1999; Long et al., 2010). With the exception of the eel, these patterns have been observed in fishes with cartilaginous vertebral columns or notochords, and are perhaps due to the mechanical properties of cartilaginous fishes (Porter et al., 2014; Porter et al., 2016; Long et al.,

2004; Long et al., 2002). It has been suggested that anterior body flexion may be a result of recoil from body undulation, or that it may control the driving frequency of undulation; however, recent studies show that movement of fishes heads increases their sensitivity to external stimuli (McHenry et al., 1995; Webb, 1988, Akanyeti et al., 2016).

Additionally, fish may have morphological adaptations associated with damping or selectively alter damping coefficients to minimize the effect recoil and higher order harmonics (Lighthill, 1977; Long, 1988; Root et al., 1999; Webb, 1988). We suggest that bonnethead and scalloped hammerhead sharks increase anterior body frequency independently from the rest of the body to increase sensory perception (i.e., 28 electroreception and vision) (Kajiura and Holland, 2002; McComb et al., 2009).

Increased anterior body frequency (yaw defined in McComb et al., 2009) is particularly important for the bonnethead and scalloped hammerhead because it increases the area covered by the cephalofoil when scanning for bioelectric fields, a critical hunting behavior in benthic animals (Kajiura, 2001; Kajiura and Holland, 2002). Furthermore, head yaw increases the visual binocular overlap by upwards of 15o in both species

(McComb et al., 2009). The greater anterior body flexion frequency for both species may allow bonnethead and scalloped hammerhead sharks to increase sensory perception without increasing whole-body frequency and the associated energetic cost (Fig. 1.6B).

Finally, the double oscillation system shown here, in which the anterior body undulates at a different frequency than the posterior body, may be specific to cartilaginous fishes

(Long, 1995). Shark vertebral columns are non-linear, viscoeleastic systems that store and transmit energy behaving as both a spring and a brake, which allows for the differential transmission of power depending on the undulatory amplitude and frequency

(Porter et al., 2016; Porter et al., 2014). We hypothesize that the variable mechanical behavior of the cartilaginous vertebral column allows for high frequency undulation in the anterior body without disrupting lower frequency, posterior body undulation.

Finally, we did not support our hypothesis that morphological variables would correlate with swimming kinematics within each species. We found no relation between cephalofoil area and anterior body amplitude, anterior body fineness ratio and anterior body amplitude, or pectoral fin are and mid body amplitude. These results may be due to the high variability associated with volitional swimming kinematics and narrow range of morphological variability within our individuals. Future studies may examine the relation of morphological variables and swimming kinematics of hammerhead shark 29 where differences may be evident when examining more species or a greater variation in sizes.

We show that bonnethead and scalloped hammerhead morphology varies along the length of the whole body dorsally and in cross-section (Fig.2.2-4). We propose that the differences in undulatory amplitude observed between these species are a result of trunk stiffness (as measured by second moment of area and anterior body fineness ratio) and cephalofoil width (Fig. 2.2-4). We were not able to correlate any of the 2D morphological variables examined here with swimming kinematic outputs. We hypothesize that the dorso-ventrally compressed anterior trunk of the bonnethead may be more resistant to high frequency bending and rather compensates with high amplitude undulation. Future studies should focus on examining the relation between second moment of area variations along the body with differences in flexion amplitude and frequency.

Conclusions

Previous literature focuses primarily on morphology and function of the cephalofoil of hammerhead sharks. This is the first study to examine whole body morphology and swimming kinematics of two hammerhead species. We found that morphologically different scalloped hammerhead and bonnethead sharks swim at similar volitional velocities and Strouhal numbers, but modulate the amplitude of undulation differently. The scalloped hammerhead has a larger cephalofoil, laterally compressed anterior trunk, and undulates at a lower flexion amplitude in comparison the bonnethead which has a smaller cephalofoil and dorso-ventrally compressed anterior trunk. We found that both species exhibit greater flexion frequency in the anterior body setting up a double oscillating system, which we propose may be specific to fishes with cartilaginous 30 skeletons. We hypothesize that high frequency head yaw increases sensory perception without the added energetic costs increasing undulatory frequency throughout the whole body.

31 Chapter 2 Figures

Figure 2.1: Eight anatomical landmarks were digitized on each individual for both species. For regional flexion frequency and amplitude analyses, four isolated regions are outlined in brackets as the anterior body (angle ABC), mid-body (angle BCD), posterior- body (angle CDE), and caudal peduncle (angle DEF).

32

Figure 2.2: Morphological differences between bonnethead (black) and scalloped hammerhead (white) bodies. (A) The cephalofoil (standardized by dorsal body area) of the scalloped hammerhead shark was 39% larger than the bonnethead though the standardized pectoral fin area (B) of the bonnethead was 38% larger. (C) Standardized planning surface area (cephalofoil area + pectoral fin area) of the scalloped hammerhead was 5% greater than the bonnethead. (D) Anterior body fineness ratio was 30% greater in the scalloped hammerhead. Error bars represent standard error. Stars denote significant statistical differences.

33

Figure 2.3: Regional variation in second moment of area of the body between bonnethead

(black) and scalloped hammerhead sharks (white). (A) Second moment of area (Ix) was greater in the bonnethead compared to the scalloped hammerhead at all five regions. (B)

Ix:Iy was greater in the scalloped hammerhead at four of the five regions, demonstrating that the scalloped hammerhead has a more laterally compressed trunk. The biggest differences in shape between the two species was observed in the anterior trunk region.

34

Figure 2.4: Variations in anterior body cross-sectional morphology, quantified as Ix:Iy at third gill slit. (A) There was no difference in cross sectional shape across body sizes for either species. (B) Cross sectional shape was approximately circular (Ix:Iy = 1) in the bonnethead in comparison to the scalloped hammerhead in which the body was laterally compressed (Ix:Iy > 1). Error bars represent standard error. Stars denote significance.

35

Figure 2.5: Comparison of performance variables between the bonnethead (black) and scalloped hammerhead (white). (A, B) Neither velocity (cm∙s-1) nor standardized velocity (body lengths∙s-1) was significantly different between species. (C, D) Tail beat frequency and standardized tail beat amplitude did not vary between species. Error bars represent standard error.

36

Figure 2.6: Flexion variables differed between bonnethead (black) and scalloped hammerhead (white) sharks. (A) Midline amplitude increased significantly along the length of the body with the largest amplitudes produced in the caudal region. Midline amplitude differed between species at MB and PB regions where bonnethead amplitude was on average 28% greater than the scalloped hammerhead. (B) AB had significantly higher frequencies than the three posterior regions (MB, PB, CF) in both species and was not different between species. The frequency of flexion was consistent across the three posterior regions (MB, PB, CF) for both species. Error bars represent standard error. Lower case letters denote significant statistical differences.

37 CHAPTER 3: THREE-DIMENSIONAL MOVEMENTS OF THE PECTORAL FIN

DURING YAW TURNS IN THE PACIFIC SPINY DOGFISH, SQUALUS SUCKLEYI

Reproduced with permission: Hoffmann, S. L., Donatelli, C. M., Leigh, S. C., Brainerd,

E. L., & Porter, M. E. (2019). Three-dimensional movements of the pectoral fin during yaw turns in the Pacific spiny dogfish, Squalus suckleyi. Biol. Open, 8(1), bio037291.

Abstract

Fish pectoral fins move in complex ways, acting as control surfaces to affect force balance during swimming and maneuvering. Though objectively less dynamic than the fins of their actinopterygian relatives, shark pectoral fins undergo complex conformational changes and movements during maneuvering. Asynchronous pectoral fin movement is documented during yaw turning in at least two shark species but the three- dimensional (3D) rotation of the fin about the body axes is unknown. In this study, we quantify the 3D actuation of the pectoral fin base relative to the body axes. We hypothesized that Pacific spiny dogfish rotate pectoral fins with three degrees of freedom relative to the body during volitional turning behaviors. We found that the pectoral fin on the inside of the turn is consistently protracted, supinated, and depressed. Additionally, turning angular velocity increased with increasing fin rotation. We found that estimated drag on the fin increased and the shark decelerated during turning. Based on these findings, we propose that Pacific spiny dogfish uses drag-based turning during volitional swimming. Post-mortem muscle stimulation revealed depression, protraction, and supination of the pectoral fin through stimulation of the ventral and cranial pterygoideus

38 muscles. These data confirm functional hypotheses about pectoral fin musculature and suggest that Pacific spiny dogfish actively rotate pectoral for drag-based turning.

Introduction

The morphology and movement of control surfaces (structures that adjust an organism’s position in space) in swimming vertebrates have profound effects on stability and maneuverability (Webb and Weihs, 2015; Fish and Lauder, 2017). Paired fins and flippers are particularly important in balancing forces during steady swimming and reorienting force during maneuvering (Harris, 1936; Nursall, 1962; Fish, 1997; Fish and

Shannahan, 2000; Fish, 2002; Wilga and Lauder, 2000; Fish et al., 2018). Despite the vast diversity of whole body morphology and swimming styles (i.e. median paired fin

[MPF] vs. body caudal fin [BCF]), the pectoral fins of fishes are widely acknowledged as dynamic control surfaces generating thrust, lift, and drag critical to maneuvering (Webb,

1984; Drucker and Lauder, 2002; Drucker and Lauder 2003; Lauder and Drucker, 2004;

Webb and Weihs, 2015; Fish and Lauder, 2017). Synchronous pectoral fin rotation symmetrically alters force generation such that the horizontal swimming trajectory is unaffected. For example, sunfish rotate both pectoral fins to direct forces anteriorly, resulting in reactional braking directed through the center of mass (Drucker and Lauder,

2002). Alternatively, asynchronous pectoral fin rotation generates an imbalance of forces and initiates yawing (horizontal maneuvering). Sunfish and trout rotate the outside fin to generate a laterally oriented force and turn the body horizontally, while the inside fin directs thrust posteriorly moving the fish forward (Drucker and Lauder, 2001; 2004).

Asynchronous pectoral fin movement is also observed in shark yaw turning (Kajiura et

39 al., 2003; Domenici et al., 2004), but the 3D kinematics and their effect on turning have not been quantified.

The volitional swimming behavior of sharks has been documented in a few species but is limited by use of 2D video (Lowe, 1996; Kajiura et al., 2003; Domenici et al., 2004; Porter et al., 2009; Porter et al., 2011; Hoffmann et al., 2017). Studies examining yaw maneuvering in sharks used dorsal video and focused on whole body kinematics; but asynchronous pectoral fin movement has also been noted during turning

(Kajiura et al., 2003; Domenici et al., 2004). In a dorsal view of the bonnethead shark, the visible surface area of the pectoral fin area inside to body curvature is significantly smaller than the outside fin, suggesting the pectoral fins may play different roles during turning (Kajiura et al., 2003). Similarly, the spiny dogfish differentially moves the pectoral fins to create tight turning radii during escape maneuvers (Domenici et al.,

2004). In these instances, fin movement is hypothesized to increase drag, thereby creating a turning moment (Kajiura et al., 2003). Despite observations that they are dynamic control surfaces rotating in at least two axes, the role of pectoral fin movement in yaw maneuvering remains unclear (Pridmore, 1994; Wilga and Lauder 2000; 2001; Kajiura et al., 2003; Domenici et al., 2004; Oliver et al., 2013).

The pectoral fins of fishes become increasingly mobile and flexible through evolutionary time, yet the fins of basal clades are also described to have some range of motion in relation to the body (Wilga and Lauder, 1999; Lauder 2015). The kinematics and morphology of shark pectoral fins are described in a few species, and are generally stiffer than those of ray finned fishes and lack jointed fin rays. Though shark pectoral fins undergo substantial conformational changes during swimming, they are not collapsible to

40 the same degree as most actinopterygian fins (Fish and Shannahan, 2000; Wilga and

Lauder 2000, 2001; Lauder 2015). Despite this major difference in flexibility and structure, shark pectoral fins are mobile at the insertion and have associated musculature that is well situated to actuate 3D rotation of the fin in relation to the body (Marinelli and

Strenger, 1959; Fish and Shannahan 2000; Wilga and Lauder 2000,2001). Squalids have three pectoral fin muscles associated with the pectoral fin: the cranial pterygoideus, dorsal pterygoideus, and ventral pterygoideus, which are hypothesized to protract, elevate, and depress the fin, respectively (Marinelli and Strenger, 1959). Previous data on leopard sharks, semifaciata, demonstrate that during vertical maneuvering, the dorsal and ventral fin muscles are active during rising and sinking as the fin elevates and depresses, respectively (Maia et al., 2012). We hypothesize that active fin rotation about the pectoral girdle plays a major role in reorienting the fin, and thus force generation, during maneuvering.

One factor that confounds the role of pectoral fin rotation in shark maneuvering is the use of differing anatomical and rotational terminology (Pridmore, 1994; Liem and

Summers, 1999; Goto et al., 1999; Fish and Shannahan, 2000; Wilga and Lauder, 2000,

20001; Kajiura et al., 2003; Oliver et al., 2013). During vertical maneuvering, the pectoral fins are described as “ventrally rotated” or changing angle of attack to flow, which may refer to both depression or pronation/supination of the fin (Fish and

Shannahan, 2000; Wilga and Lauder 2000, 2001). Additionally, depression/elevation are often used interchangeably with abduction/adduction to describe pectoral fin rotation about the rostro-caudal axis (Table 3.1; Marinelli and Strenger, 1959; Liem and

Summers, 1999; Wilga and Lauder, 2001; Oliver et al., 2013). For example, Oliver et al.

41 (2013) noted the pelagic thresher, Alopias pelagicus, adducts both pectoral fins to initiate a breaking moment during tail slaps. Put in context, the fins are likely depressed, though fin depression has also been referred to as abduction (Table 1; Wilga and Lauder 2001;

Oliver et al., 2013). Thresher fin movement is further described as “laterally rotated”, potentially referring to either rotation of the fin about the rostro-caudal axis (elevation) or the dorso-ventral axis (protraction/retraction) (Oliver et al., 2013). Qualitative observations of pectoral fin movement describe fins as being “tucked” under the bonnethead shark during turning, and “swinging” during walking in the epaulette shark

(Hemiscyllium ocellatum), but the 3D movement of the fin remains unclear (Pridmore,

1994; Goto et al., 1999; Kajiura et al., 2003). Resolving the terminology used to describe pectoral fin movement specific to sharks will greatly increase our understanding of their functional roles and associated musculature.

The goal of the present study is to describe 3D movement of Pacific spiny dogfish pectoral fins during routine yaw turning and under targeted muscle stimulation. We aimed to (1) quantify the 3D rotations of pectoral fins in relation to the body, (2) investigate the effects of pectoral fin movement on whole body maneuvering kinematics, and (3) describe pectoral fin rotation in response to targeted stimulation of pectoral girdle musculature. In free-swimming sharks, we targeted yaw turns since previous studies documented pectoral fin movement during horizontal maneuvering and proposed that pectoral fin depression generates turning momentum (Kajiura et al., 2003; Domenici et al., 2004). Similarly, we hypothesized that the fin inside to body curvature would be depressed to generate torque during turning. Swimming trials were followed with targeted post-mortem muscle stimulation to determine the role of pectoral girdle

42 musculature in fin actuation. We hypothesized that post-mortem muscle stimulation of the dorsal pterygoideus (DP), ventral pterygoideus (VP), and cranial pterygoideus (CP) would result in elevation, depression, and protraction of the fin, respectively.

Methods

Pacific spiny dogfish, Squalus suckleyi (n=3, 51.2 cm – 56.3 cm fork length), were collected via otter trawl in Friday Harbor, WA. All husbandry and procedures were approved under University of Washington IACUC protocol 4239-03. Individuals acclimated in a 2 m diameter tank for a minimum 7 days prior to swimming trials.

Volitional swimming trials

Individuals were anesthetized via submersion in a 190 L aquarium with a 0.133 g∙L-1 MS-222 solution buffered with NaOH via recirculating aquarium powerhead. White rectangular plastic beads (4 x 3.5 x 3.5 mm) were affixed with cyanoacrylate along both the anterior trunk and pectoral fins of the shark (Fig. 3.2A). Beads were placed so that each region had a minimum of five markers evenly spaced along the semi-rigid parts of the fin and body. After bead placement, which lasted less than five minutes, the individual recovered in a 1.5 m diameter holding tank until normal ventilation resumed.

The individual was then transferred to a 2 m diameter, semicircular filming arena with water depth approximately 1.5 m.

Two GoPro Hero3+ light cameras were mounted on cement blocks and positioned along the diameter of the tank, approximately 45o to one another, with the lenses fully submerged (Fig. 3.2B). Cameras were synchronized with a flashing light, filmed at 30 fps with 1080 p resolution, and were set to narrow field of view. A 1 m3 volume was

43 outlined using corks affixed to bricks in the view of both cameras and was calibrated for

3D analyses with a 31.5 cm x 40.5 cm checkerboard (Fig. 3.2C). A minimum of twelve images of the checkerboard calibration object from different regions throughout the volume of interest were tracked for both camera views in XMALab v.1.3.9 to generate a

3D calibration (Knorlein et al., 2016). Any remaining video distortion was corrected in

ProDrenalin v.1.0 camera optimization software designed to remove barrel distortion specific to the GoPro Hero 3+ cameras (proDAD, Inc.). After distortion correction, additional images of the calibration object from different regions of the volume of interest were tracked in XMALab and the distance between known points on the checkerboard were calculated and compared to known distances to ensure that any barrel distortion was removed (Knorlein et al., 2016). The difference between the known distance on the checkerboard and calculated distance from tracked points in XMALab did not exceed 0.5 mm.

After resuming normal swimming behavior, individuals were enticed to execute yaw turns in the calibrated volume by placing an object in the path of the shark

(Domenici et al., 2004). Total filming time lasted no longer than three hours, and videos were later clipped into trials in which the shark turned in the calibrated volume. Three trials were selected for each of the three individuals, for a total of n = 9 trials. Trials were selected in which the fin interior to body curvature (hereafter referred to as the inside fin) and trunk markers are clearly visible, limited change in altitude was observed, and the shark executed a clear yaw turn.

44 Muscle stimulation trials

Following volitional swimming trials, individuals were euthanized via submersion in a 2 g∙L-1 MS-222 solution buffered with NaOH. Bipolar electrodes made from 57 μm diameter insulated alloy wire were implanted sub-dermally in post mortem individuals via 21gauge needles (Flammang, 2010). We targeted pectoral fin muscles and one lead was placed in each of the following: cranial pterygoideus (protractor), dorsal pterygoideus (levator), and ventral pterygoideus (depressor) (Fig. 3.5). Individuals were fully submerged and suspended using a mesh sling in a 190 L aquarium filled with seawater. Two Panasonic Lumix cameras were positioned 45o to one another and approximately 60 cm away from the tank wall. Cameras were synchronized with a flashing light and the tank volume was calibrated with a 3D calibration object made from

Lego bricks (Knorlein et al., 2016). We conducted stimulation trials on one fin for each individual, with one trial per each of the three muscles. Trials were filmed at 120 fps and

1080 p resolution. Electrodes were stimulated individually using a BK Precision 4052 signal generator. A continuous 30 Hz, 10 V square wave pulse was generated until the fin came to rest in the rotated position at which point the pulse was discontinued and the fin returned to resting position (Flammang, 2010). Leads were placed as close to the middle of the muscle as possible and we used a relatively high stimulation to capture maximal muscle recruitment. All pulses lasted less than 2 s, and we observed that the fin was at rest before further stimulation trials on subsequent muscles were conducted.

3D marker tracking

Bead markers were tracked in 3D using XMALab v.1.3.9. Rigid bodies were created from a minimum of five beads on the inside fin and trunk. The precision of

45 marker tracking in XMALab is calculated as the standard deviation of the distance between markers in a rigid body, and precision is ± 0.1 mm or better in marker based X- ray Reconstruction of Moving Morphology (XROMM) studies (Brainerd et al., 2010;

Knorlein et al., 2016). Rigid bodies are typically generated from one bone; however, sharks lack rigid skeletal elements for the formation of true rigid bodies and pectoral fins undergo conformational changes during swimming (Wilga and Lauder, 2000). Even so, the cartilaginous elements of the fin base are well calcified, and we assume that the fin base acts roughly as a rigid body. Further, markers were attached to the skin and there may be some artifact of soft tissue movement that increases marker tracking error

(Leardini et al., 2004). For the purposes of this study, we positioned beads at the proximal pectoral fin base towards the leading edge, where the radials support the fin and are tightly associated with a network of collagenous fibers (Fig. 3.1; Marinelli and

Strenger, 1959). Beads were placed in a constellation pattern with a minimum of four beads per rigid body to best describe the mobility of the pectoral fin base relative to the body axes (Knorlein et al., 2016). We treated the fin and trunk region as rigid bodies as an estimation of whole fin movement, since shark pectoral fins change conformation during swimming (Maia et al., 2012; Wilga and Lauder 2000,2001). The average standard deviation of inter-marker distance in the fin and trunk rigid bodies in this study was 0.68 mm (Table 3.2), setting an upper limit of less than 0.02% of body length for the amount of non-rigidity displayed by the fin and trunk in the region of our bead sets. The inter-marker distance errors measured here are larger than the 0.1 mm error that is standard in marker based XROMM (Brainerd et al., 2010) and could be due to the combined effects of non-rigidity and non-homogeneity of material in the fin and body,

46 external placement of markers to skin, use of larger markers, and an increase in the volume of interest and associated increase in calibration error.

VROMM animation

In XMALab, rigid body movement was calculated from the XYZ coordinates of the constellations of markers on the fin and body, and the rigid body transformations were filtered using a low-pass Butterworth filter with a 10 Hz cut-off. Rigid body transformations were applied to polygonal mesh shapes representing the fin and trunk in

Autodesk Maya 2016 (San Rafael CA, USA) (Fig. 3.3A). To measure the motion of the pectoral fin relative to the body, we used the XROMM Maya Tools (xromm.org) to assign a joint coordinate system (JCS) to the articulation point between the fin base and body (FB-JCS) (Camp and Brainerd, 2014; Camp and Brainerd, 2015). FB-JCSs were placed to minimize translation and measure the three degrees of rotational freedom of the inside fin in relation to the body. FB-JCSs were oriented so that the Z-axis was directed cranio-caudally, the Y-axis medio-laterally, and the X-axis dorso-ventrally (Fig. 3.3A).

The FB-JCS rotations were calculated as Euler angles with the rotation order Z,Y,X.

JCSs measure rotation according to the right-hand rule so that positive rotation about the axes represents the following anatomical movements of the inside fin: Z-axis elevation,

Y-axis pronation, and X-axis protraction (Fig. 3.3A).

Data analysis

Pectoral fin movement was described as the range of Euler angle rotation (α; deg) about the three FB-JCS axes when the individual executed yaw turns. In addition to reporting the three angles separately, we also summed them (β; deg) to get an approximate sense of overall turning effort of the fin base. This method provides only an

47 approximate measure because Euler angles depend on rotation order and are subject to magnitude distortions near the poles of Euler space (i.e. gimbal lock). However, given that all the fin motions were fairly small (< 25 deg) and were zeroed near the equator, the sum of the three Euler angles likely provides a useful measure of overall turning effort of the fin base. All variables reported were taken from the frame of video where maximum total rotation was calculated. We compared the magnitude of inside fin rotation about three axes using a one-way ANOVA. Post-hoc Tukey’s tests were used to compare variables if the ANOVA was significant. We report the mean ± the standard error of the mean for all variables.

As a metric of turning performance, we calculated the angle of turning for each trial (n = 9). A turn was defined as a change in heading in which one fin beat occurred.

We measured the distance of the initial heading leading into the turn (Hi), the distance of the final heading exiting the turn (Hf) and calculated the hypotenuse (H) between the initial and final headings. Turning angle (θ; deg) was calculated using the law of cosines where

푯 ퟐ+푯 ퟐ−푯ퟐ 퐜퐨퐬(휷) = 풊 풇 (Eq. 3.1; Porter et al., 2011) ퟐ푯풊푯풇

-1 Angular velocity of the turn (wt; deg·s ) was calculated as the turning angle (θ; deg) divided by the total time of the turn (t; s)

훉 풘 = (Eq. 3.2) 풕 풕

To estimate the hydrodynamics of the pectoral fin, we estimated drag forces by digitizing the 2D fin shape and virtually rotating it. We created a 3D polygon object in

Matlab using the patch function, which was then rotated using the 3D rotation values derived from the kinematics. The area of the fin to flow (A; m2) was calculated using the 48 polyarea function by projecting the polygon object on to the XY plane, which was assumed to be orthogonal to flow. A was calculated for each frame based on the frame by frame rotation of the fin. Drag force (Fd) was then estimated as

ퟏ 푭 = 푪 푨흆푽ퟐ (Eq. 3.3) 풅 ퟐ 풅 where Cd is the drag coefficient of a streamlined shape based on wetted area,

ퟑ 풅 ⁄ 풅ퟑ 푪 = ퟏ + ퟏ. ퟓ ퟐ + ퟕ (Eq. 3.4) 풅 풍 풍 and d is the depth of the fin (m), l is the length (m), p is the density of saltwater at 10o C

(kg·m-3), and V is instantaneous linear velocity of the shark (m·s-1) (Hoerner, 1965).

These estimates treat the fin as a flat plate do not consider conformational changes that are previously shown to affect force balance in shark dorsal fins (Maia and Wilga, 2013;

Maia and Wilga, 2016).

To describe the effect of fin rotation on turning performance, we used simple linear regressions to examine the relations among the turning angular velocity (wt) and pectoral fin rotation in all three axes (α), total pectoral fin rotation (β), the area of the fin to flow (퐴), and the estimated drag (Fd).

Results

Pectoral fin kinematics during routine turns

For all trials (n = 9), the inside fin was protracted, supinated, and depressed while the shark executed yaw turns (Fig. 3.2). We report all variables from the frame of maximum total rotation. Maximum pectoral fin rotation about the X axis ranged from 2o to 13o (Fig 3.2B, 3.3; 6.6 ± 1.57o). Y axis rotation ranged from -0.3o to -7o (Fig. 3.2C,

3.3; -4.5 ± 0.83o); and the greatest rotation was about the Z axis, which ranged from -6o

o o to -20 (Fig. 3.2D, 3.3; -15.6 ± 1.47 ; F2,24=20.6537, P < 0.0001). Total fin rotation (the

49 sum of the absolute value of rotation in all three axes) ranged from 8o to 34o (27.6 ±

1.98o)

Inside fin depression and protraction were both positively related to shark turning angular velocity (Fig. 3.4A; R2=0.5339, P=0.0253; R2=0.81662, P=0.0008; respectively).

Total fin rotation (β), calculated as the sum of rotation in all three axes, was positively related to shark turning angular velocity (Fig. 3.4B; R2=0.8077, P=0.0010). To estimate drag, we used fin rotation to determine the projected area of the fin to flow. The mean area of fin to flow, derived using fin rotation, was 1.5 ± 0.25 cm2 (Fig. 3.4C). We also calculated instantaneous velocity throughout the trial to estimate force (Eq. 3.3). In every trial, we found that velocity (ΔV) decreased during the turn (-10.0 ± 1.30 cm·s-1). We estimated instantaneous drag throughout each trial (Fig. 3.4D; 6.32 ± 0.16 mN). Area of the fin to flow (A) and drag (Fd; Eq. 3.3) were also significantly positively related to shark turning angular velocity (Fig. 3.4C, D; R2=0.4517, P=0.0474; R2=0.6379,

P=0.0098).

Pectoral fin muscle stimulation

Targeted stimulation of the dorsal pterygoideus (DP) muscle resulted in 16.1 ±

2.16o of retraction, 4.1 ± 2.50o of pronation, and 15.9 ± 1.58o of elevation (Fig. 3.5).

Similarly, stimulation of the ventral pterygoideus (VP) resulted in 10.0 ± 2.12o of retraction; however, the fin was also supinated (7.0 ± 1.61o) and depressed (8.2 ± 2.03o;

Fig. 3.5). The only muscle stimulation to result in protraction was the cranial pterygoideus (CP; 21.9 ± 5.98o; Fig. 3.5). Stimulation of the CP also resulted in 11.5 ±

3.00o of supination, and no definitive trend for depression or elevation. The CP originates on the scapula-coracoid and inserts on both the dorsal and ventral portion of the

50 propterygium. Dissections confirmed that leads were placed in the CP for all three individuals, but placement within the CP was variable. In one individual, lead placement was slightly dorsal to the propterygium, and in the two other sharks, leads were slightly ventral to the propterygium. Variable placement in the CP resulted in 14.43o of elevation in the dorsally placed lead, while stimulation in the other two individuals resulted in depression (8.1 ± 3.27o SEM).

Discussion

We found that Pacific spiny dogfish rotate the inside pectoral fin substantially about all three axes in a consistent manner while executing yaw turns (Fig. 3.3). These rotations turned the fin to increase the area to the flow, likely increasing drag. Targeted stimulation of pectoral fin muscles further suggests that pectoral fin rotation is under muscular control (Fig. 3.5). These three results together suggest that sharks actively protract, supinate, and depress the inside pectoral fin to facilitate drag based yaw turning

(Fig. 3.3, 3.4).

Pectoral fin rotation during volitional turning

Shark pectoral fins are dynamic control surfaces that adjust position and conformation during vertical maneuvering (Daniel, 1922; Harris, 1936; Alexander, 1965;

Fish and Shannahan, 2000; Wilga and Lauder, 2000; Maia et al., 2012). Our data further document pectoral fin rotation, now in the context of yaw maneuvering (Fig. 3.3, 3.4).

We found that the inside fin is depressed up to 20o in all trials and both pectoral fin depression and protraction (up to 12o) were significantly related to the turning angular velocity of the shark (Fig. 3.3, 3.4A). Total pectoral fin rotation was also significantly related to turning angular velocity, suggesting that fin rotation may contribute to the

51 turning speed of the shark (Fig. 3.4B). Total pectoral fin rotation doubled between the slowest and fastest turns recorded, with a fivefold increase in the turning angular velocity

(Fig. 3.4B). This study describes volitional turning and the variability in these data likely reflects the range of behaviors seen in a natural environment. The range of data presented here demonstrates that pectoral fins rotate about all three axes and there was a strong relationship between total fin rotation and turning angular velocity (Fig. 3.4).

From the maximally-rotated 3D pose of the fin in each trial we calculated the area of the fin to flow, which was positively related to turning angular velocity (Fig. 3.4C).

We found a significant relation between the turning angular velocity, total fin rotation, and estimated drag, consistent with the hypotheses that fin rotation generates drag to aid in yaw maneuvering (Fig. 3.4D; Kajiura et al., 2003; Domenici et al., 2004). We also noted a decrease in velocity during turning in each trial, which in combination with high drag estimates, suggests that pectoral fin rotation contributed to drag based turning (Kato et al., 2002; Fish and Nicastro, 2003; Fish and Lauder, 2017). The pectoral fin drag we estimate in this study is similar to that previously measured for bamboo sharks during vertical maneuvering and slightly less than drag measured on robotic flapping fins (Wilga and Lauder 2001; Tangorra et al., 2010). We propose that increasing the area of the fin to flow generates drag, causing the fin to act as a pivot about which the body rotates (Fig.

3.4C). In this study, we describe pectoral fin rotation in relation to turning performance; but axial bending, caudal fin movement, and dorsal fin stiffening may all play substantial role in yaw maneuvering (Kajiura et al., 2003; Domenici et al., 2004; Porter et al., 2009;

Maia and Wilga, 2013). Further studies considering whole body kinematics would greatly increase our understanding of the forces acting on the body during yaw maneuvering.

52 Pectoral fin movement and function has been previously described in many ways but lacks standardized terminology. For example, epaulette sharks are described as

“swinging” their pectoral fins during walking, bonnethead sharks “tuck” their fins during turning, and pelagic thresher sharks are noted to “laterally rotate” their fins during braking (Pridmore, 1994; Kajiura et al., 2003; Oliver et al., 2013). Additionally, the use of “adduction” and “abduction” is interchanged throughout the literature, and it is less applicable to the position and movement of shark pectoral fins compared to ray finned fishes (Table 3.1; Marinelli and Strenger, 1959; Wilga and Lauder, 2001; Oliver et al.,

2013). Our findings demonstrate that shark pectoral fins rotate about three axes, and we propose the use of protraction/retraction, pronation/supination, and depression/elevation to standardize the description of movements, similar to terminology used to describe wing rotation in bird flight (Fig. 3.3; Dial et al., 1988; Tobalske et al., 2003; Tobalske,

2010). These motions are all alluded to in previous studies with varying terminology. For example, epaulette sharks use sequential fin protraction/retraction in a walking gate

(Pridmore, 1994; Goto et al., 1999). Shark fin depression and elevation are described previously as the change in their negative dihedral angle of to the body and may also be referred to as adduction/abduction (Table 3.1; Alexander, 195; Ferry and Lauder 1996;

Fish and Shannahan, 2000; Wilga and Lauder 2000, 2001). Differences in fin elevation/depression and pronation/supination is the least clear since many previous studies are describing changes in fin angle of attack, which may be a result of either of these motions or some combination. Here, we propose the use of pronation/supination to describe the long axis rotation of the fin where pronation results in the dorsal movement of the trailing edge relative to the leading edge and supination vice-versa. In this instance,

53 ninety degrees of rotation in either direction would result in the trailing edge being directly in line with the leading edge.

In this study, we examined one species assuming that the base of the pectoral fin acted roughly as a rigid body. Shark pectoral fin morphology is variable and fins undergo behavior mediated conformational changes during swimming and maneuvering that would substantially affect force generation (Moss, 1972; Wilga and Lauder, 2000; Wilga and Lauder, 2001; Maia et al., 2012). We suggest caution should be taken when generalizing functional roles among all species and fin morphologies. Further endeavors to validate this role would benefit from integrating volitional swimming with particle image velocimetry, and computational fluid dynamics in a comparative context.

Pectoral fin musculature

We showed that pectoral fins rotate during yaw maneuvering, and we used post- mortem muscle stimulation experiments to show that fins are under muscular control. We found that contraction of the ventral pterygoideus (VP) resulted in 8o of pectoral fin depression and contraction of the dorsal pterygoideus (DP) resulted in 16o of elevation

(Fig. 3.5F). These experimental results support functional hypotheses derived from anatomical descriptions of pectoral fin muscles (Marinelli and Strenger, 1959; Liem and

Summers, 1999; Goto et al., 1999; Wilga and Lauder, 2001). Volitional swimming trials showed that the inside fin is depressed up to 27o, and post-mortem stimulation resulted in

8o of depression in the VP and also in the cranial pterygoideus (CP), when the lead was placed in the ventral propterygium. We hypothesize that simultaneous activation of the

VP and CP resulted in greater pectoral fin depression during volitional swimming.

54 In addition to fin depression, we found that stimulation of the CP was solely responsible for 22o of pectoral fin protraction (Fig. 3.5C). The CP fans out over the anterior portion of the propterygium, located on both the dorsal and ventral margins of the fin with muscle fibers oriented orthogonally to the body axis (Marinelli and Strenger,

1959; Liem and Summers, 1999; Goto et al., 1999; Wilga and Lauder, 2001). In volitional turning, the maximum amount of pectoral fin protraction we observed was 18o, and we hypothesize that the CP is the only muscle responsible for controlling pectoral fin protraction. Alternatively, stimulation of the DP and VP resulted in 16o and 10o of pectoral fin retraction; respectively (Fig. 3.5C). The muscle fibers of the DP and VP are at an oblique angle to the body fanning out posterolaterally, resulting in pectoral fin retraction, along with rotation in other planes (Marinelli and Strenger, 1959; Liem and

Summers 1999; Wilga and Lauder 2001; Fig. 3.5). Stimulation of all three pectoral fin muscles resulted in long axis rotation: stimulation of the CP and VP resulted in 7o and 9o supination; respectively; and stimulation of DP resulted in 4o of pronation (Fig. 3.5D).

Previous studies note changes in pectoral fin angle of attack, resulting from depression/elevation, pronation/supination, or some combination thereof, but the resulting fin rotation in these experiments is unclear (Fish and Shannahan, 2000; Wilga and Lauder, 2000, 2001).

We targeted intrinsic pectoral fin muscles contained within the fin, but there is also extrinsic axial musculature associated with the fin that may contribute to rotation

(Marinelli and Strenger, 1959; Liem and Summers, 1999; Wilga and Lauder, 2001). The cucullaris originates on the scapular process of the pectoral girdle and runs longitudinally to insert on anterior epaxial muscles, and it is hypothesized to protract the scapula

55 (Marinelli and Strenger, 1959; Liem and Summers, 1999; Wilga and Lauder, 2001).

Additionally, a portion of the hypaxialis inserts on the posterior margin of the scapular process and may play a role in retracting the pectoral girdle (Marinelli and Strenger,

1959; Liem and Summers, 1999; Wilga and Lauder, 2001). Recent work demonstrated that the pectoral girdle of white-spotted bamboo sharks is mobile during suction feeding, and it is hypothesized that this movement may also occur during locomotion (Camp et al.,

2017). Future studies on fin actuation should consider the role of the pectoral girdle and associated musculature.

3D volitional kinematics

Swimming studies are often conducted in flumes to minimize non-steady locomotion and calibration error, but the unidirectional flow and working volume constraints limit the study of maneuvering and larger-bodied animals. The 3D kinematics of maneuvering behaviors are especially understudied, likely due to problems with calibrating large volumes. Volitional swimming and maneuvering has been studied in a number of large aquatic vertebrates, but is limited by the use of 2D video, which may oversimplify or disregard movements not visible in the filming plane (Lowe, 1996; Blake et al., 1994; Fish, 1997; Fish and Shannahan, 2000; Rohr and Fish, 2002; Kajiura et al.,

2003; Domenici et al., 2004; Porter et al., 2009; Porter et al., 2011; Seamone et al., 2014;

Hoffmann et al., 2017). Recent studies have successfully demonstrated the use of multi- camera systems calibrated for 3D analysis in large volume environments (Ros et al.,

2011; Sellers and Hirasaki, 2014; Jackson et al., 2016; Fish et al., 2018). Many advances in these techniques were developed using consumer-grade cameras and free, open source software, making the ability to study 3D kinematics increasingly accessible (Hedrick,

56 2008; Brainerd et al., 2010; Jackson et al., 2016; Knorlein et al., 2016). With our growing understanding of the importance of control surfaces in swimming, data on the 3D kinematics of fins and flippers will greatly benefit fields such as movement ecology and ecomorphology.

In this study, we adapted Video Reconstruction of Moving Morphology

(VROMM) in two major ways: for use with low-cost, underwater light cameras and fully submerged in a large volume environment (Fig. 3.1; Brainerd et al., 2010; Knorlein et al.,

2016; Jimenez et al., 2018). Marker tracking error, which was on average 0.684 mm (less than 0.02% of the animal’s TL), demonstrates that this method is capturing these larger scale underwater movements with good precision for the size of the arena and size of the animals (Table 3.2). Further development of this technique has the potential to eliminate size and behavioral constraints that have previously limited the study of volitional movements and prevented 3D motion analysis.

Conclusion

The control surfaces of sharks have been largely thought to play a major role in balancing forces on the body during swimming to maintain vertical position in the water column. Here, we demonstrate that pectoral fins move substantially in ways that facilitate maneuvering. Though to a lesser degree, this is similar to highly mobile pectoral fins of ray finned fishes that are well documented to play a major role in maneuvering. The pectoral fins of sharks and basal actinopterygians also undergo conformational changes during pitch adjustment, further exemplifying the complex movement that fins are capable of in these groups. As with ray finned fishes, physiological and ecological demands have led to a vast diversity of body and fin shape among sharks. Future

57 endeavors should examine the relationship between fin shape and function to better understand the evolution of sharks.

58 Chapter 3 Figures and Tables

Figure 3.1: Video reconstruction of moving morphology (VROMM) experimental design.

(A) Pacific spiny dogfish were outfitted with white bead markers along the anterior body and leading edge of the pectoral fin. (B) Two fully submerged Go-Pro cameras were angled approximately 45o to one another and focused on the same 1m3 volume outlined with bricks. (C) Cameras were time synchronized with a flashing light and calibrated for

3D analyses with a 7 x 9 checkerboard calibration object.

59

Figure 3.2: Pectoral fin rotation relative to the body axes. (A) The 3D shapes represent the body and inside fin and the white dots represent tracked points. A joint coordinate system (FB-JCS) was placed at the proximal fin base to measure relative fin rotation about the dorso-ventral (X; red), medio-lateral (Y; green), and cranio-caudal (Z; blue) axes. (B-D) Sample rotation trace of one turn highlighted in the gray box. The pectoral fin was (B) protracted, (C) supinated, and (D) depressed during the turn. This pectoral fin

60 rotation pattern was observed in all nine trials. Protraction rotates the fin cranially, supination causes the trailing edge of the fin to translate ventrally, increasing the angle of attack, and depression makes the negative dihedral angle of the fin more negative.

61

Figure 3.3: We found significantly greater depression of pectoral fins during turning

(F2,24=20.6537, P < 0.0001). For all nine trials, the inside pectoral fin was protracted, supinated, and depressed. The absolute values of rotation were used in an ANOVA to compare the range of rotation in each axis and lower-case letters denote significant differences. The box represents the mean (middle line) ± standard error of the mean.

Whiskers represent the upper and lower extremes.

62

Figure 3.4: Relation among pectoral fin movement and turning kinematics. (A) Both depression and protraction were positively related to turning angular velocity while supination was not (P=0.253, P=0.0008). (B) When considering the sum of rotations about all three axes, there was a significant positive relation with turning angular velocity

(P=0.0010). (C) The area of fin to flow was significantly positively related to turning angular velocity (P=0.0474). (D) Drag was also significantly positively correlated with turning angular velocity (P=0.0098).

63 Figure 3.5: Skeletal and muscular morphology of the pectoral fin of the Pacific spiny dogfish. Three basal cartilages (propterygium, mesopterygium, and metapterygium) articulate with the scapulo-coracoid. Radial elements fan out distally and support the fin web. (A) On the dorsal side of the fin, the dorsal pterygoideus (DP) originates on the scapulo-coracoid and inserts distally on the intermediate radials. The cranial pterygoideus

(CP) originates on the dorsal margin of the scapula-coracoid and inserts on both the dorsal and (B) ventral portions of the propterygium. The ventral pterygoideus (VP) originates on the ventral margin of the scapula-coracoid and inserts ventrally on the intermediate radials. Black dots mark the target of lead placement for each muscle (C-E)

Post mortem muscle stimulation resulted in fin rotation about the three body axes described in Fig. 2. Only one lead was placed per muscle, but placement in the CP was along the anterior margin of the leading edge and can be seen in the dorsal and ventral view. (C) The cranial pterygoideus was the only muscle to result in protraction when 64 stimulated. (D) Stimulation of the ventral and cranial pterygoideus resulted in supination whereas stimulation of the dorsal pterygoideus pronated the fin. (E) The dorsal pterygoideus elevated the fin when stimulated and the ventral pterygoideus depressed the fin, supporting the hypothesis that they are antagonistic muscles.

65 Table 3.1: Pectoral fin muscle terminology as previously described in the literature.

Cranial Ventral Dorsal Species pterygoideus pterygoideus pterygoideus Citation S. Cranial Ventral Dorsal Marinelli and acanthias pterygoideus pterygoideus pterygoideus Strenger, 1959 S. acanthias - Flexor of fin Extensor of fin Gilbert, 1973 S. Depressor, Levator, Liem and Summers, acanthias - adductor abductor 1999 H. Depressor Levator ocellatum - pectoralis pectoralis Goto et al., 1999 C. Depressor, Levator, Wilga and Lauder, plagiosum Protractor abductor adductor 2001 M. Depressor Depressor Levator pelagios pectoralis pectoralis pectoralis Tomita et al., 2014

66 Table 3.2: Precision of point tracking and mean rigid body error for three volitional trials per individual. Precision is measured by the standard deviation of the distances between markers within a rigid body and was on average 0.684 mm. Rigid body error, calculated as the error between the optimized marker constellation from all frames and the reconstructed location of the markers, averaged 0.24 mm over all trials and rigid bodies

(Knorlein et al., 2016).

Mean SD inter-marker distance (mm) Mean rigid body error ± SD (mm) Pectoral Fin Body Pectoral Fin Body Individual 1 0.750 0.863 0.217 ± 0.113 0.265 ± 0.125 Individual 2 0.549 0.667 0.282 ± 0.158 0.287 ± 0.175 Individual 3 0.473 0.800 0.182 ± 0.076 0.233 ± 0.111

67 CHAPTER 4: BODY AND PECTORAL FIN KINEMATICS DURING YAW

TURNING IN BONNETHEAD SHARKS (SPHYRNA TIBURO)

Abstract

Maneuvering is a crucial locomotor strategy among aquatic vertebrates, common in routine swimming, feeding, and escape response. Combinations of body bending and fin movements generate an imbalance of forces resulting in deviation from an initial path.

Body form largely controls an animal’s inherent stability (or instability) and has substantial consequences for stability and maneuverability. Fusiform body shapes are considered best for accelerating or cruising, whereas shorter, rigid bodies with mobile planing surfaces (fins) lend to maneuverability. Sharks have elongate bodies that bend substantially in combination with pectoral fin rotation during yaw (horizontal) turning.

The most derived hammerhead species, the bonnethead shark (Sphyrna tiburo) has a laterally expanded head that may act as additional planing surface. In fact, hammerhead species are more agile than their fusiform counterparts, and it is hypothesized that they may use planing surfaces to cause an imbalance of forces for greater maneuverability. In this study, we use VROMM to describe the three-dimensional (3D) pectoral fin and body kinematics during routine yaw turning in bonnethead sharks. Body bending and pectoral fin rotation correlate positively with turning performance metrics, and we propose that bonnetheads use body bending and fin rotation to reorient lift and maintain speed throughout a turn. We observed a second turning strategy in which the fin makes substrate contact, serving as pivot about which the body turns. This strategy is similar to

68 that previously documented for the Pacific spiny dogfish. We also describe the anatomy of bonnethead pectoral fins and use muscle stimulation to confirm functional hypotheses about their role in actuating the fin. We propose that differences in maneuvering strategies and pectoral fin muscle function reflect the ecological and physiological differences among species.

Introduction

The ability to maneuver is essential for locating prey, predator avoidance, and routine navigation. Aquatic vertebrates use body bending and/or fin movements during yaw (horizontal) turning as their primary means of maneuvering (Webb 1997; Fish 1997;

Kajiura et al., 2003; Domenici et al., 2004; Gotlieb et al., 2010; Porter et. al., 2009, 2011;

Maia and Wilga, 2013; Webb and Weihs, 2015; Hoffmann et al., 2019). Cetaceans rely largely on posteriorly oscillated flukes for thrust production, and anterior flippers to produce stabilizing or maneuvering forces (Fish, 2002; Fish et al., 2003; Weber et al.,

2009; Fish et al., 2014). In this instance, yawing is achieved mainly through longitudinal rolling of the body axis that leads to banking (Fish, 2002; Fish et al., 2006; Goldbogen et al., 2013; Segre et al., 2016). Banking is an energetically efficient turning mechanism that allows for the maintenance of speed during a turn, and are carried out by the reorientation of lift by control surfaces, in this case the flippers (Fish, 2002, Fish et al., 2003; Fish and

Lauder, 2017). Alternatively, many fishes and semi-aquatic mammals use asynchronous appendage movements to produce an imbalance of forces resulting in turning about the center of mass (Fish and Nicastro, 2003; Drucker and Lauder, 2001,2004; Hoffmann et al., 2019).

69 Body shape is well documented to affect swimming and maneuvering performance among fishes (Webb, 1984; Wardle et al., 1995; Sfakiotakis et al., 1999;

Blake, 2004; Webb and Weihs, 2015). Some sharks are often categorized as cruising specialists, yet a number of species demonstrate exceptional turning performance (Webb and Keyes, 1982; Webb, 1984; Kajiura and Holland, 2002; Kajiura et al., 2003; Domenici et al., 2004; Porter et al., 2009). Sharks have moderately flexible, elongate bodies that bend substantially during maneuvering, which is related in part to vertebral and cross- sectional trunk morphology (Kajiura and Holland, 2002; Kajiura et al., 2003; Domenici et al., 2004; Porter et al., 2009). Interestingly, hammerhead species have increased body flexibility and maneuverability during turning in comparison to fusiform species (Kajiura and Holland 2002; Kajiura et al., 2003; Porter et al., 2009). There are conflicting hypotheses about the potential advantage of the laterally expanded hammerhead cephalofoil in maneuvering: the wing like head shape may generate turning forces during banking thereby increasing maneuverability, but two hammerhead species are not observed to bank during prey searching, while a third species may bank during routine swimming (Thomson and Simanek,1977; Nakaya, 1995; Kajiura and Holland, 2002;

Kajiura et al., 2003; Payne et al., 2016). Instead, it appears that during turning, the pectoral fin located on the inside of the body curvature (hereafter referred to as the inside fin) may be moved to create a pivot about which the body bends, creating a smaller turning radius (Kajiura et al., 2003).

At least two shark species move their pectoral fins asynchronously during yaw maneuvering, and they are hypothesized to play a role in turning (Kajiura et al., 2003;

Domenici et al., 2004; Hoffmann et al., 2019). During vertical maneuvering, pectoral fins

70 generate thrust to reorient the body, and asynchronous pectoral fin movement may create an imbalance of forces that increase maneuverability, similar to maneuvering mechanisms in actinopterygian fishes (Drucker and Lauder, 2001, 2004; Wilga and

Lauder, 2000, 2001). Pacific spiny dogfish (Squalus suckleyi) protract, supinate, and depress the inside fin during yaw turning (Hoffmann et al., 2019). Turning angular velocity and estimates of drag produced by the fin increase with increasing fin rotation, and it is hypothesized that the fin generates drag about which the body rotates (Hoffmann et al., 2019).

Fins vary greatly among shark species, and previous studies document functional differences among species with varied fin morphologies (Wilga and Lauder, 2000, 2001;

Fish and Shannahan, 2000; Maia and Wilga 2013; Flammang 2010). Despite differences in swimming mode, body flexibility, and physiology, both Pacific spiny dogfish and bonnethead sharks use pectoral fin rotation during yaw turning (Kajiura et al, 2003;

Dominici et al., 2004; Hoffmann et al., 2019). The Pacific spiny dogfish is a fusiform, bottom dwelling shark that buccal pumps to respire and may spend time resting on the bottom (Compagno, 1984). In contrast, the bonnethead shark has an additional planing surface at the head, is a continuously swimming obligate ram ventilator, and is more agile than non-hammerhead species (Compagno, 1984; Kajiura et al., 2003). The potential drawbacks of two species comparisons notwithstanding, these species afford an opportunity to use 3D kinematics to compare turning mechanism and performance among similarly sized individuals with varied morphology and physiology (Garland and Adolph,

1994).

71 The goals of this study were to describe the 3D kinematics and turning performance of bonnethead sharks. We quantified the rotation of the inside pectoral fin as well as body bending kinematics and assessed their impact on turning performance. We also describe the anatomy of the bonnethead pectoral fin and used post mortem muscle stimulation to confirm hypotheses about the function of the pectoral fin musculature.

Finally, we compared these kinematic variables to a previous study on Pacific spiny dogfish to assess maneuvering performance and strategy. We hypothesized that the bonnethead shark would protract, supinate, and depress the fin inside to body curvature, and that increasing fin rotation would correlate with turning angular velocity as previously described for the Pacific spiny dogfish (Hoffmann et al., 2019). However, we predicted that the bonnethead turns at greater speeds than the Pacific spiny dogfish since they are an active, obligate ram ventilating species rather than a buccal pumper like the

Pacific spiny dogfish.

Methods

Bonnethead sharks (Sphyrna tiburo, n=4) were captured via gill net in Long Key,

FL and transported to the Florida Atlantic University Marine Research Laboratory in

Boca Raton, FL where they were cared for under FAU IACUC protocol A15-43.

Animals were housed in a 6 m diameter tank with 1.5 m water depth and flow-through seawater, and individuals were acclimated for minimum seven days prior to filming trials.

Marker placement

Individuals were anesthetized via submersion in a 0.133 g · L-1 MS-222 solution buffered with NaOH. Once ventilatory gill movement slowed to indicate anesthesia was in effect, individuals were placed on a surgical platform and intubated with fresh flow-

72 through seawater. Black, hemispherical beads were affixed to the trunk and pectoral fins with VetBond (3M Company, St. Paul, MN). At least five beads were positioned along the rigid anterior trunk and the proximal fin base near the leading edge (Fig. 4.1A). Bead placement lasted less than three minutes, and then the individual was returned to the semicircular 6 m diameter filming arena for a recovery period of approximately one hour until normal ventilation and swimming behavior resumed.

Volitional swimming trials

Methods described here were modified from Hoffmann et al., 2019. Three GoPro

Hero 5 Black cameras were mounted to cement blocks and positioned along the lateral edge of the filming arena (GoPro, Inc., San Mateo, CA; Fig. 4.1B). Cameras were time synchronized using a flash of light and filmed at 1080 p, 60 fps, and we used a linear field of view, which removes the effect of fish-eye barrel distortion. A 31.5 cm x 40.5 cm checkerboard (7 squares x 9 squares) was used to calibrate the cameras for 3D analyses

(Fig. 4.1C). Individuals were enticed to maneuver through the calibrated space by placing an object in their path (Domenici et al., 2004). Three trials per individual were chosen for three individuals (n = 9), where a clear yaw turn with minimal pitch adjustment occurred, and minimum 5 markers along each the inside pectoral fin and anterior body were clearly visible in two of the cameras. For the fourth individual, only two trials met these criteria, due to variable conditions within this flow through seawater system. In this study, we analyzed movement from only the inside fin during a turn because the body occluded the outside fin in the video reconstructions.

73 Muscle stimulation trials

Upon completion of volitional swimming trials, individuals were euthanized via submersion in a 2 g · L-1 MS-222 solution buffered with NaOH. Post mortem, individuals were fully submerged and suspended in a 190 L tank. Bipolar leads made from 57 μm diameter insulated alloy wire were placed in three pectoral fin muscles hypothesized to control maneuvering (dorsal pterygoideus [DP], ventral pterygoideus

[VP], cranial pterygoideus [CP], Fig 8). A 10V, 30Hz square wave pulse was applied to the targeted muscles one at a time via BK Precision 4052 signal generator (BK Precision

Corporation, Yorba Linda, CA) to stimulate contraction. Stimulation lasted no longer than 2 s per muscle and we ensured that the fin returned to a resting position between trials. Muscle stimulation experiments were recorded with two GoPro Hero 5 Black cameras positioned approximately 45o to one another and focused on the tank with overlapping fields of view. Cameras were time synchronized with a flashing light and calibrated for 3D analysis using a checkerboard calibration object (Knorlein et al., 2016;

Hoffmann et al., 2019).

Following muscle stimulation trials, the fin and pectoral girdle were dissected to confirm lead placement and describe the muscle arrangement and articulations between the fin and girdle. Fin skeletal and muscle morphology differs among species and there are limited data on pectoral fin anatomy specific to the bonnethead (Wilga and Lauder,

2000, 2001; Maia et al., 2012; Da Silva and De Carvalho, 2015)

3D marker tracking

For both volitional swimming and muscle stimulation trials, markers along the fin and body were tracked in 3D using XMALab v. 1.5.1 (Knorlein et al., 2016). Rigid

74 bodies were created from markers on the leading edge of the pectoral fin and anterior trunk of the body to quantify the fin rotation relative to the body. Movement of the fin and trunk as rigid bodies were calculated in XMALab using five or more markers distributed in a constellation pattern to describe their relative motion. Rigid body transformations were applied to polygons that served as estimations of the fin and body in

Autodesk Maya 2017 (San Rafael CA, USA). A joint coordinate system (JCS) was assigned to the proximal insertion at the base of the pectoral fin at the body axis (Fig.

4.2A; Camp and Brainerd, 2015; Hoffmann et al., 2019). The Euler angle rotation (α; deg) was calculated for each of the three axes of rotation. Because individual Euler angle rotations were small (< 25o) and were zeroed at the equator, we report total fin rotation as the scalar sum of rotation in all three axes (β; deg; Hoffmann et al., 2019).

2D whole body kinematics

Whole body kinematics were quantified using the X, Z coordinates representing the dorsal plane. Instantaneous linear velocity (cm·s-1) was calculated as the change in distance of a point at the first dorsal fin insertion over time, which was standardized by fork length (FL; cm) to derive swimming speed (U; body lengths·s-1). The change in velocity (maximum velocity – minimum velocity) over a turn was also compared among trials (Δ velocity; cm·s-1). Turning angle (θ; deg) was calculated as the angle between the initial (Hi) and final (Hf) heading, where H is the hypotenuse between the two:

푯 ퟐ+푯 ퟐ−푯ퟐ 퐜퐨퐬(휷) = 풊 풇 (Eq. 4. 1) ퟐ푯풊푯풇

Turning angular velocity (wt; deg·s-1) was calculated as the change in angle over time:

훉 풘 = (Eq. 4.2) 풕 풕

Body curvature is represented by the bending coefficient (BC) calculated as: 75 푳 푩푪 = ퟏ − (Eq. 4.3) 푻푭 where L is the minimum distance between the head and the caudal peduncle during the turn (cm) and FL is the fork length of the individual (cm) (Brainerd and Patek, 1998;

Azizi and Landberg 2002; Kajiura et al., 2003; Porter et al., 2009). Finally, the maximum bending angle of the body (MBA; deg) was calculated as the change in angle formed at the first dorsal fin between two segments: the eye to the first dorsal fin insertion and the first dorsal fin insertion to the caudal peduncle (Porter et al., 2009). Previous studies show that MBA overestimates body curvature in the anterior body and BC is a better whole body index, yet both are useful to compare whole body kinematics among species described in other studies (Kajiura et al., 2003; Porter et al., 2009).

Data analysis

We report the fin rotation angle about each axis (α) from the frame of maximum total rotation (β) as a range and the mean ± standard error of the mean. The magnitude of rotation in each axis was compared using a one-way ANOVA. The effect of fin rotation on turning performance was examined using simple linear regressions between body curvature measurements, and fin rotation, turning angle (θ), and turning angular velocity

(wt). To determine the effect of whole body kinematics on turning angular velocity, we applied a generalized linear model with total fin rotation (β), change in velocity (ΔV), bending coefficient (BC), and maximum bending angle (MBA) as predictor variables.

Models were evaluated for best fit by considering P values (P< 0.05), lowest AICc value, and greatest adjusted coefficient of determination (R2).

Fin rotation and turning performance variables for the bonnethead were compared to the same variables measured for Pacific spiny dogfish in a previous study (Hoffmann

76 et al., 2019). Fin rotation about each axis, total fin rotation, change in velocity, turning angle, and turning angular velocity were compared between species in a One-way

ANOVA.

Results

Pectoral fin kinematics

In all turning trials analyzed, the pectoral fin rotated about all three body axes

(Fig. 4.2). For ten of the eleven trials, the inside fin was protracted, pronated, and depressed (Fig. 4.2E). In one trial, the individual first retracted, supinated, and elevated the fin, quickly followed by fin pronation and depression to contact the substrate during turning (Fig. 4.2F). We removed this trial from further analyses as it represents an alternative maneuvering strategy.

We observed positive X axis rotation, representing fin protraction, ranging from

3o to 16o (Fig. 4.2B, 4.3; 8.5o ± 1.1o). Y axis rotation was also positive, indicating fin supination, ranging from 3o to 12o (Fig. 4.2C, 4.3; 5.0o ± 0.9o). Rotation about the Z axis was negative, representing fin depression, ranging from -18o to -2o (Fig. 4.2D, 4.3; 10.4o

± 1.9o). The magnitude of rotation did not differ between axes (Fig. 4.3). Total fin rotation ranged from 15o to 35o (24o ± 2.3o).

Point tracking precision was calculated as the standard deviation of the intermarker distance within a rigid body (Knorlein et al., 2016). In this study, we assume that the fin base and body are rigid, despite lacking true rigid elements (i.e. bones).

Marker based XROMM studies report mean SD of intermarker distance less than 0.1 mm, and a previous VROMM study on Pacific spiny dogfish reports the mean SD of intermarker distance less than 0.7 mm, which is less than 0.02% of the animal’s total

77 length (Hoffmann et al., 2019). In this study, there was no difference in the SD of intermarker distance between rigid bodies (fin base vs. body) or among individuals. Mean point tracking precision for all trials and rigid bodies was 2.19 mm. We hypothesize that the increase in precision error observed here compared to the previous shark VROMM study is the result of a 4x increase in the volume of interest and larger study organisms

(Hoffmann et al., 2019). Even so, precision error in this study is still less than 0.3 % of the bonnethead shark’s total body length.

Whole body kinematics

Whole body bending was analyzed by measuring the maximum bending angle

(MBA; deg) and the bending coefficient (BC; Fig. 4.4). MBA ranged from 41.1o to 88.9o

(60.9o ± 4.4o). BC ranged from 0.29 to 0.53 (0.38 ± 0.02). MBA and BC were significantly positively related (R2 = 0.7923, P = 0.0004).

Bonnethead shark turning angle (θ; deg), turning angular velocity (wt; deg·s), and overall change in velocity (Δ velocity; cm·s-1) were examined as turning performance metrics. Turning angle ranged from 7.3o to 61.0o (37.8o ± 5.7o). Turning angular velocity

(deg·s) ranged from 33.5o to 177.6o (113.9o ± 15.4o). Instantaneous linear velocity (Δ velocity) increased during every trial and the change in velocity range from 18.7 cm·s-1 to

40.8 cm·s-1 (27.0 cm·s-1 ± 2.6 cm·s-1).

Turning performance

To examine the relationship between fin rotation and turning performance, we regressed rotation variables with turning performance (Fig. 4.5). When separated by axes of rotation, only fin depression was significantly related to the angular velocity of the turn (Fig. 4.5A; R2 = 0.4295, P = 0.0397). Total fin rotation was positively related to

78 turning angular velocity (Fig. 4.5B; R2 = 0.6576, P = 0.0044). Total fin rotation was also positively related to Δ velocity (Fig. 4.5C; R2 = 0.6701, P = 0.0038).

The effect of body flexion on turning performance was analyzed using simple linear regressions between whole body bending and turning performance. Neither whole body variables were related to the turning angle. Bending coefficient (BC) was significantly positively related to turning angular velocity (Fig. 4.6A; R2 = 0.4418, P =

0.0360). Maximum bending angle (MBA) was positively related to turning angular velocity (Fig. 4.6B; R2 = 0.4976, P = 0.0227). MBA was also positively related to the change in velocity (Fig. 4.6C; R2 = 0.5578, P = 0.0131).

To determine the effect of pectoral fin and whole body bending kinematics on turning performance, we modeled turning angle and turning angular velocity with total rotation, BC, MBA, and Δ velocity as factors. No models significantly predicted turning angle, but the top five best models for turning angular velocity were all significant (Table

4.1). All top five best fit models for turning angular velocity contain total rotation as a significant factor. All top five best-fit models also consider one of the whole body bending variables. The best fit model predicts 67% of the variation in the data with total fin rotation and bending coefficient as variables.

Muscle stimulation

Stimulation of each targeted muscle resulted in fin rotation about all three body axes. Negative X axis rotation (Fig. 4.2B: fin retraction) occurred with stimulation of the

DP (Fig. 4.7D; -8.2o ± 2.1o) and the VP (Fig. 4.7D; -3.9o ± 2.0o). The CP was the only muscle to protract the fin (X axis; Fig. 4.7D; 15.9o ± 3.4o). The DP and CP pronated the fin (Y axis; Fig. 4.7E; 20.2o ± 5.7o, 9.4o ± 2.7o, respectively), while the VP was the only

79 fin supinator (Fig. 4.2C: Y axis; -5.9o ± 1.5o). Finally, the DP was the only muscle to produce fin elevation (Fig. 4.2D: Z axis; Fig. 4.7F; 22.9o ± 1.7o). Both the VP and CP acted as fin depressors (Fig. 4.7F; -9.3o ± 2.5o, -12.9o ± 4.6o).

Comparison to Pacific spiny dogfish

Fin rotation and turning performance values for the bonnethead were compared to previous data on the Pacific spiny dogfish (Table 4.2; Hoffmann et al., 2019). The polarity of Y axis rotation differed between the bonnethead (pronation) and Pacific spiny dogfish (supination), but the absolute value of rotation did not differ. The only significant difference between the two species was in the Δ velocity (Table 4.2). The absolute value of Δ velocity was three times greater in the bonnethead shark than the Pacific spiny dogfish. Further, the bonnethead accelerated through all turns while the Pacific spiny dogfish decelerated during turning.

Discussion

Bonnethead sharks use combinations of pectoral fin rotation and body bending to execute two types of yaw turns (Fig. 4.2, 4.4; Table 4.1). Sharks either protract, pronate, and depress the inside fin to accelerate through yaw turns while maintaining their position in the water column (Fig. 4.2E); or, they first retracted, supinated, and elevated the fin, followed by fin pronation and depression to make contact with the substrate, creating a pivot about which the body turns (Fig. 4.2F). Post-mortem stimulation shows that the three muscles directly associated with the pectoral fin produce actuation about all three-body axes, as observed in these volitional turning trials (Fig. 4.7).

80 Pectoral fin kinematics during volitional turning

Previous studies demonstrate that shark pectoral fins rotate about the body axes and undergo conformational changes during maneuvering (Wilga and Lauder 2000; Fish and Shannahan 2000; Hoffmann et al., 2019). In this study, we describe pectoral rotation relative to the body axes with the caveat that the fin may also be undergoing conformational changes, which are not captured in these data (Wilga and Lauder, 2000,

2001). For ten of eleven trials, we observed that the inside pectoral fin was protracted, pronated, and depressed during yaw turning in the water column (Fig. 4.2B-D, Fig. 4.3).

Fin depression changes the negative dihedral angle of the fin to the body, which is previously shown to decrease stability and initiate roll (Wilga and Lauder, 1999, 2000).

Though we were unable to capture the motions of the fin outside to body curvature in this study, previous studies note significant differences in the fin area presented from a dorsal view during yaw turning in bonnetheads (Kajiura et al., 2003). Assuming that is the case in this study, we hypothesize that pectoral fin instead pronates, reorienting lift to initiate banking, comparable to the flipper orientation and turning mechanism in cetaceans (Fish,

2002; Fish et al., 2003; Goldbogen et al., 2013; Segre et al., 2016; Fish and Lauder,

2017).

In addition to increasing maneuverability by changing in the dihedral angle through fin depression, pronation changes the fin angle of attack. Assuming that pectoral fin angle of attack, as previously described in the literature, is largely a factor of long axis rotation (y axis), we report a similar range of fin pronation in some species (leopard, sandbar, sand tiger, spiny dogfish, and white sturgeon; Fig. 4.3; Wilga and Lauder, 1999;

Wilga and Lauder, 2000; Fish and Shannahan, 2000). During vertical maneuvering,

81 sturgeon and leopard shark pectoral fins are rotated synchronously generating thrust to reorient the anterior body to rise (Wilga and Lauder 2000). Asynchronous fin depression and pronation would destabilize the body, initiate roll along the longitudinal body axis, and result in a banking turn (Fish, 2002; Fish et al., 2006; Goldbogen et al., 2013; Segre et al., 2016).

Banking in hammerhead species is further complicated by the laterally expanded cephalofoil, which may generate additional turning forces as it is rolled (Nakaya, 1995).

A previous study on bonnethead yaw turning hypothesized that the cephalofoil increases stability by minimizing banking (whole body rolling) during prey seeking maneuvers near the benthos (Kajiura et al., 2003). Minimized banking while turning may prevent the cephalofoil from hitting the substrate, and increases electroreceptive capability by maintaining a small uniform distance between the cephalofoil and substrate; two advantages that are less applicable to routine turning higher up in the water column

(Kajiura et al., 2003). One trial in the present study is a turn in which the individual made pectoral fin contact with the substrate (Fig. 4.2F). In this turn, the inside fin was retracted

(Fig 4.2B; X axis), supinated (Fig. 4.2C; Y axis), and elevated (Fig. 4.2D; Z axis), and this was the only trial where velocity decreased during turning. We hypothesize that this turning mechanism is comparable to that previously described for prey seeking behavior in the bonnethead (Kajiura et al., 2003).

In contrast, the great hammerhead (Sphyrna mokarran) rolls substantially during steady swimming, and is hypothesized to reorient the dorsal fin and one pectoral fin as primary lifting surfaces to decrease the cost of transport (Payne et al., 2016). However,

Fish and Lauder (2017) noted that the asymmetrical size of the dorsal and pectoral fins

82 would instead induce longitudinal rolling of the body axis, returning the body to a normal swimming orientation. We hypothesize that bonnethead sharks rotate pectoral fins to generate an imbalance of forces, rolling the body for banking. Rolling would also alter cephalofoil orientation to generate additional forces anterior to the center of mass, where steering is most effective (Nakaya, 1995; Fish and Lauder, 2017). Variability in maneuvering strategy is described both within and among various aquatic species, and we hypothesize that the two strategies presented here only capture a small subset of this volitional behavior (Tytell and Lauder 2002; Hale, 2002; Hale et al., 2002; Domenici et al., 2004).

Whole body kinematics

Body bending is also a major factor in turning performance (Kajiura et al., 2003;

Domenici et al., 2004; Porter et al., 2009). Both bending coefficient (BC) and maximum bending angle (MBA) are positively related to turning angular velocity, and MBA is positively related to the change in velocity (Fig. 4.6). Similar to total fin rotation, the change in velocity, MBA, and BC nearly doubled from the slowest to the fastest trials. As hypothesized, there was a significant positive relation between MBA and BC (Fig. 4.4).

Previous studies show that MBA differs between species while BC may not, demonstrating that different body curvature metrics have varied impacts on the results, even though the two variables are related (Porter et al., 2009). BC is traditionally calculated as the chord distance between the head and the tip of the caudal fin, but this has been shown to overestimate curvature in the body due increased body and caudal fin flexibility (Brainerd and Patek, 1998; Azizi and Landberg, 2002; Kajiura et al., 2003;

Porter et al., 2009). We calculated BC as the distance between the head and the caudal

83 peduncle (Porter et al., 2009), resulting in small differences between our values and those of other studies. Even still, the MBA and BC we calculate for bonnetheads are comparable to values previously described for yaw turning near the substrate in other shark species: bonnethead, scalloped hammerhead, sandbar, and leopard (Fig. 4.4;

Kajiura et al., 2003; Porter et al., 2009).

Turning performance

The values we document for turning angle and turning angular velocity are comparable to Pacific spiny dogfish but are substantially less than those previously reported for other shark species (Kajiura et al., 2003; Domenici et al., 2004; Table 4.2).

Previous studies document yaw maneuvering in the context of prey locating or escape responses, and we hypothesize that the turning behavior captured in our data represents slow, steady maneuvering rather than reacting to stimuli. Neither turning angle nor turning angular velocity differed between the bonnethead and Pacific spiny dogfish, but Δ velocity throughout a turn was three times greater in the bonnethead (Table 4.2). In addition to the significant difference in magnitude, velocity increased when bonnetheads turned in the water column and decreased in all Pacific spiny dogfish (Table 4.2). Despite these differences, turning angle and turning angular velocity was similar between species

(Table 4.2).

Turning angular velocity was the best indicator of turning performance in the present study and was related to a number of fin and whole body kinematic variables.

Similar to the relationships previously described for the Pacific spiny dogfish, Z axis and total fin rotation are both significantly related to turning angular velocity (Hoffmann et al., 2019; Fig. 4.5A-B). Total fin rotation is also significantly related to the change in

84 bonnethead shark velocity (Fig. 4.5C). We found a greater than fivefold increase between

-1 -1 the fastest (wt = 177.6 deg · s ) and slowest (wt = 33.5 deg · s ) trials, and total fin rotation doubled and fin depression more than tripled with angular velocity. Together, these data suggest that increasing fin rotation plays a role in creating tighter, faster turns in the bonnethead shark.

Turning angular velocity was significantly predicted by the top five best fit models in a generalized linear model (Table 4.1). The top five best fit models consider total fin rotation as a significant factor. All models also include one of the body bending variables, suggesting that fin rotation and whole body kinematics together best predict turning angular velocity. Our top five best fit models account for at least 60% of the variation in our system. Other kinematic factors known to contribute to swimming performance include changes in pectoral fin conformation, caudal fin displacement and stiffness, and dorsal fin movements, which may account for the variation not explained in the present study (Wilga and Lauder, 2000,2001; Flammang, 2010; Maia et al., 2012;

Maia and Wilga, 2013; Maia and Wilga, 2016).

Muscle stimulation

Bonnethead sharks rotate their inside fin relative to all three body axes and post- mortem stimulation confirmed functional hypotheses of the associated pectoral fin muscles. Three muscles are directly affiliated with the fin itself and were previously shown to play a role in actuation: the dorsal pterygoideus (DP), ventral pterygoideus

(VP), and cranial pterygoideus (CP; Fig. 4.7; Maia et al., 2012; Hoffmann et al., 2019).

Our experiments showed that stimulation of each individual muscle resulted in fin rotation about all three axes (Fig. 4.7).

85 The DP originates on the scapulo-coracoid and axial musculature posterior to the pectoral girdle and fans out distally over the three basal cartilages to insert on the intermediate radials (Fig. 4.7). Stimulation of the DP resulted in fin retraction, pronation, and elevation (Fig. 4.7). Fin elevation is previously attributed to DP activity, and this pattern of rotation relative to the body axes is the same as the Pacific spiny dogfish

(Marinelli and Strenger, 1959; Maia et al., 2014; Hoffmann et al., 2019). The VP also produced the similar rotation patterns in the bonnethead and the Pacific spiny dogfish: retraction, supination, and depression (Fig. 4.7; Hoffmann et al., 2019). On the ventral side of the fin, the VP also originates on the scapulo-coracoid and the axial musculature, and it inserts on the intermediate radials (Fig. 4.7). In two axes (Y and Z), the DP and VP are antagonistic muscles: the DP pronates and elevates the fin while the VP supinates and depresses the fin (Fig. 4.7). Both muscles retracted the fin, likely resulting from the muscle fibers fanning out distally at an oblique angle to the body axis (Fig. 4.7).

The only muscle to produce a different pattern of rotation in the bonnethead compared to the Pacific spiny dogfish was the CP. In the bonnethead, the CP originates antero-medially to the scapulo-coracoid (Fig. 4.7). Unlike squalids where the CP has insertions on both sides of the fin, the bonnethead CP is localized to the anterior margin of the scapulo-coracoid and does not fan out distally into the fin (Marinelli and Strenger,

1959; Hoffmann et al., 2019). Stimulation of the CP protracted, pronated, and depressed the fin (Fig. 4.7C-F). In the Pacific spiny dogfish, the CP supinated the fin, and varied

EMG lead placement resulted in both depression and elevation of the fin (Hoffmann et al., 2019). Interestingly, Y axis rotation was the only difference in the pattern of fin rotation between the two species during turning, where bonnethead sharks pronated the

86 fin and Pacific spiny dogfish supinated the fin (Fig. 4.7; Hoffmann et al., 2019). We hypothesize that the dissimilar muscle morphology and function between these two species represent differences in maneuvering strategies associated with varied habitat use.

Ecomorphological differences in maneuvering strategy

The bonnethead shark and Pacific spiny dogfish turn at similar angles and turning velocities during slow swimming (Table 4.2), despite differences in fin rotation and anatomy. Pectoral fin movement during yaw turning contributes significantly to maneuvering, but we hypothesize that the mechanism of turning differs between the two species (Kajiura et al., 2003; Domenici et al., 2004; Hoffmann et al., 2019). Fin rotation was comparable in the X (protraction) and Z axes (depression), but bonnethead demonstrated positive Y axis rotation (pronation) while the Pacific spiny dogfish consistently supinated the fin (Fig. 4.2E, Table 4.2; Hoffmann et al., 2019). Pacific spiny dogfish supinated fins to increase the area to flow and thus, drag, which created a pivot about which the body rotates (Hoffmann et al., 2019). Additionally, Pacific spiny dogfish decelerated during turning whereas bonnethead sharks accelerated, and the magnitude of

Δ velocity was three times greater in the bonnethead. We hypothesize that rather than increasing the area of fin to flow, thereby generating drag, bonnethead sharks pronate the inside fin to reorient lift, causing the body to bank while accelerating through the turn.

Longitudinal rolling of the body axis is especially advantageous because it changes the cephalofoil angle to act as an additional force generating surface in the anterior body where steering is most effective (Nakaya, 1995). We also document one turn in which the individual made pectoral fin contact with the substrate, which is comparable to yawing

87 during prey seeking behavior previously described in the bonnethead (Kajiura et al.,

2003).

One major difference between the bonnethead and Pacific spiny dogfish pectoral fin anatomy is the extent of radials into the fin. Pacific spiny dogfish have aplesodic fin morphology, in which basals are long and sometimes fused, and the radials support less than half of the fin web, and the distal region is instead supported by ceratotrichia

(Marinelli and Strenger, 1959; Compagno, 1984; Da Silva and De Carvalho, 2015). The bonnethead shark has plesodic fin morphology with shorter basals, and the radials support the majority of the fin (Compagno, 1984). Only a small portion of the distal and trailing edge of the fin have only ceratotrichia support, and the propterygium does not support any radials (Maia et al., 2012; Hoffmann et al., 2019). Previous studies suggest an inverse relationship between radial support and fin muscle size where aplesodic fins are hypothesized to have a greater range of motion and control compared to plesodic fins

(Compagno, 1984; Maia et al., 2012). We did not find substantial differences in range of motion during muscle stimulation experiments or volitional yaw turning. Rather, we note differences in CP morphology and function, which we hypothesize facilitates the varied pattern of fin rotation observed during turning in these two species.

Conclusions

In this study, we found that the bonnethead shark rotates the inside pectoral fin about three axes during turning. For one turning strategy (n = 10), the bonnethead accelerates through the turn and the inside fin is protracted, pronated, and depressed; and whole body movements (BC and MBA) were correlate positively with both turning angular velocity and Δ velocity. For the second strategy, the fin underwent a more

88 complex rotation pattern: first retracting, supinating and elevating, followed by a period of fin depression and pronation to contact the substrate; which slowed the turn. Post mortem muscle stimulation experiments confirmed that the pectoral fin rotates positively and negatively about all three axes as observed during volitional turning. The top five best-fit models all suggest that some combination of fin rotation and body bending explain at least 65% of the variability in these data. Finally, our comparative analyses showed that pectoral fins of bonnethead sharks mostly accelerated through turns and Y axis rotation was positive (fin pronation) while Pacific spiny dogfish decelerated during turn and used fin supination instead.

89 Chapter 4 Figures and Tables

Figure 4.1: Camera set-up and bead placement for VROMM. (A) Black hemispherical beads were placed along the body and pectoral fins of four bonnethead sharks. (B) Three

GoPro Hero 5 Black cameras (outlined in yellow) were mounted to cement blocks and angled at a common volume of interest, marked with white coral fragments. (C) The trial arena was calibrated for 3D analyses by taking images of a 7 square x 9 square checkerboard at various regions throughout the volume.

90 Figure 4.2: Fin rotation relative to the body axes in two sample trials demonstrating two turning strategies employed by the bonnethead shark. (A) JCS placement at the proximal fin base denotes three axes of fin rotation relative to the body. Rotation about the dorso- ventral body axis (X; red) represents (B) fin protraction and retraction, medio-lateral body axis (Y; green) represents (C) fin pronation and supination, and cranio-caudal body axis (Z; blue) represents (D) fin elevation and depression. (E) An exemplar trial demonstrating the pattern of fin rotation during turning in the water column, where the turning period is outlined in the light gray box. (F) Using this turning strategy fin rotation is more complex, where the fin is first retracted, supinated, and elevated, and then the fin

91 is pronated and depressed to contact the substrate (darker gray box) before it returns to neutral.

92

Figure 4.3: Range of pectoral fin rotation about the body axes during turns, in which the fin did not contact the substrate. The magnitude of rotation did not differ among axes.

Boxes represent the mean (middle line) ± the standard error of the mean, and whiskers represent the minimum and maximum values. During turning, the fin was protracted (X), pronated (Y), and depressed (Z).

93

Figure 4.4: Whole body flexion kinematics. Individuals demonstrated a range of body bending coefficients (BC) and maximum bending angles (MBA), which were significantly related to each other. Significance (P < 0.05) is denoted by the corresponding trendline and R2 value.

94

Figure 4.5: Fin rotation relative to turning performance. (A) Fin depression was the only axis of rotation significantly related to turning angular velocity. Total fin rotation was significantly positively related to turning angular velocity (B) and the change in velocity

(C). Significance (P < 0.05) is denoted by the corresponding trendline and R2 value.

95

Figure 4.6: Whole body kinematics relative to turning performance. Body bending coefficient (BC; A) and maximum body angle (MBA; B) were significantly positively related to turning angular velocity. (C) MBA was also significantly related to the change in velocity. Significance (P < 0.05) is denoted by the corresponding trendline and R2 value.

96

Figure 4.7: Fin rotation relative to the body from stimulation of three target muscles in the pectoral fin. dorsal pterygoideus (A; orange), ventral pterygoideus (B; purple), and cranial pterygoideus (C; yellow). Lead placement targeted the middle of the muscle body, represented by black dots. (D-F): Fin rotation relative to the X (D), Y (E), and Z (F) axes described in Fig. 4.2. The CP was the only muscle to produce fin protraction (D) whereas the DP and VP both resulted in retraction. The DP and CP both pronated the fin (E) and the VP supinated the fin. The DP was the only fin elevator (F) and stimulation of the VP and CP both resulted in fin depression. Error bars represent standard error of the mean.

97 Table 4.1: Top five best fit models predicting turning angular velocity. All top five models included total fin rotation as a significant variable and the top two also included one of the body bending variables (BC or MBA).

2 2 Adj R R AIC P < 0.05 Variables 0.6659 0.7401 107.5001 0.0089 Total rotation BC 0.6594 0.7351 107.6914 0.0096 Total rotation MBA 0.6113 0.7409 116.4718 0.0342 Total rotation Δ velocity BC 0.6151 0.7434 116.3728 0.0332 Total rotation Δ velocity MBA 0.6158 0.7439 116.3550 0.0330 Total rotation BC MBA

98 Table 4.2: Fin rotation and turning performance variables compared between the bonnethead and Pacific spiny dogfish (Hoffmann et al., 2019). Values represent the mean

± standard error and significance is denoted by *. The absolute values of X, Y, Z axis rotation and the change in velocity were used for statistical tests. Only the absolute value of change in velocity during turning was significantly different between the two species.

Pacific spiny Bonnethead dogfish F1,17 P < 0.05 X rotation (deg) 8.55 ± 1.28 6.57 ± 1.35 1.1236 0.304 Y axis rotation (deg) 5.03 ± 0.83 -3.2 ± 0.87 2.2935 0.1483 Z axis rotation (deg) -10.38 ± 1.75 -15.16 ± 1.84 3.5472 0.0769 Total rotation (deg) 23.96 ± 2.59 24.93 ± 2.69 0.0671 0.7987 Turning angle (deg) 37.81 ± 5.48 43.26 ± 5.78 0.4683 0.503 Angular velocity (deg · s- 1) 113.86 ± 15.08 92.35 ± 15.89 0.9648 0.3398 *Δ velocity (cm · s-1) 27.00 ± 2.04 -9.97 ± 2.16 32.9003 < 0.0001

99 CHAPTER 5: COMPARATIVE MORPHOLOGY OF SHARK PECTORAL FINS

Abstract

Sharks vary greatly in morphology, physiology, and ecology. Differences in whole body shape, swimming style, and physiological parameters have previously been linked to varied habitat uses. Along with whole body morphology, shark pectoral fins are also previously described to vary in both shape and skeleton; however, there are limited comparative data on external and skeletal morphology. Further, fins are previously categorized into two discrete groups based on the amount of skeletal support present: (1) aplesodic, where less than half of the fin is supported and (2) plesodic where greater than half of the fin is supported. The classifications have been used to phylogenetically place species, though the methodology of classification is infrequently described and the comparisons may not be valid. Additionally, our understanding of shark pectoral fin ecomorphology is limited by access to samples from a broad variety of species. In this study, we sampled fins from various families, orders, and ecological classifications. We examined the external morphology, skeletal extent, and cross-sectional shape of the cartilaginous elements. Using phylogenetic comparative methods, we show that fin morphology does not differ significantly when considering the level of relatedness between species, suggesting there may be some mechanical constraint. We also describe a range of skeletal extent, rather than two discrete categories. We find that fins are shaped like hydrofoils in cross-section, supporting hypotheses that fins may be lift producing structures in sharks. Finally, we find that a number of morphological variables such as

100

number of radials, radial calcification and shape, and fin taper all correlate with skeletal extent. Within these morphospaces, we also describe that some orders/families tend to occupy certain areas with limited overlap. With this study, we demonstrate that there is some mechanical constraint limiting variations in shark pectoral fin morphology, but there are subtle differences that appear to occur within shark groups that share close phylogenetic relationships and similar biological parameters.

Introduction

Control surfaces, or structures that affect an organism’s position in space, vary greatly in morphology among aquatic vertebrates (Fish and Lauder, 2017). For many open ocean swimmers (some marine mammals, sea turtles, oceanic fishes), control surfaces are shaped like hydrofoils (thicker leading edges that taper posteriorly) which tend to be less flexible to promote lift generation (Fish and Lauder, 2017). Aquatic vertebrates that are associated with more architecturally complex environments (reef associated fishes) have more flexible fins that may be used for increased maneuverability

(Lauder and Drucker, 2004; Blake, 2004). External fin shape is also shown to change among habitat use where open ocean species have long, thin fins and benthic associated species have shorter, wider fins (Wainright et al., 2002; Fulton, 2005; Fish and Lauder,

2017). These comparisons are often made using aspect ratio, the ratio of fin length to area, which affects the amount of lift produced relative to drag (Webb, 1975; Vogel,

1994; Weber et al., 2009, 2014). In contrast, long, stiff fins are likely less maneuverable and may not be ideal for animals swimming in complex environments.

The ecomorphology of the body, caudal fin, swimming style, sensory structures, and feeding apparatus are all documented to vary among shark species (Thomson and

101 Simanek, 1977; Webb and Keyes, 1982; Motta and Wilga, 2001; Kajiura et al., 2005;

Litherland et al., 2009; Meredith and Kajiura, 2010; Flammang, 2014). As is demonstrated in other fishes, it is hypothesized that variations in shark pectoral fin morphology may reflect ecological differences (Shirai, 2011; Maia et al., 2012). There are also varied descriptions of shark pectoral fin function that may be the result of different study species (Daniel, 1922; Harris, 1936; Wilga and Lauder, 2000, 2001; Fish and Shannahan, 2000). Historically, shark pectoral fins are hypothesized to generate lift that balances the body during steady swimming (Daniel, 1922; Harris, 1936; Ferry and

Lauder, 1996; Fish and Shannahan, 2000). For at least one benthic shark species, negligible lift is generated by the pectoral fins during steady swimming and the anterior body generates the balancing lift force (Wilga and Lauder, 2000). In general, our understanding of the comparative morphology of sharks is limited to species that are easy to access and/or are successful in captivity, making an ecomorphological assessment of pectoral fins challenging.

In addition to external variations in morphology, shark pectoral fin skeleton is also documented to vary among species. In general, three basal elements (propterygium, mesopterygium, and metapterygium) articulate with the scapulocoracoid at the proximal fin base (Fig. 5.1; Marinelli and Strenger, 1959; Liem and Summers, 1999). Three series of radial elements extend distally into the fin web from the basals (Fig. 5.1; Marinelli and

Strenger, 1959; Liem and Summers, 1999). Thin, flexible ceratotrichia are embedded in the dense connective tissue that anchors the skeleton to the fin (Marinelli and Strenger,

1959; Liem and Summers, 1999). Differences in the relative amount of radial support in the fin have been described throughout the literature as a diagnostic characteristic that

102 was historically used in phylogenetic classification (Compagno, 1977; Maisey, 1984;

McEachran, 1989; Compagno, 1990; Shirai, 1996). Fins with less than 50% radial support are considered aplesodic while fins with greater than 50% radial support are plesodic (Compagno, 1977; Maia et al., 2012). These classifications vary within families and orders, though the method of classification is often undescribed and may not be comparable among all studies (Fig. 5.2; Bendix-Almgreen, 1975; Compagno, 1973,

1977; 1988; Zangerl, 1973; Maisey, 1984; Maia et al., 2012; Crawford, 2014).

Differences in skeletal extent would likely affect the mechanical behavior of the fin. Fins with more skeletal support may be stiffer, and thus, more efficient as hydrofoils. In contrast, flexible fins may be better suited for maneuverability. An inverse relationship between skeletal extent and muscle mass has been described among a few species, further suggesting that there may be a finer degree of control over flexible, maneuverable fins

(Maia et al., 2012).

The goal of this study was to describe the external and skeletal morphology of sharks from various families, orders, and ecological classifications. We hypothesized that there would be an ecomorphological gradient in fin morphology from benthic to oceanic species. We predicted that oceanic species would have high aspect ratio fins with extensive skeletal support that maximize hydrodynamic efficiency. In contrast, we predicted that benthic species would have low aspect ratio fins with limited skeletal support for increased flexibility and maneuverability. We assessed the external and skeletal morphology of pectoral fins from 18 species (five families, three orders) among four ecological classifications. We used phylogenetic comparative methods to test our ecomorphological hypotheses in phylogenetic context.

103 Methods

Fins were examined from 18 species: five families, three orders (Table 1). Shark pectoral fins were opportunistically sampled from various strandings, at vessel mortality, fishing tournaments, and donated from various researchers from species in the Western

Atlantic. Due to the numerous methods and people used to obtain fins, there was variability in the way they were removed from the trunk, and we standardized the fin

“base” as a perpendicular line from leading edge to the posterior end of the curvature of the trailing edge lobe (Fig. 5.3A). The data presented here represent the radial cartilages and do not include the most proximal portion of the pectoral fin including the three basal cartilages that articulate with the scapula-coracoid at the proximal body axis (Marinelli and Strenger 1959; Liem and Summers 1999; Maia et al., 2012).

External morphology

Scaled images of the dorsal and ventral side of the fin were captured with a Nikon

D3500 DSLR mounted and leveled perpendicular to the fin (Nikon, Tokyo, Japan). Fin area (Af), length (Lf), and width (Wb; Wf) were measured in ImageJ (Schneider et al.,

2012). Fin length was measured as the perpendicular straight-line distance from the base to the distal fin tip (Fig. 5.3A). Fin width was measured at (1) the base as the perpendicular straight-line distance from the leading edge to the posterior margin of the trailing edge lobe (Wb) and (2) the fork as the perpendicular straight-line distance from the leading edge to the most posterior portion of the trailing edge lobe (Wf) (Fig. 5.3A).

Fin aspect ratio (AR) was calculated as:

104 푳 ퟐ 푨푹 = 풇 (Eq. 5.1) 푨풇

Geometric morphometrics of fin shape

We used geometric morphometrics to quantitatively describe and compare the pectoral fin shape of each species in our dataset. We identified five putatively homologous locations on the pectoral fin to serve as landmarks and recorded the

Cartesian coordinates of these locations on each shark fin using the digital images described above with the program tps-Dig v.2.2 (Fig. 5.4; Rohlf, 2007). We performed a

Procrustes superimposition on the landmark arrays of all specimens to remove the effects of scale, position, and rotation (i.e., non-shape variables) on the coordinate positions of our variables using functions in the R package “geomorph” (Adams & Otárola-Castillo,

2013; Adams et al., 2016; R Core Team, 2017). We created species averages of the landmark arrays using the custom R script from (Buser, Burns & López, 2017).

Skeletal morphology

Skin, connective tissue and ceratotrichia were carefully removed to expose the pectoral fin skeleton (Fig. 5.3A). Scaled images of the dorsal and ventral side of each fin skeleton were taken with a Nikon D3300 (Nikon, Tokyo, Japan). The number of radials

(nr), skeletal area (Ask) and the length of the longest radial (Lsk) were measured in ImageJ

(Schneider et al., 2012). The amount of skeletal support was calculated as the ratio of areas (skeletal extent area; SEa)

푨풔풌 푺푬풂 = (Eq. 5.2) 푨풇

and the ratio of the lengths (skeletal extent length; SEl)

105 푳풔풌 푺푬풍 = (Eq. 5.3) 푳풇

Cross sectional morphology

Cross-sectional morphology was measured along the most proximal radial for three regions in the fin: the leading edge, longest, and trailing edge radial (Fig. 5.3B).

Scaled images of the cross sections were taken using a Leica EZ4 W Stereo Microscope when small enough, and a Nikon D3300 when radial size exceeded the size of the microscope stage (Nikon, Tokyo, Japan; Leica Microsystems, Buffalo Grove, IL). The areas of calcification (Ac) and total area (At) were measured in ImageJ to calculate percent calcification of the radial cross section (% Ar) as

푨풄 % 푨풓 = × ퟏퟎퟎ (Eq. 5.4) 푨풕

Radial shape (Sr) was characterized by calculating the second moment of area (I) along both the x and y neutral axes

횷 풓 풓 ퟑ 푰 = 풚 풙 (Eq. 5.5) 풚 ퟒ

횷 풓 풓 ퟑ 푰 = 풙 풚 (Eq. 5.6) 풙 ퟒ

(Fig. 5.3B; Mulvany and Motta, 2013). Radial shape (Sr) was calculated as

푰풚 푺풓 = (Eq. 5.7) 푰풙

106 where values greater than one represent dorso-ventrally compressed radials, less than one represent laterally compressed radials, and equal to one represents a perfect circle

(Mulvany and Motta, 2013.

Whole fin cross-sectional morphology was analyzed by examining the amount of taper, or change in width, from the leading edge to the longest radial (leading edge taper,

TLE) and from the longest radial to the trailing edge (trailing edge taper; TTE). Taper was calculated as

푫풍풐풏품−푫풍풆풂풅풊풏품 푻푳푬 = (Eq. 5.8) 푾풔

where Dlong is the lateral radial diameter of the longest radial, Dleading is the lateral radial diameter along of the leading edge radial, and Ws is the segment width from the leading edge radial to the longest radial. TTE was calculated using the same equation substituting leading edge for trailing edge. The taper angle (α) was calculated as

푻 휶 = 퐭퐚퐧−ퟏ( ) (Eq. 5.9) ퟐ

Data analysis

We used phylogenetic multivariate analysis of variance (phyMANOVA) to test for differences in average pectoral fin morphology across each of our discreet habitat guilds. We used a phylogenetic hypothesis of the relationships of all species included in this study (Buser, unpublished), which closely matches the phylogenetic hypothesis of these species published in the most current and extensive phylogenetic study of

107 elasmobranchs (Naylor et al., 2012). We quantified pectoral fin morphology in two ways:

1) using the landmark-based dataset described above and 2) using the measured variables described above. In both datasets, we calculated species averages of each trait. For the measured variables, we visually assessed normality using a quantile-quantile (QQ) plots in the R statistical environment (R Core Team, 2017). For each dataset, we visualized the variance therein using a principle components analysis (PCA), and overlaid the phylogenetic relationships of the species using the phylomorphospace method of

(Sidlauskas, 2008) using basic functions in R as well as functions from “geomorph”

(Adams & Otárola-Castillo, 2013; Adams et al., 2016). We used functions from the R package GEIGER (Harmon et al., 2008) to perform the phyMANOVA. We also examined the relationships between morphological variables using simple linear regressions. Preliminary results indicated that Squatina is a major outlier in the dataset, potentially skewing the results of our comparative analyses, so we removed the taxon for all phylogenetic comparative methods.

Results

When considering the phylogenetic relationships among 18 species, there was no significant difference in pectoral fin shape (both linear morphological measurements and

3D geometric morphometrics) or skeletal morphology (Fig. 5.5, 5.6). Since there are no significant phylogenetic results, we describe the following trends in fin morphology with the caveat that there is no significant evidence to suggest that these differences are not the product of evolutionary history when accounting for relatedness.

108 Geometric morphometrics and phylogenetic comparative methods

For the landmark-based dataset, the first two principal components capture approximately 62% and 17% of the observed variance in our dataset, respectively (Fig.

5.5). Principal component 1 captures variation in the elongation of the leading edge of the pectoral fin, such that high values of PC1 are associated with a relatively short leading edge and negative values are associated with a relatively long leading edge. The second principal component captures variation in the length of the trailing edge and degree of forkedness of the fin, such that high values of PC2 are associated with fins with a relatively wide base and virtually no fork, while low values of PC2 are associated with fins with a relatively narrow base and a deep fork (Fig. 5.5).

For the dataset of measured variables, the first two principal components capture approximately 60% and 28% of the observed variance in our dataset, respectively (Fig.

5.6). The first principal component is strongly associated with variation in the percent area of skeletal extent, such that high values of PC1 are associated with pectoral fins with relatively low percent area of skeletal extent, and high values of PC1 are associated with relatively large percent area of skeletal extent. Principal component 2 is strongly associated with variation in the percent of radial support as well as the skeletal extent of the longest radial, such that high values of PC2 are associated with a high percent of radial support but a low extent of skeletal support for the longest radial and low values of

PC2 are associated with a relatively low percent of radial support and a high extent of skeletal support for the longest radial (Fig. 5.6).

For both the landmarks and measured traits datasets, there is no statistically significant difference in the mean morphology across the three ecological groups tested

109 herein (p > 0.6 for landmark dataset; p > 0.3 for measured traits dataset). The substantial overlap of the three groups is visually obvious in the morphospace derived from each dataset (Fig. 5.5, 5.6). Though there is some separation of the “Oceanic” and “Inshore” groups, the “Offshore/Inshore” group co-occupies a great deal of morphospace with the other two groups. Furthermore, the “Oceanic” group is made up entirely of members of

Lamniformes, and in fact all lamniform species in this study are classified as “Oceanic.”

This phylogenetic bias is accounted for using the phyMANOVA, but suggests that any commonalities among the “Oceanic” classified species may be due simply to shared evolutionary history, rather than shared habitat. However, this does not rule out the possibility that the morphology shared by lamniform sharks confers an adaptive advantage to living in oceanic habitats and allowed that group to dominate. The physical properties of certain morphological features observed in this study would in fact confer seemingly adaptive advantages to life in oceanic vs inshore habitats and we discuss these below.

Gross morphology

Fin shapes and skeletal extent vary among the species documented in this study

(Fig. 5.7). For most species, the leading edge lobe is longer than the trailing edge lobe and, it is generally more supported by the radial elements. The relative length of the leading edge lobe accounted for the majority of differences (60%) among species in the geometric morphometric analysis (PC1, Fig. 5.5. The relative width of the fin base accounted for another 28% of the variation (PC 2, Fig. 5.5). Overall, A. vulpinus had the longest, thinnest fins while two hammerhead species, S. lewini and S. tiburo, had the shortest, widest fins (Fig. 5.8A). We hypothesized that Lamniformes species would have

110 the greatest aspect ratio fins, but this characterization of shape differed greatly among all species (Fig. 5.8A).

Differences in the areas of radial support occurred at the distal fin web and trailing edge (Fig. 5.7). For example, almost the entirety of the A. vulpinus fin is supported by radials, the C. carcharhias fin also has extensive radial support towards the distal region but the trailing edge lacks skeletal elements. In contrast, the S. tiburo fin lacks support both in the distal end and the trailing edge (Fig. 5.7). The exception to this pattern is the S. dumeril fin, which is highly supported throughout the proximal fin area, while only the distal edge of the fin is unsupported.

When considering the relative area of skeletal support by the radials, we find a range from aplesodic to plesodic (Fig. 5.8B). Half of the Carcharhiniformes species were considered aplesodic, with less than 50% of the fin area supported. In general, aplesodic fins were found in the smaller bodied species (R. terraenovae, C. acronotus, S. tiburo).

In contrast, larger bodied species, known to make larger scale migrations, had plesodic fins (P. glauca, G. cuvier, C. limbatus) (Compagno, 1977, 1984; Kajiura and Tellman

2015). C. falciformis had the greatest skeletal extent of the Carcharhiniformes species, and it was comparable to the Lamniformes species. All Lamniformes species examined in this study had plesodic fins, ranging from 66% support (C. maximus) to 96% skeletal support (A. vulpinus) (Fig. 5.8B). S. dumeril, the single representative from the

Squatiniformes, also had a high degree of skeletal fin support (Fig. 5.8B).

When grouped by family, there was very little variation in gross morphological characteristics, particularly for Lamniformes (Fig. 5.9). The Lamniformes species sampled here have similar body types, habitat use, and locomotor style, excluding the

111 (C. maximus). The Carcharhiniformes species sampled here cover a broader range of body sizes, body types, and habitat uses, and we found there was broader variation in morphology within this order (Table 1). The major detail that is lost in this generalization is the variation in skeletal extent in Carcharhinidae and Sphyrnidae

(Fig. 5.9B). We found that species within these families had both aplesodic and plesodic fins, but when averaged they tended to be considered plesodic for both families (Fig.

5.9B). Aspect ratio of pectoral fins ranged from 2.1 at the least (Squantinidae) to 3.5

(Alopiidae). The number of radials varied among species examined in this study: 22-28

(Carcharhinidae), 24-28 (Sphyrnidae), 24 (Cetorhinidae), 29-33 (Lamnidae), 35

(Alopiidae), and was greatest in Squatinidae (50) (Fig. 5.9C).

Cross-sectional morphology

To describe cross-sectional fin morphology, we measured the shape and calcification of the proximal radial at three points throughout the fin: leading edge, longest radial (near the middle), and the trailing edge (Fig. 3B). In order to estimate the flexibility of a fin in cross section, we measured the distribution of material (I) in the dorso-ventral (x) and lateral (y) planes (Fig. 3B; Mulvany and Motta, 2013). We used

Iy:Ix to describe the shape of a radial such that a value of one represents equal resistance to bending in all planes, less than one represents greater resistance to bending in the dorso-ventral plane, and greater than one represents greater resistance to bending in the lateral plane (Vogel, 2003; Mulvany and Motta, 2013). Thus, the larger the Iy:Ix, the more flexible a fin is in bending in the dorsoventral plane. For all families, the trailing edge radial was the most dorso-ventrally compressed, followed by the leading edge radial, while the longest radial was the most laterally compressed (Fig. 5.10A). Only Lamnidae

112 and Alopiidae had radials that were more laterally compressed than a perfect circle (Fig.

5.10A).

The amount of calcification among the leading edge, longest, and trailing edge radial was less consistent than shape (Fig. 5.10B). In general, Lamnidae and Alopiidae had less calcification (15%) overall than the other three families, while Sphyrnidae appears to have the greatest (40%).

Taper angle describes the change in cross-sectional height from the leading edge radial to the longest radial (leading edge taper) and from the longest radial to the trailing edge radial (trailing edge taper). For all but one species, trailing edge taper was greater than leading edge taper (Fig. 5.10C). Sphyrnidae was the only family where this was not the case, and the two degrees of taper were not different. Both Lamnidae and Alopiidae had substantially greater trailing edge taper (2x, 4.5x, respectively), while trailing edge taper was greater than leading edge taper in Carcharhinidae and Squatinidae, but to a lesser degree (1.8x, 1.4x, respectively).

Relationships among morphological variables

To describe variations in pectoral fin morphology that have been previously overlooked, we examined the relationships between external and cross-sectional shape to skeletal support, which is historically used to classify fin shape, and thus, phylogeny and habitat use. Considering all species, there was no relationship between aspect ratio and skeletal extent, though we suspect increasing the scope of species examined, especially in the Carcharhiniformes may elucidate this trend (Fig. 5.11A; P =0.0641, R2 = 0.1981).

Skeletal extent was positively related to the number of radials (Fig. 5.11B; P =

0.0051, R2 = 3965). There was no significant relationship between trailing edge

113 morphology and skeletal extent. Radial calcification was negatively related to skeletal extent along both the leading edge and longest radial (Fig. 5.11C; P = 0.0133, R2 =

0.4120; P = 0.0311, R2 = 0.3101; respectively). Similarly, radial shape was negatively related to skeletal extent, but only along the longest radial (Fig. 5.11D; P = 0.0031, R2 =

0.5033). Among all species, the longest radial was significantly more calcified and more laterally compressed than the leading edge radial (F1,90 = 3.9469; P = 0.0420; F1,90 =

3.9469; P < 0.0001). Finally, trailing taper angle was positively correlated with skeletal extent (Fig. 5.11D; P = 0.0017, R2 = 0.5453).

We further analyzed species within Carcharhiniformes because the range of body sizes, migratory behavior, ecological classifications, and number of species sampled provides a broader insight to morphological variation within an order (Table 1). There was a significant relationship between skeletal support and aspect ratio and trailing edge taper within this order (Fig. 5.12; P = 0.0201, R2 = 0.4325; P = 0.0061, R2 = 0.6307).

Discussion

The classification of fins as aplesodic (less than half of the fin supported by skeleton) and plesodic (greater than half) is referred to sporadically throughout the literature; including as speculation about habitat use and these terms been used as a diagnostic character in phylogenetic analyses (Fig. 5.2; Compagno, 1977, 1984; Maisey,

1985; Wilga and Lauder, 2001; Sakai, 2011; Maia et. al., 2012; Crawford, 2014). In this study, we demonstrate that skeletal support is more appropriately described as a continuum that varies within families and orders; rather than the binary classification that is historically relied upon. Though there is limited variance within families, we do find that differentiation between aplesodic/plesodic designations is lost when evaluating at the

114 family level, which may have resulted in over-generalizations in previous studies.

Further, we describe a suite of morphological variables that, when considered with fin support, demonstrate that skeletal extent alone is not a comprehensive diagnostic of fin morphology. Interestingly, none of the morphological characteristics in this study are significantly different among species from different habitats when considering phylogenetic relationships, suggesting that there are mechanical constraints of fin shape in sharks.

Geometric morphometrics and phylogenetic comparative methods

An analysis of fin shape using geometric morphometrics reveals that the two greatest axes of variation are the leading edge shape (62% of the observed variance in the data set) and trailing edge shape considering the curvature of the fork (17%; Fig. 5.5).

Lamniform species tend to have long pectoral fins with deep forks, while

Carcharhiniform species are much more variable in their pectoral fin shape (Fig. 5.5). We also examined linear fin measurements in phylogenetic context (Fig. 5.6). PC1 describes variation in the area of fin skeletal support (60% observed variance) and PC 2 describes relative length of the longest radial (28%; Fig. 5.6). Lamniformes and Carcharhiniformes appear to separate along PC1, with Lamniformes all tending to have a high extent of skeletal support in the pectoral fins, while there is much more variability within

Carcharhiniformes. For all species, the leading edge is well supported by radials, likely making this region stiff. This may be advantageous for force generation, especially considering the hydrofoil cross-sectional shape of the fin (Fig. 5.10A). The major differences in skeletal support appear to occur at the trailing edge (Fig. 5.7). The

Lamniform species have skeletons that support the majority of the fin web, including the

115 trailing edge, while Carcharhiniform species have more variation in skeletal extent occurring at the distal fin tip and trailing edge (Fig. 5.7, 5.8B). Flexibility at the distal fin tip and trailing edge may allow for greater changes in fin conformational shape, and thus, a finer degree of control over trim (Wilga and Lauder, 2000, 2001). There is some separation of the oceanic species from the inshore/offshore and inshore species in skeletal support, but this trend was not significant (Fig. 5.6).

Aspect ratio

퐿 2 We compared aspect ratio (AR; 푓 ) among species as a proxy for hydrodynamic 퐴푓 efficiency, and hypothesized that AR would also be greatest in open ocean species (order

Lamniformes); however, this trend is less conspicuous (Fig. 5.8A). The species documented in this study fall within a small AR range from 1.8 - 3.5, which is comparable to AR described previously for a number of fish caudal and pectoral fins

(Sambilay, 1990; Fulton et al., 2005; Binning and Fulton, 2011). Low AR fins are typically characteristic of species that use drag-based swimming, such as pectoral fin paddling, while greater AR fins may be indicative of species using lift-based swimming

(Vogel, 1994; Fulton et al., 2005; Wainright et al., 2002). In labrid fishes, AR is related to fin attachment to the body: fins with greater AR attach at a shallower angle allowing greater motion in the dorso-ventral axis, suggesting lift-based locomotion (Wainwright et al., 2002). Though sharks rely primarily on body-caudal fin swimming, differences in fin

AR and attachment to the body may indicate a difference between drag-based and lift- based thrust production, but in maneuvering rather than straight swimming. The Pacific spiny dogfish (which has relatively low AR, flexible fins) uses pectoral fin to generate drag during yaw turning (Hoffmann et al., 2019). In contrast, the bonnethead shark has

116 somewhat greater AR, stiffer pectoral fins that are used to reorient lift to maintain speed throughout yaw turns (Hoffmann and Porter, in review). These data warrant further analyses similar to the comprehensive studies of fin aspect ratio and swimming performance in actinopterygian fishes, which would greatly benefit our understanding of the relationship between shark fin shape, locomotor performance, and habitat use

(Wainwright et al., 2002; Walker and Westneat, 2002; Lauder and Drucker, 2004; Fulton and Bellwood, 2004; Fulton et al., 2005).

Skeletal extent

The analysis of shark fin morphology and function often centers on their classification into aplesodic or plesodic fins, referring to the percentage of the fin supported by the cartilaginous skeletal elements (Sakai, 2011; Maia et al., 2012). In this comparative study using phylogenetic morphometrics, we describe a continuum of radial support rather than groupings into discrete categories (Fig. 5.8B). As we hypothesized, species in the order Lamniformes have extensive skeletal support while

Carcharhiniformes have less, with about half of those species falling into the “aplesodic” category (Fig. 5.8B). Since we document differences in skeletal extent within families, we suggest that fin morphology should be evaluated at the species level. When considered at the family level, variation is low but the resolution at the delineation between aplesodic and plesodic is lost. Perhaps even more precise would be to refer to skeletal extent continuously, rather than as binary categories. The hypothesis that aplesodic fin support is the ancestral condition from which plesodic fins arose may lend too much credence to the idea that these are two discrete states (Compagno et al. 1989).

Our data demonstrate that differences in fin shape and skeletal anatomy are explained by

117 phylogenetic relationships, thus considering skeletal extent as a continuous variable rather than a categorical condition may be more applicable (Fig. 5.5, 5.6).

These differences in skeletal extent are not significant when considering phylogeny, but may have functional consequences nonetheless. More skeletal support is hypothesized to increase overall fin stiffness, since the anatomical components that make up the non-skeletonized portion of the fin are flexible ceratotrichia, skin, and connective tissue (Shirai, 2011; Maia and Wilga, 2012; Fig. 5.1). The Lamniformes and

Squatiniformes both have extensive skeletal support, and would be hypothesized to have stiffer fins, that serve different functions based on the habitats of these groups.

Lamniformes are larger bodied, open ocean species, of which Lamnidae and Alopiidae could be considered high performance predators (Compangno, 1984). Stiffer fins in this environment likely minimize drag and generate more lift, thereby increasing swimming efficiency. Squatiniformes, on the other hand, are benthic sharks that commonly interact with the substrate (Compagno, 1984). In this environment, fins with more extensive skeletal support may be beneficial for weight bearing behaviors such as burrowing, resting, or punting from the substrate (Compagno, 1984).

Of the orders examined, Carcharhiniformes had the most variation in skeletal extent, ranging from 40% to 70% (Fig. 5.8B). This was the only order with variability in habitat use, migratory behavior, and size at maturity, making it a good candidate for comparative analyses among species (Table 1). When separated, there was a significant relationship between skeletal extent and aspect ratio in this order, which was not true for the whole dataset (Fig. 5.11A, 5.12A). The larger bodied, migratory species (G. cuvier, P. glauca, C. obscurus, C. falciformis, S. mokarran) had fins with greater skeletal support

118 and AR which are likely more efficient for sustained swimming (Fig. 5.12A, Table 1). In contrast, smaller bodied, non-migratory species (R. terraenovae, C. acronotus, C. isodon,

S. tiburo) had fins with less skeletal support and AR (Fig. 5.12A, Table 1). We hypothesize that this difference in fin morphology exemplifies a functional tradeoff between fin flexibility and hydrodynamic efficiency. Larger bodied migratory species may benefit more from hydrodynamically efficient body design for sustained cruising, but smaller bodied species that are reef associated may use flexible fins for maneuvering in more architecturally complex environments (Table 1; Thomson and Simanek, 1977;

Hoffmann and Porter, in review). This hypothesis is further supported by the inverse relationship between skeletal extent and the amount of pectoral girdle musculature: more muscle is need for a finer degree of control over flexible fins (Maia et al., 2012).

Cross sectional morphology

The extent of skeletal support in a fin gives some insight into its mechanical behavior, but the distribution of material and calcification within the radials may also affect overall stiffness (Vogel, 2003; Mulvany and Motta, 2013). For all families, the longest radial (in the middle of the fin) was the least dorso-ventrally compressed while the leading and trailing edges tapered (Fig. 5.10A). This gives the fin a characteristic foil shape, with greater thickness in the middle and tapering at the edges. Only Lamnidae and

Alopiidae had radials that were laterally compressed (Iy:Ix < 1), suggesting that these fins may resist bending in the dorsoventral plane better than the other families. Species within

Lamnidae and Alopiidae are larger bodied, oceanic sharks, for whom stiff lift producing fins would maximize lift to drag for more efficient swimming (Table 1). Further, among all species we found a significant relationship between radial shape and skeletal extent,

119 demonstrating that fins with greater skeletal support also have radials that resist dorsoventral bending, increasing overall stiffness (Fig. 5.11D). Contrary to our hypothesis, Lamnidae and Alopiidae had the least amount of radial calcification, and there was an inverse relationship between skeletal extent and radial calcification (Fig.

5.10B, 5.11C). We hypothesize that radials in these families are shaped to resist bending and have extensive skeletal support, and the costly addition of calcification is unnecessary.

The shape of radials along the leading edge, middle, and trailing edge of all families examined in this study demonstrate a characteristic foil shape as has been hypothesized throughout the literature (Harris, 1936; Alexander, 1965; Ferry and Lauder,

1996; Maia et al., 2012). In general, foils induce less drag at high speeds if the thickness is low, making tapering an effective shape strategy to maximize lift to drag (von Mises,

1945; Webb, 1975; Vogel, 1994;

Weber et al., 2009, 2014). For all families examined, the angle of taper between the leading edge and longest radial was less than or equal to the taper at the trailing edge

(Fig. 5.10C). Shark pectoral fins are shown to produce lift, either during steady swimming or vertical rising, making cross sectional hydrofoil an efficient design (Harris,

1936; Wilga and Lauder, 2000; Fish and Shannahan, 2000). Further, we found a positive relationship between skeletal support and trailing edge taper (Fig. 5.11E). These data suggest that fins with greater skeletal support, mostly Lamniformes, maximize lift to drag ratios, thereby increasing hydrodynamic efficiency. The Sphyrnidae were the only family where leading and trailing edge tapers did not differ substantially, suggesting the fin lacked a true hydrofoil shape. It is hypothesized that the laterally expanded head of

120 Sphyrnids may be the major lift producing foil in these species, and the pectoral fins may not be used for balancing (or imbalacing, during maneuvering) forces (Nakaya, 1995;

Kajiura et al., 2003, Payne et al., 2016).

Conclusion

We found that shark pectoral fin morphology did not vary significantly among 18 species using phylogenetic methods suggesting that there must be mechanical constraints that maintain a certain range of fin shape. Here, we describe a spectrum in both gross and cross-sectional morphology that may confer functional advantages in different habitats.

Taken together, these relationships between gross and cross sectional morphology suggest that fins with extensive skeletal support are designed to resist bending in the dorsoventral plane. At one end of the continuum, Lamniformes appear to have fins designed to maximize hydroxamic efficiency: greater AR and skeletal extent, more radials which are laterally compressed, and a high degree of taper. At the other end,

Carcharhiniformes have fins that may be better designed for complex fin movements: lesser AR and skeletal extent, and fewer dorsoventrally compressed radials. Within

Carcharhiniformes, we observe another gradation between the migratory and nonmigratory species consistent with the above patterns. Further consideration of more species from a greater diversity of families would greatly benefit our understanding of shark ecomorphology.

121 Chapter 5 Figures and Tables

Figure 5.1: Gross anatomy of cartilaginous skeletal elements in shark pectoral fins. Fins generally have a longer, tapered leading edge (anterior) and a trailing edge (posterior) fork that terminates in a lobe. Internally, three basal cartilages (the propterygium, mesopterygium, and metapterygium) articulate at the proximal fin base with the scapulo- coracoid. Three sets of radials (proximal, intermediate, and distal) extend distally from the basals to support the fin web. Thin, flexible ceratotrichia are embedded in the connective tissue that overlays the skeleton and attaches it to the fin. Fins have been categorized based on the extent to which the skeletal elements (radials) extend into the fin web. If less than 50% of the fin web is supported by the skeletal elements, the fin is termed “aplesodic” (A), whereas greater than 50% skeletal support is considered

“plesodic” (B). For both fin types, the leading edge lobe is generally more supported by the radials, and thus more rigid, than the trailing edge lobe.

122

Figure 5.2: Shark fin classification by family (Phylogeny: Velez-Zuazo and Agnarsson,

2011; Classifications: Compagno, 1977; Maisey, 1985; Wilga and Lauder, 2001; Maia et. al., 2012; Sakai, 2011; Crawford, 2014).

123

Figure 5.3: Meristics and morphometrics used to quantify pectoral fin differences among species. A) External fin morphology was measured as the fin area, length, and width at the base and fork. Skin and connective tissue were removed to reveal the skeletal anatomy, comprised of three sets of radials that extend distally into the fin. The leading edge radial, longest radial, and trailing edge radials were further dissected to examine cross sectional morphology (B). In general, radials were more dorso-ventrally compressed on the leading and trailing edges than at the longest radial. C) The radii along the dorso-ventral (NAx; rx) and lateral (NAy; ry) neutral axes were measured to calculate the second moment of area (I) in each axis. Total and calcified areas were measured for each cross section as well.

124

Figure 5.4: Landmarks used for shape analysis. Outline from lateral photograph of the left pectoral fin of Carcharhinus limbatus (fork length 67 cm, fin length 13.8 cm) used in this study. Five putatively homologous landmarks are indicated on the outline of the pectoral fin: 1) proximal insertion of the trailing lobe, 2) distal tip of trailing lobe, 3) inflection of the trailing edge fork, 4) distal fin tip, 5) proximal base of leading edge as described in Fig. 5.3.

125 Figure 5.5: Phylomorphospace of the first two principal components of pectoral fin morphology in Lamniform and Carcharhiniform sharks from Oceanic, Inshore, and

Inshore/Offshore habitats, captured using measures of gross anatomy and the internal skeleton. The morphospace occupied by members of each habitat is indicated by color- coded convex hulls (outlines): purple indicates Oceanic species, bright green indicates

Inshore species, bright blue indicates Inshore/Offshore species. Phylogenetic tips represent the average morphology of each species. The color of the symbols on tips indicate the taxonomic order to which the species belongs: green indicates Lamniformes, blue indicates Carcharhiniformes. The symbols themselves represent the taxonomic family in which the species belongs: green triangles represent Alopiidae, green circles represent Lamnidae, blue triangles represent Carcharhinidae, blue hexagons represent

Cetorhinidae, blue circles represent Sphyrnidae. The shape change described by each PC

126 axis is shown at the extreme ends of each axis. An outline of the shape of the pectoral fin of each species with the most extreme value of each PC axis is indicated at the location of said species in morphospace: PC1+, Carcharhinus isodon; PC1-, Alopias vulpinus; PC2+,

Sphyrna lewini; PC2-, Prionace glauca. For each pectoral fin outline, the extent of skeletal support is shaded in grey.

127 Figure 5.6: Phylomorphospace of the first two principal components of pectoral fin shape in lamniform and Carcharhiniform sharks from Oceanic, Inshore, and Inshore/Offshore habitats, captured using geometric morphometrics. The morphospace occupied by members of each habitat is indicated by color-coded convex hulls (outlines): purple indicates Oceanic species, bright green indicates Inshore species, bright blue indicates

Inshore/Offshore species. Phylogenetic tips represent the average morphology of each species. The color of the symbols on tips indicate the taxonomic order to which the species belongs: green indicates Lamniformes, blue indicates Carcharhiniformes. The symbols themselves represent the taxonomic family in which the species belongs: green triangles represent Alopiidae, green circles represent Lamnidae, blue triangles represent

Carcharhinidae, blue hexagons represent Cetorhinidae, blue circles represent Sphyrnidae.

128 The shape change described by each PC axis is shown at the extreme ends of each axis.

Shape change is represented using an outline sketched from a photograph of a pectoral fin from nasus. The outline is warped here to show the shape change associated with the most extreme positive and negative values of each PC axis observed in the dataset. A

Thin Plate Spline deformation grid is overlaid to illustrate the interpolated shape change between landmark locations.

129

Figure 5.7: Exemplar fin overlays for five shark families demonstrating fin shape and the extent of skeletal support among families. Squatinidae (A) and Alopiidae (B) had the greatest amount of radial support, Lamnidae (C) was intermediate, and Carcharhinidae

(D) and Sphyrnidae (E) had the least amount of radial support.

130

Figure 5.8: Fin classification and shape by species. (A) Aspect ratio varied greatly among orders and there was no real trend among the groups. (B) Five species, all

Carcharhiniformes, had aplesodic fins. All Lamniformes and the one Squatiniformes species had plesodic fins. In the observed species, skeletal extent ranged from 40% -

86%. The majority of the unsupported fin web, particularly in the plesodic fins, was along the trailing edge (see Fig. 5.7). Error bars represent the standard error of the mean.

131

132 Figure 5.9: External and skeletal morphology by family and order. (A) Alopiidae had on average the greatest aspect ratio and (B) skeletal support. On average, all families observed in this study can broadly be classified as having plesodic fins, despite the differences observed by species (see Fig. 5.1). (C) The number of radials was substantially greatest in Squatinidae and least in Carcharhinidae, Sphyrnidae, and

Cetorhinidae. Lamnidae and Alopiidae were intermediate. Error bars represent the standard error of the mean.

133

134 Figure 5.10: Cross-sectional morphology of the fin and radials. (A) For all species, both the leading and trailing edge proximal radials were substantially more dorso-ventrally compressed than the longest proximal radial, but only in the Lamniformes was the longest radial laterally compressed. (B) The amount of calcification was highly varied among radials and families, but in general the Lamniformes had less radial calcification than other groups. (C) For all species except Sphyrnidae, taper angle was greater along the trailing edge. When considering the means of all species, trailing edge taper angle was significantly greater than leading edge taper angle by a factor of two (P = 0.0032).

135 Figure 5.11: Relationship between skeletal extent and cross-sectional morphology. (A)

There was no relationship between skeletal support and aspect ratio. (B) Radial support was positively related to the number of radials (P = 0.0051, R2 = 0.3965). (C)

Calcification of the proximal radial was negatively correlated to skeletal extent for both leading edge and proximal radials (P = 0.0133; R2 = 0.4120; P = 0.0031; R2 = 0.3101, respectively). The proximal longest radial was significantly more calcified than the leading edge radial (F1,90 = 3.9469; P = 0.0420). (D) Proximal radial shape was also

136 negatively correlated to skeletal extent, but only along the longest radial (P = 0.0031; R2

= 0.5033). Further, the proximal leading edge radial was significantly more dorso- ventrally compressed than the longest radial (F1,90 = 3.9469; P < 0.0001) (E) Trailing edge taper is positively correlated with skeletal extent (P = 0.0017; R2 = 0.5453).

137

Figure 5.12: Relationship between skeletal extent and morphology in order

Carcharhiniformes, separated by migratory behavior. Both aspect ratio (A) and trailing edge taper (B) were positively related to skeletal extent (P = 0.0201, R2 = 0.4325; P =

0.0061, R2 = 0.6307).

138 Table 5.1: Sample size, habitat use, ecological designation, and biology of species encompassed in the present study (Compagno, 1984).

Ecological Size at maturity Order n Habitat classification Migratory? (cm) Squatiniformes Squatinidae Squatina Benthic 4 dumeril Bathydemersal N 92 - 107 Carcharhiniformes Carcharhinidae Carcharhinus Reef Inshore 1 acronotus associated N 103 - 137 Carcharhinus Reef Inshore/Offshore 6 limbatus associated Y 120 - 194 Carcharhinus Reef Inshore/Offshore 4 obscurus associated Y 220 - 300 Carcharhinus Inshore 1 isodon Demersal N 150 - 139 Carcharhinus Inshore/Offshore 2 plumbeus Benthopelagic Y 126-183 Carcharhinus Reef Inshore/Offshore 1 falciformis associated Y 202 - 260 Prionace Oceanic 2 glauca Oceanic Y 170 - 221 Inshore 8 terraenovae Demersal N 85-90 Galeocerdo Inshore/Offshore 1 cuvier Benthopelagic Y 210-350 Sphyrnidae Reef Inshore 5 Sphyrna tiburo associated N 80-90 Coastal- Inshore/Offshore 3 Sphyrna lewini oceanic Y 140-273 Sphyrna Coastal- Inshore/Offshore 2 mokarran oceanic Y 210-300 Lamniformes Cetorhinidae Cetorhinus Oceanic 1 maximus Oceanic Y 500-980 Lamnidae 5 Lamna nasus Oceanic Oceanic Y 170-180 Carcharodon Oceanic 4 Oceanic Y 450-500 Oceanic 4 oxyrinchus Oceanic Y 275-285 Alopiidae Alopias Oceanic 5 vulpinus Oceanic Y 226-400

139

CHAPTER 6: SYNTHESIS, FUTURE DIRECTIONS, AND SIGNIFICANCE

Synthesis

The diversity of shark morphology, physiology, behavior, and habitat use make them an ideal study group to examine ecomorphological adaptations and the associated differences in function. Yet, the logistical constraints of studying large bodied, pelagic animals with limited access to samples has greatly limited our understanding of these relationships in sharks. Much of our understanding of the biomechanics of shark swimming comes from studies on benthic species that are easy to keep in aquaria, but are likely not representative of all species (Webb and Keyes, 1982; Kajiura et al., 2003;

Donley et al., 2003; Domenici et al., 2004; Porter et al., 2009; Porter et al., 2011). In situ swimming data exist for one pelagic species, the shortfin mako, Isurus oxyrinchus, and exemplify large variations in morphology and physiology between this open ocean swimmer and a benthic species, the leopard shark, Triakis semifiasciata (Donley and

Shadwick, 2003; Donley et al., 2004). Further, swimming studies are frequently conducted in flumes which have been shown to significantly affect swimming behavior and physiology in comparison to volitional swimming (Lowe, 1996). The few existing volitional swimming studies on sharks are limited by the use of two-dimensional (2D) video (Kajiura et al., 2003; Domenici et al., 2004; Porter et al., 2009; Porter et al., 2011;

Hoffmann et al., 2017). With this study, we describe differences in the functional morphology of volitional swimming between two closely related hammerhead species

(Chapter 2), provide the first quantification and comparison of three-dimensional (3D)

140

kinematics of maneuvering in free swimming sharks (Chapter 3,4), and discuss the ecomorphological differences in control surfaces governed by overarching mechanical constraints on pectoral fin shape in sharks (Chapter 4).

Function and ecomorphology

Body shape affects undulatory kinematics in sharks (Thomson and Simanek,

1977; Webb and Keyes, 1982; Donley and Shadwick, 2004). In this study, we find that in addition to axial morphology, differences in the morphology of accessory structures also correlate with variations in undulatory kinematics (Chapter 2; Hoffmann et al., 2017).

Morphological differences in the laterally expanded head of hammerhead species

(Family: Sphyrnidae) correlate with variations in regional undulatory frequency and amplitude, which we propose may be related to sensory structures located within the cephalofoil. These data suggest that a whole body approach, rather than focusing on primarily axial morphology, should be taken when considering the functional differences in swimming mechanics among species.

To address the literature gap on the functional morphology of shark appendicular structures, we quantified the 3D movement of pectoral fins during routine turning in two species of sharks (Chapters 3, 4; Hoffmann et al., 2019; Hoffmann and Porter, in review).

Previous studies disagree on the role of shark pectoral fins in swimming, which we hypothesize may results from the study of ecomorphologically diverse species (Daniel,

1922; Harris, 1936; Alexander, 1965; Ferry and Lauder, 1996; Wilga and Lauder, 2000,

2001; Fish and Shannahan, 2000; Maia et al., 2012). The long-standing hypothesis that all shark pectoral fins are relatively fixed, rigid hydrofoils that function only to balance forces on the body is previously discounted for at least one species, though this result

141 should not be generalized for all species that display large ranges in whole body morphology, physiology, and ecology (Wilga and Lauder, 2000; Maia et al., 2012). We focused on the articulation of the pectoral fin relative to the body axis since changes in fin conformation were already described and the lack of standardized terminology confounds the understanding of pectoral fin movement throughout the literature as it is sporadically described (Pridmore, 1994; Goto et al., 1999; Wilga and Lauder, 2000,

2001; Kajiura et al., 2003; Domenici et al., 2004; Maia et al., 2012). We demonstrate that pectoral fins for two species move in all three axes of rotation during yaw maneuvering

(Chapters 3, 4; Hoffmann et al., 2019; Hoffmann and Porter, in review). Post mortem muscle stimulation confirms that pectoral fin musculature produces similar rotation of the pectoral fin, suggesting that the movement we quantified during volitional swimming trials is likely under muscular control. Further, we demonstrate the pectoral fin rotation correlates with turning performance for both species studied, demonstrating that pectoral fin movement plays a role in maneuvering.

This study was the first to compare 3D maneuvering kinematics between two morphologically, ecologically and physiologically distinct species. We hypothesize that the Pacific spiny dogfish, Squalus suckleyi, uses drag based yaw maneuvering in comparison to the Bonnethead shark, Sphyrna tiburo, which may instead rely on lift- based turning. Pacific spiny dogfish are demersal species that frequently rest on the benthos, thus drag based maneuvering allows for more precise control over turning and the associated loss of momentum is likely negligible to this slow swimming species

(Compagno, 1984; Fish and Nicastro, 2003). In contrast, bonnethead sharks are a pelagic species that swim continuously therefore maintaining momentum may be more beneficial

142 for minimizing energetic losses during routine maneuvering (Compagno, 1984; Fish and

Nicastro, 2003). This proposed difference highlights the importance in examining functional morphology on a species level basis when considering differences in ecology and physiology before generalizing function among species.

In addition to differences in rotation quantified during yaw maneuvering, we describe a continuum of external and skeletal morphology among shark species (Chapter

5; Hoffmann et al., in prep). Despite variation in shape and skeletal anatomy, pectoral fin anatomy as a whole was not significantly different among species, families, or ecological groups when considering phylogeny. This indicates that there is some mechanical constraint acting on shark pectoral fins, suggesting that fins may generally be serving similar functions among species. We hypothesize that shark fins act as control surfaces among all species, balancing (or unbalancing) forces on the body during swimming and maneuvering (Fish and Lauder, 2017). Species specific differences in external shape, skeletal anatomy (and therefore mechanical behavior), and movement of pectoral fins likely confer advantages in different habitats, but our data suggest that these variations are the product of evolutionary history. For example, all but one species classified as

Oceanic in this study fall within Lamniformes, which are often cited as high performance, pelagic swimmers. The morphological variations observed between orders, families, and species are intrinsically tied to phylogenetic relationships, and we were unable to capture species from a broad range of ecological designations due to the lack of access to specimens. This study provides a comparative insight to the diversity of pectoral fin morphology within sharks but is also limited by the sampling challenges that historically accompany the comparative study of sharks.

143 Volitional swimming kinematics

The overarching goal of this study was to describe the relationship between morphology and function of volitional swimming mechanics in sharks. Previous studies demonstrate that the mechanics and energetics of swimming in flumes differs from free swimming behavior (Lowe, 1996). Flumes apply size, velocity, and behavior constraints on the study animal. For example, the study of yaw maneuvering is precluded by the unidirectional flow and small target volume. The volitional swimming and maneuvering behavior of sharks has been quantified in a number of species, but has been limited by the use of 2D analyses. In this study, we provide the first 3D kinematic analyses of maneuvering in free swimming sharks. We adapted 3D motion capture techniques (Video

Reconstruction of Moving Morphology [VROMM]) for use with fully submerged cameras in a large volume environment (Knorlein et al., 2016; Jimenez et al., 2018). For both studies, the maximum error of point tracking did not exceed 0.3% of the total body length of the observed animal. Additionally, these methods were developed with low- cost, consumer-grade cameras and free, open-source software, making this technique accessible to researchers worldwide. Further development of this technique will increase our ability to study animals and behaviors in natural conditions that were previously not possible.

Future directions

We document differences in morphology and function among species from various ecological designations. As with many shark swimming studies, we were limited by access to study animals from a broad range of habitats and we make two species comparisons (Chapters 2, 4; Wilga and Lauder, 2001; Donley and Shadwick, 2004; Maia

144 and Wilga, 2013, 2016; Hoffmann et al., 2017; Hoffmann and Porter, in review). From two species comparisons, we are unable to make significant inferences about ecomorphological adaptions (Garland and Adolph, 1994). However, we provide baseline knowledge about the 3D actuation of pectoral fins and possible ecomorphological differences among species. Future studies should describe the maneuvering performance of species with various whole body morphology, physiology, and habitat use.

Additionally, we used post mortem muscle stimulation to hypothesize about the role of pectoral fin musculature in actuating the fin. In situ electromyography experiments are needed to confirm pectoral fin muscle activity to determine muscular control over fin rotation. Further, we focused solely on the pectoral fin inside of body curvature during turning due to camera occlusion, and future studies should examine movements of the outside fin, the other paired and caudal fins, and whole body movements for a comprehensive understanding of maneuvering mechanics.

In addition to ecomorphological differences in maneuvering, we quantified external and skeletal fin shape among 18 species. We propose that there is a continuum of skeletal support, rather than two discrete categories as is previously described

(Compagno, 1984, 1988; Maia et al., 2012). We found no significant differences in morphology among ecological designations considering phylogenetic relatedness, but our access to specimens had limited ecological variability within orders and families. Future studies should target species from different ecological designations within phylogenetic groups to explore variations in ecomorphology. Additionally, we had a single benthic representative in this study which greatly overgeneralizes the diversity of species with this ecological designation. Ecomorphological analyses of sharks as a whole are

145 confounded by challenges in accessing samples from a comprehensive representation of species, which is a challenge that accompanied the present study as well. Future studies on the comparative morphology of shark control surfaces will benefit greatly from the use of standardized terminology and analysis methods to build a comprehensive comparative dataset.

Significance and broader impacts

The role of control surfaces in swimming sharks has been confounded by the use of differing methods and study species. In this study, we make direct comparisons of control surface kinematics between two species and propose that functional variations are related to differences in morphology, physiology, and ecology. Further, we propose that there is a mechanical constraint on fin morphology that maintains a degree of similarity in fin shape and function among species. Subtle variations in morphology and function may confer advantages in various habitats, but we propose that shark pectoral fins are generally purposed to balance (and unbalance) forces on the body during swimming and maneuvering, as is well described among other aquatic vertebrates (Fish and Lauder,

2017).

The understanding of shark swimming, especially compared to the vast datasets on actinopterygian swimming, is limited by the challenges associated with studying large bodied, free swimming animals. We adapted a technique to capture 3D kinematics in free swimming sharks for use with low-cost, consumer grade cameras and free, open source software. This technique makes the study of 3D kinematics in large, aquatic vertebrates a new possibility that is accessible to researchers among many disciplines. Additionally, this technique is relatively mobile and, with further development, will facilitate the study

146 of 3D kinematics in wild environments. Ecomorphological analyses are contingent upon our ability to capture animal-environment interactions and increasing our understanding of natural behaviors among as broad a variety of species as possible.

The study of swimming ecomorphology has broad applications among the design of aquatic vessels. The specificity of design among naval vessels and autonomous underwater vehicles (AUVs) that serve as ocean monitoring systems will greatly increase their functionality and efficiency. The United States Department of Defense (DOD) is one of the worlds’ largest consumers of fuel (Schwartz et al., 2012). In fiscal year (FY)

2014, the DOD consumed 87.4 million barrels of fuel, accounting for 80% of U.S. government energy expenditure, amounting to roughly $17 billion dollars. For perspective, second on the list for United States governmental energy usage is the postal service at 4% (Department of Defense 2016 Operational Energy Strategies, 2014).

Furthermore, naval vessels account for 28% of overall petroleum use by the DOD

(Schwartz et al., 2012). The staggering use of petroleum, along with the price tag it carries, makes increasing efficiency one of the top DOD priorities. The 2016 Operational

Energy Strategy identifies the “enhance[ment] of mission effectiveness of the current force through updated equipment” as one of the main objectives. Bio-inspired design is a growing field that combines natural strategies with engineering principles to increase overall efficiency. Studies such as this that consider the functional ecomorphology of structures associated with movement are an ideal starting to point for the inspiration specific, effective, efficient vessel design moving forward.

147

APPENDICES

148 Appendix A: Permission To Reproduce Hoffmann et al., 2017

149

Appendix B: Permission To Reproduce Hoffmann et al., 2019

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