THE ORIGIN and EVOLUTION of SNAKE EYES Dissertation

Home , Alethinophidia, Anilius, Anomochilus, Bipedidae, Caenophidia, Cylindrophis

CONQUERING THE COLD SHUDDER: THE ORIGIN AND EVOLUTION OF SNAKE EYES

Dissertation

Presented in Partial Fulfillment for the Requirements for

the Degree of Doctor of Philosophy in the Graduate

School of The Ohio State University

Christopher L. Caprette, B.S., M.S.

****

The Ohio State University 2005

Dissertation Committee:

Thomas E. Hetherington, Advisor Approved by Jerry F. Downhower

David L. Stetson Advisor The graduate program in Evolution, John W. Wenzel Ecology, and Organismal Biology

ABSTRACT

I investigated the ecological origin and diversity of snakes by examining one complex structure, the eye. First, using light and transmission electron microscopy, I contrasted the anatomy of the eyes of diurnal northern pine snakes and nocturnal brown treesnakes. While brown treesnakes have eyes of similar size for their snout-vent length as northern pine snakes, their lenses are an average of 27% larger (Mann-Whitney U test, p = 0.042). Based upon the differences in the size and position of the lens relative to the retina in these two species, I estimate that the image projected will be smaller and brighter for brown treesnakes. Northern pine snakes have a simplex, all-cone retina, in keeping with a primarily diurnal animal, while brown treesnake retinas have mostly rods with a few, scattered cones. I found microdroplets in the cone ellipsoids of northern pine snakes. In pine snakes, these droplets act as light guides. I also found microdroplets in brown treesnake rods, although these were less densely distributed and their function is unknown. Based upon the density of photoreceptors and neural layers in their retinas, and the predicted image size, brown treesnakes probably have the same visual acuity under nocturnal conditions that northern pine snakes experience under diurnal conditions.

Second, I quantified the orbital area, binocular overlap, eye size, lens size, and the refractive powers of the lens and spectacle within and among colubrid snakes and pit vipers. Among colubrid snakes, the size-adjusted orbital area fit preditions based upon

ecology, with nocturnal arboreal species having the largest orbits (p < 0.001). My results on the distribution of binocular overlap among colubrid snakes, however, contradicted earlier studies. Diurnal arboreal species had the smallest angle of overlap, while terrestrial nocturnal species had the greatest degree of overlap (one-way ANOVA, p <

0.001). Among pit vipers, the eastern cottonmouth had a much greater average orbital area (one-way ANOVA, p < 0.001) for its body size than other species. This species is the only aquatic pit viper, and forages for a wide variety of food, including ectothermic prey under dim light, which may explain its relatively large eyes. Pit vipers had smaller orbital areas than colubrid snakes, but significantly greater binocular overlaps (Mann-

Whitney U test, p < 0.001). The eyeballs of nine species were significantly subspherical, as were the lenses of four species. The lens contributed significantly more to the total refraction (paired t-tests, p < 0.05) than the spectacle in all but two species, the brown treesnake, in which the spectacle had greater refractive power (paired t-tests, p < 0.001), and the northern copperhead, in which the two elements did not differ in their refractive powers.

Snakes evolved from lizards but have dramatically different eyes. These differences are cited widely as compelling evidence that snakes had fossorial and nocturnal ancestors. Snake eyes, however, also exhibit similarities to those of aquatic vertebrates. I used a comparative analysis of ophthalmic data among vertebrate taxa to evaluate alternative hypotheses concerning the ecological origin of the distinctive features of snake eyes. In parsimony and phenetic analyses, eye and orbital characters retrieved groupings more consistent with ecological adaptation rather than accepted phylogenetic relationships. Fossorial lizards and mammals cluster together, whereas

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snakes are widely separated from these taxa and instead cluster with primitively aquatic vertebrates. This indicates that snakes eyes most resemble those of aquatic vertebrates, and suggests that the early evolution of snakes occurred in aquatic environments.

ACKNOWLEDGMENTS

I am grateful to my committee for their patience, understanding, and substantial assistance in revising this document. I am particularly thankful to Tom Hetherington, my advisor, for his continued support well above and beyond any conceivable call of duty.

Jerry Downhower provided frequent sparks to ignite the fire of curiosity about snake eyes. My discussions concerning phylogenetic analyses with John Wenzel were vital to the completion of the most significant chapter of my dissertation, and quite entertaining as well. Dave Stetson’s expertise in light and electron microscopy were invaluable to the completion of two of my dissertation chapters.

The Borror Laboratory of Bioacoustics (BLB) was most generous in “employing” me throughout a number of quarters, despite my dissertation research having no bearing on animal communication whatsoever. I want to thank Abbott Gaunt for bringing me to

OSU in the first place, Sandra L.L. Gaunt for first providing me with an RA at the BLB, and Doug Nelson and Jill Soha for going so far as to letting me manage the digital project during its final year. My experiences at the BLB were some of the best of my graduate career.

Cathy Drake and the EEOB (Zoology) office staff, despite overwhelming responsibilities, remain outstanding examples of efficiency to which every bureaucratic organization should aspire. I am grateful to José Diaz for taking time out of his busy days

to assist me in preparing specimens for TEM and training me in the finer points of said preparation. Kathy Wolken and Brian Kemmenoe of the Campus Imaging and

Microscopy Facility trained me in using the TEM. The staff of the Biological Sciences and Pharmacy Library, under the superb leadership of Bruce Leach, were always helpful, considerate, and reliable.

I thank the California Academy of Sciences for funding my visit to their herpetological collections, their helpful staff, Jens Vindum, Rhonda Lucas, and Ricka

Stoelting, in particular; the National Museum of Natural History for granting me access to their herpetolocical collections, Addison Wynn and Roy McDiarmid, in particular;

John Condit for helping me locate specimens in the OSU Museum; and the USGS BRD for funding the work on brown treesnakes through the former Ohio Cooperative Fish and

Wildlife Research Unit.

People too numerous to count contributed to many discussions, criticisms, and witticisms concerning my dissertation research. They are responsible for much of the good and none of the bad in this document. The late Garth Underwood provided my earliest exposure to snake eyes through his chapter in Biology of Reptilia. Al Savitsky encouraged me in my work and got me thinking seriously about snake eye development.

Brady Porter, Brad Coupe, Nancy Anderson, Chris Shulse, Hitesh Khanna, Earl

Campbell, Robin Taylor, Kurt Pickett, and Karen Hallberg represent some of the more significant friends that contributed to my thinking in this effort. Science Club provided a wealth of relief after each week of frustration, and for that (and the great beer) I am most grateful. Lastly, but certainly not least, I thank my wife, Heather Caprette, for her love and support throughout most of this endeavor.

VITA

1966...... Born

1988...... B.S., Biology, Cleveland State University

1993...... M.S., Biology, Cleveland State University

1999 – present...... Ph.D Candidacy Department of Evolution, Ecology, and Organismal Biology The Ohio State University

PUBLICATIONS

Caprette, C.L., Lee, M.S.Y., Mokany, A., Shine, R.A. and Downhower, J.F. 2004.The origin of snakes (Serpentes) from the perspective of vertebrate eye anatomy. Biological Journal of the Linean Society, 81: 469-482.

Caprette, C.L. and Gates, M.A. 1994. Quantitative analyses of interbreeding in populations of vannus-morphotype Euplotes, with special attention to the nominal species E. vannus and E. crassus. Journal of Eukaryotic Microbiology, 4: 316-324.

FIELDS OF STUDY

Evolution, Ecology and Organismal Biology

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TABLE OF CONTENTS

Page:

Abstract...... ii

Acknowledgments...... v

Vita...... vii

List of Tables...... x

List of Figures...... xi

Chapters:

1. Introduction...... 1

What are snakes?...... 2

The origin of snakes...... 4

The significance of snake eyes...... 9

2. Comparative ophthalmic anatomy of two ecologically distinct colubrid snakes, the

northern pine snake (Pituophis melanoleucus) and the brown treesnake (Boiga

irregularis)...... 20

Materials and methods...... 24

Results...... 25

Discussion...... 28

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3. Geometric optics and visual ecology among snakes...... 44

Materials and Methods...... 46

Results...... 48

Discussion...... 50

4. The origin of snakes (Serpentes) from the perspective of vertebrate eye anatomy.... 70

Materials and Methods...... 73

Results...... 74

Discussion...... 77

Appendix A. Specimens...... 89

Appendix B. Character State Descriptions...... 95

List of References...... 97

LIST OF TABLES

Table Page:

3.1 Mean differences among combined habitat and light categories in sOrA (upper cells) and angle of binocular overlap (lower cells) for colubrid snakes. In each cell, the value indicates the mean for the category in the row heading minus the mean for the category in the column heading. Significant differences are indicated by symbols as follows: * – p < 0.001, † – p = 0.001, ‡ – p = 0.012...... 62

3.2 Correlations between snout-vent length (mm) and refractive power (diopters) for eight species of colubrid snakes and two species of pit vipers. Values are Pearson correlations followed by p-values (two-tailed). Significant correlations are indicated by an asterisk (*)...... 66

4.1 Character by taxon data matrix...... 80

4.2 Pairwise distances comparing Alethinophidia (advanced snakes) and scolecophidia (blindsnakes) to other vertebrate taxa. Distances shown in ascending order (i.e., decreasing similarity) for advanced snakes...... 87

4.3 Medians of ranked pairwise distances from blindsnakes andadvanced snakes for vertebrate taxa grouped by ecology...... 88

LIST OF FIGURES

Figure: Page: 1.1 Skull of Python molurus, illustrating cranial kinesis and major components of the jaw suspension. A) Articulated skull, showing the position of the jaw elements with the mouth closed. B) Skull with the jaws disarticulated. Each element articulates with a moveable joint. C) Articulated skull, showing the position of the jaws during a strike. Abbreviations: art – articular, col – columella, den – dentary, ect – ectopterygoid, fro – frontal, max – maxilla, pal – palatine, par – parietal, prm – premaxilla, por – postorbital, prf – prefrontal, pte – pterygoid, qua – quadrate, sup – supratemporal...... 15

1.2 The distribution of snakes around the world. Black areas represent terrestrial, amphibious, and aquatic species. Grey areas indicate the seasnakes, seakraits, and acrochordid snakes...... 16

1.3 Three possible phylogenies given Vidal and Hedges (2004) findings. See the text for an explanation. (A) The first of two phylogenies that refute a marine origin hypothesis for snakes. (B) Second of two phylogenies that refute a marine origin hypothesis for snakes. (C) The only hypothesis supported by Vidal and Hedges (2004) data...... 17

1.4 Photoreceptors of a basal reptile, the red-eared slider, Trachemys scripta. (A) A single cone, (B) a double cone, and (C) a rod. Abbreviations: el – ellipsoid, fp – foot piece, lim – limitans, my – myoid, nu – nucleus, od – oil droplet, os – outer segment, pa – paraboloid. The arrow indicates the direction of light as it passes through the retina...... 18

1.5 Representative morphology of snake photoreceptors: (A) double cone, (B) large single cone, (C) small single cone, (D) single cone with a long myoid, (E) small single cone with an extremely long myoid, (F) double cone with an extremely long myoid, (G) long rod, (H) short rod. Abbreviations: el – ellipsoid, fp – foot plate, lim – limitans, my – myoid, nu – nucleus, os – outer segment. The arrow indicates the direction of light as it passes through the retina...... 19

2.1 (A) Light micrograph of a radial section (methacrylate 10 µm, toluidine blue, 100x) through the anterior portion of the eye of a pine snake, Pituophis melanoleucus. Bar = 100 µm. (B) Radial section (methacrylate10 µm, toluidine blue, 1000x) through the ciliary body of a brown treesnake, Boiga irregularis. Bar = 1 µm.(C) Circumferential section (methacrylate10 µm, toluidine blue, 400x) through the ciliary body and iris of a brown treesnake. Bar = 5 µm. Abbreviations: co – cornea, cb – ciliary body, cs – scleral-venous sinus (canal of Schlemm), ir – iris, ma – muscle of accommodation, re – retina, sc – sclera...... 35

2.2 (A) Retina of a pine snake, Pituophis melanoleucus, with a large single cones visible at the center and a small single cone to its left (methacrylate 12-µm section, toluidine blue, 1000x). Bar = 10 µm. (B) Thin section (Spurr resin 1- µm section, methylene blue, 1000x) showing a double cone. Bar = 10 µm. Abbreviations: ael – accessory cell ellipsoid, anu – accessory cell nucleus, aos – accessory cell outer segment, el – ellipsoid layer, lim – limitans, lsc – large single cone, onu – outer nuclear layer, os – outer segment layer, pel – principle cell ellipsoid, pnu – principle cell nucleus, pos – principle cell outer segment, ssc – small single cone. The arrow indicates the direction of light as it passes through the retina...... 36

2.3 Radial sections through the retina of a Brown treesnake, Boiga irregularis. (A) A 10-µm section (methacrylate, toluidine blue, 400x) showing the densely packed rods with long, thin outer segments, and two cones, whose large ellipsoids are at the base of the rod outer segment layer. (B) A 1- µm section (Spurr resin, methylene blue, 400x) showing the rod nuclei, ellipsoids, outer segments, and a single, large cone, with a large ellipsoid and long, tapering outer segment. Bars = 10 µm. Abbreviations: cel – cone ellipsoid, co – cone, cos – cone outer segment, lim – limitans, nu – nucleus, os – outer segment layer, pe – pigmented epithelium. The arrow indicates the direction of light as it passes through the retina...... 37

2.4 Radial retinal sections of (A) pine snake, Pituophis melanoleucus and (B) brown treesnake, Boiga irregularis. Both sections: Spurr resin, 1-µm section, methylene blue, 400x, Bars = 20 µm. Abbreviations: gcl – ganglion cell layer, inl – inner nuclear layer, onl – outer nuclear layer, rel – rod ellipsoids, ros – rod outer segments, rv – retinal vessel. The arrow indicates the direction of light as it passes through the retina...... 38

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2.5 Tangential section (methacrylate 12 µm, toluidine blue, 400x) through the ellipsoid region of the retina of a pine snake, Pituophis melanoleucus. This section passes through the base of the outer segments in the upper half and through the middle of the ellipsoids in the lower half. Light transmitted through the retina funnels through the periphery of the ellipsoids (el) of large and small single cones, and the principle members of double cones, and is focused at the base of the outer segments (os). The accessory cells (ac) of double cones also are indicated. Bar = 50 µm...... 39

2.6 Electron micrographs (6300x) of a large single cone from a pine snake, Pituophis melanoleucus. A. Outersegment (os) and ellipsoid (el) containing microdroplets. B. Enlargment of the ellipsoid in (A), providing a closer look at the microdroplets. C. Distal portion of the ellipsoid of the principle member of a double cone, showing the mitochondria (mt) and microdroplets. Bar = 1 µm.. 40

2.7 Transmission electron micrographs of brown treesnake, Boiga irregularis, retinas showing characteristics of the rods. (A) Three rods showing irregular mitochondria in their ellipsoids. Bar = 5 µm. (B) A section of a rod outer segment showing membranous disks within and independent of the plasma membrane. Bar = 2 µm. (C) The distal ends of rod outer segments are adjacent to, but not embedded in the pigmented epithelium. Bar = 2 µm...... 41

2.8 (A) Transmission electron micrograph of a brown treesnake, Boiga irregularis, rod at the junction of the inner and outer segments. Bar = 2 µm. (B) An enlargement of the rod ellipsoid between the mitochondria, showing small droplets. Bar = 500 nm...... 42

2.9 Comparison of refractive powers of the spectacle and lens of brown treesnakes (BTS) and northern pine snakes (NPS). The p-values are based upon paired t- tests, 1-tailed...... 43

3.1 A scatterplot of the total refractive power as a function of the radius of the eyeball for three species of snakes from a study done by Sivak (1977). The fit line was graphed for the combined data and has a Pearson correlation of -0.97. 56

3.2 Measurements obtained from snake heads used to calculate the angle of binocular overlap in the frontal plane, illustrated using the head of an urutu, Bothrops alternatus, a South American pit viper Abbreviations: HL – head length, NW – width between the nares, ORH – height of the orbit, ORV – width of the orbit, SBW – snout base width, SL – snout length, θ – the angle of binocular overlap...... 57

3.3 Plot of the size-adjusted OrA (mm) for colubrid snakes (A) and pit vipers (B), grouped by typical habitat. Filled boxes are means. Whiskers are one standard error of the mean in length...... 58

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3.4 Plot of the size-adjusted OrA (mm) for colubrid snakes, grouped by typical light environment. Filled boxes are means. Whiskers are one standard error of the mean in length...... 59

3.5 Plots of the angle of binocular overlap (degrees) for colubrid snakes (A) and crotalid snakes (B), grouped by typical habitat. Boxes are means. Whiskers are one standard error of the mean in length...... 60

3.6 Plot of the angle of binocular overlap (degrees) in colubrid snakes, grouped by light environment. Boxes are means. Whiskers are one standard error of the mean in length...... 61

3.7 Comparison of the size-adjusted orbital area and binocular overlap among ecological groups of snakes, based upon the data in Table 3.1...... 63

3.8 Boxplots of comparing axial diameters (clear boxes) and equatorial diameters (filled boxes) of the eyeballs (A) and lenses (B) for eight species of colubrid snakes and two pit viper species. P-values are based upon paired t-tests. Abbreviations: Ag_co – eastern cottonmouth, Ag_pi – northern copperhead, Bo_ir – brown treesnake, Co_co – northern black racer, El_ob – black rat snake, He_pl – eastern hognose snake, La_tr – eastern milk snake, Ne_si – northern water snake, Op_ae – rough green snake, Th_si – eastern garter snake...... 64

3.9 Boxplots of refractive powers (diopters) of the spectacles (open boxes) and lenses (closed boxes) for eight species of colubrid snakes and two species of pit vipers. P-values are based on paired t-tests. Abbreviations: Ag_co – eastern cottonmouth, Ag_pi – northern copperhead, Bo_ir – brown treesnake, Co_co – northern black racer, El_ob – black rat snake, He_pl – eastern hognose snake, La_tr – eastern milk snake, Ne_si – northern water snake, Op_ae – rough green snake, Th_si – eastern garter snake...... 65

3.10 Scatter plot of axial diameters of the lenses and eyes of ten species of colubroid snakes, sorted by the typical light level during which those species are active. The solid black circles represent the only nocturnal species in the sample, brown treesnakes. The fit lines have similar slopes but the intercept is greater for brown treesnakes...... 67

3.11 Scatterplot of lens size and body size for nine colubrid species, grouped by light environment. The solid black circles represent the only nocturnal species in the sample, brown treesnakes...... 68

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3.12 Illustration of the paths of light originating from infinity and refracted through the eye of a nocturnal snake. The large pupil allows more light but requires a large lens to focus the image on the retina (thick gray lines). A diurnal snake’s lens (dotted border) is too small. Consequently, the same rays (thin black lines) fail to pass through the lens and focus well behind the retina. For such an animal only the central rays would be in focus...... 69

4.1 Functional anatomy of lizard (A) and snake (B) eyes, illustrating major differences between the two general types. C. Lizards focus by contracting large ciliary muscles ( bm, cm) anchored to scleral ossicles (so) thereby applying pressure to the lateral surface of the lens (ln) via the annular pad (ap). D. Snakes focus by moving their lens forward via increased pressure on the vitreous (vi) due to peripheral iris muscle (im) contraction. Abbreviations: an, anterior pad; bm, Brücke’s ciliary muscle; cb, ciliary body; ch, choroid; cm, Crompton’s ciliary muscle; cn, conus papilliaris; co, cornea; el, eye lid; fv, fovea; id, iris dilator muscle; is, iris sphincter muscle; ln, lens; re, retina; sc, scleral cartilage; sl, sclera; sp, spectacle; vi, vitreous; zf, zonular fibers...... 79

4.2 (A) The strict consensus of 12 most parsimonious trees from the matrix of 69 ophthalmic and orbital characters, each with length = 295, consistency index = 0.35, retention index = 0.77, shows how ophthalmic characters reflect common ecology, rather than common ancestry, among vertebrates. (B) A traditional vertebrate phylogeny synthesized from other studies and data (Sibley & Ahlquist, 1990; Helfman et al., 1997; Lee, 2000; Zug et al., 2001; Murphy et al., 2001; Scally et al., 2001): length = 405, consistency index = 0.25, retention index = 0.64. In both trees, the two groups of snakes are at the top. Gray boxes show the distribution and relationships within the lepidosaur clade for both trees. Numbers above each branch length indicate that length for optimised ophthalmic and orbital characters. Branch lengths of zero indicate nodes unsupported by ophthalmic characters...... 86

CHAPTER 1

INTRODUCTION.

The eye to this day gives me a cold shudder, but when I think of the fine known gradations, my reason tells me I ought to conquer the cold shudder (Charles Darwin, in a letter to Asa Gray, February 1860).

Darwin wrote the passage quoted above reflecting his misgivings about the ability of natural selection to explain the evolution of complex structures such as the eye. The

“fine known gradations” to which he refers are the variations in the eye present within and among species of living animals. Those variations are the fodder for natural selection, which removes those variants that fail to provide their possessors with an advantage over others in their population. Among extant animals, there is a continuum of visual complexity from the simplest patch of light sensitive skin to large, image-forming eyes with the ability to accommodate for distance and adjust the aperature for changing brightness. Within every population of eyed animals, there is variation in the quality of every measureable parameter of their eyes. Combined, the variation within and among species convinced Darwin that natural selection could explain the evolution of an image- forming eye from a light-sensitive skin patch. In this dissertation, I explore some of the variation in the eyes of one group of animals, snakes.

Snakes rely primarily on chemical and tactile cues to detect and interact with conspecifics (Ford & Burghardt, 1993). Snakes also use chemical and tactile information, as well as visual cues when foraging (Chiszar, et al., 1981; Burghardt, 1992; Schwenk,

1993; Greene, 1997). Yet, all but the most active, diurnal species of snakes are thought of as having poor eyesight (Walls, 1942; Greene, 1997; Pough et al., 1998; Zug et al., 2001).

Most lizard species, on the other hand, use primarily visual and sometimes acoustic signals to mediate social behavior and to detect prey and predators. Snakes are derived from lizards, however, and the striking differences between snake eyes and lizard eyes led Walls (1940) to propose that snakes experienced strong selection against maintaining good vision. According to Walls’ hypothesis, snakes originated from burrowing lizards with reduced eyes and evolved their novel ophthalmic anatomy with their radiation into surface habitats. That view of snake eyes, and the subordination of vision to olfaction and vibration has held sway over herpetologists’ perception of snakes ever since (Underwood,

1970; Ford & Burghardt, 1993; Greene, 1997; Zug et al., 2001). Yet vision is important to snakes, both in intraspecific (Carpenter, 1984; Ford & Burghardt, 1993) and interspecific behavior (Drummond, 1985; Greene, 1988; Herzog & Bern, 1992). Those apparent contradictions led me to investigate the evolution of snake eyes.

WHAT ARE SNAKES?

While snakes are best known for their limbless bodies, forked tongues, and unblinking eyes, there are other squamate reptiles that share those features but are only distantly related to snakes. The key features that distinguish snakes from other squamates are modifications of the skull and the vertebral column (Carroll, 1988; Lee & Scanlon,

2002). All snakes have between approximately 100 and 500 precaudal vertebrae, each of 2

which bears a pair a ribs. Snake vertebrae also have a unique set of posterior and anterior articular surfaces dorsal to the typical vertebrate zygopophyses (Carroll, 1988). In snakes, the number and articulation of their vertebrae, and their complex trunk musculature permit a remarkable degree of trunk mobility. Similarly, the skull of most snakes is extremely flexible (Fig. 1.1). In those snake species in which the skull is rigid, that rigidity is clearly derived from a more mobile ancestral state (Gans, 1961; Carroll, 1988;

Rieppel, 1988; Lee, 1998). The flexibility of snake’s skulls enables most species to consume very large prey. They can do this because individual elements of both their upper and lower jaws articulate via elastic ligaments, allowing independent mobility of each bone. In the mandible, the articular and dentary are connected loosely allowing the lower jaw to bend. Also, the distal ends of right and left dentaries are joined by a flexible ligament. Similarly, in the upper jaw the maxilla, ectopterygoid, and pterygoid each move independently, allowing them to spread. The pterygoid and articular, in turn, each connect to the distal end of the quadrate, itself articulated with the supratemporal, completing the jaw suspension. In Asian, African, and American elapids, the prefrontal is similarly mobile (Deufel & Cundall, 2003). The length of each of those elements, and particularly the supratemporal and the quadrate, determine the maximum gape of the animal (Gans, 1961). In addition to permitting an extraordinarily wide gape, that complicated set of joints allows the braincase to float above the oral cavity as the snake transports food into the esophagus. While cranial kinesis is present to some degree in many lizards (Herrel et al., 2000), in no family is it as extensive as it is in snakes.

There are approximately 2800 described species of snakes. They are found on every continent except Antarctica (Fig. 1.2). They reside in forests, grasslands and deserts

from the tropics to the arctic circle, from lowlands below sea level to high alpine slopes, and in both freshwater and marine habitats. Some snake species burrow in soil, while others dwell on its surface, still others climb trees (Greene, 1997; Zug et al., 2001), and among those some can glide (Socha & O'Dempsey, 2001). Snakes consume prey ranging in size from arthropod eggs (Webb & Shine, 1993; Webb et al., 2000; Torres et al., 2000) to mammals weighing up to 30 kg (Greene, 1997). The smallest adult snakes have a body length of a few centimeters and a mass of only a few grams, whereas the largest are nearly 10 m in length and have mass of more than 100 kg. The smallest snake species, the brahminy blindsnake, Ramphotyphlops bramina, is a parthenogenic egg-layer

(Kamosawa & Ota, 1996). One of the largest species, the anaconda, Eunectes murinus is sexual and gives birth to free-living offspring (Zug et al., 2001).

Although many lizards show no external sign of limbs, all lizard species have at least the vestiges of pectoral and pelvic girdles, except dibamids and some amphisbaenians. No snake has any remnants of the pectoral girdle, and only a few bear pelvic rudiments. Nonetheless, many lizards resemble snakes. The California legless lizard, Anniella pulchra (Anniellidae), for example, lacks external limbs and burrows in sand. In the southeastern USA, the eastern glass lizard, Ophisaurus ventralis (Anguidae), also is limbless, but it moves through dense vegetation on the surface of the ground.

Other limbless lizards include Calyptommatus (Gymnophthalmidae), pygopod gekkos

(Pygopodidae), wormlizards (Amphisbaenidae), blindskinks (Dibamidae), acontine and feyliniine skinks (Scincidae), and mole lizards (Bipedidae) (Zug et al., 2001). All of these taxa lack the synapomorphies that define snakes.

Snakes are derived from lizards, but have distinct vertebral characteristics. Those vertebral synapomorphies and extreme cranial kinesis distinguish snakes from other limbless lizards. It is those differences, however, and the absence of most anatomical features typical of lizards, that have hampered attempts to resolve the position of snakes within squamates (Coates & Ruta, 2000). Consequently, understanding the selective conditions that resulted in the origin of snakes is similarly difficult.

THE ORIGIN OF SNAKES

Nearly every herpetologist, and many biologists outside of herpetology, have an opinion on how snakes evolved from ancestral quadrupedal lizards. Notable reviews of snake origins include those of Bellairs and Underwood (1951), McDowell (1972),

Rieppel (1988), and Zaher (2000). Perhaps this question is fascinating because it requires we explain the loss of the key feature that characterizes most terrestrial vertebrates.

Snakes are tetrapods, but most are limbless. Despite lacking limbs, snakes and tetrapods share some complex features, an amnion for example, leaving no doubt that snakes are tetrapods.

Many systematic studies using morphological data agree that varanid lizards (the monitors) are the nearest living relatives to snakes (McDowell, 1972; McDowell, 1987;

Rieppel, 1988; Lee, 1997; Zaher & Rieppel, 2000; Lee, 2000), while others present evidence for a close relationship between snakes and amphisbaenians (Zaher & Rieppel,

2000; Rieppel & Zaher, 2001; Kearney, 2003). The crux of the debate regarding the origin of snakes, however, is not which taxa are snakes’ nearest relatives. Rather, the controversy focuses on the habitats occupied by snake ancestors and the environmental forces that selected for the unique features of snakes. Central to that debate are the 5

positions of the fossil taxa Pachyrachis (Caldwell & Lee, 1997), Pachyophis (Lee et al.,

1999), Podophis (Rage & Escuillie 2000), Wonambi (Scanlon & Lee, 2000), and

Haasiophis (Tchernov et al., 2000). On the one hand, those extinct taxa possessed hind limbs and occupied marine environments, thus appearing to be good marine intermediates between modern varanids and snakes. But Pachyrachis and those other fossil snakes also share many skull features in common with advanced snakes, particularly those with the greatest degree of cranial kinesis (macrostomatan snakes). Different interpretations of morphological character data and subsequent analyses resulted in different conclusions regarding the position of those fossil taxa, either as basal snakes (Caldwell & Lee, 1997;

Lee & Caldwell, 1998; Lee et al., 1999; Lee, 2000; Lee & Caldwell, 2000; Scanlon &

Lee, 2000; Lee & Scanlon, 2002), or as early invasions of the marine environment by advanced, macrostomatan snakes (Zaher & Rieppel, 1999; Greene & Cundall, 2000;

Rieppel & Zaher, 2000; Zaher & Rieppel, 2000; Rieppel & Zaher, 2001; Zaher &

Rieppel, 2002; Rieppel et al., 2003).

Taking a different approach, Greene (1997) suggested that several snake species form a ecomorphological series, or grade, of intermediates between the fossorial blindsnakes and macrostomatan snakes, based mainly upon characteristics of jaw suspension and prey types. Those putative intermediates include Anomochilus (dwarf pipesnakes), Anilius (South American pipesnakes), Cylindrophis (Asian pipesnakes),

Uropeltis (shield-tailed snakes), Loxocemus (neotropical sunbeam snake), and Xenopeltis

(Asian sunbeam snake), with boas, pythons, and their relatives as the basal groups of the macrostomatan clade. Greene and Cundall (2000) compared the feeding ecology of blindsnakes to that of most squamates and found them generally similar in that most

lizards consume many, small prey, mostly arthropods, as do blindsnakes, and reiterated

Greene’s (1997) grade hypothesis. That grade hypothesis, combined with the conclusions of Tchernov et al.(2000), led Greene and Cundall (2000) to support the terrestrial origin hypothesis. While blind snakes do share the characteristic of feeding on many, small prey with lizards, blindsnakes tend to specialize on the larvae of social insects, particularly ants and termites, and their feeding mechanics are derived, unique to the scolecophidia, rather than convergent with other squamates (Thomas, 1985; Kley, 1998; Kley &

Brainerd, 1999; Torres et al., 2000; Kley, 2001). In fact, blindsnake cranial anatomy appears to be derived from ancestors with a morphology similar to that of alethinophidian snakes rather than to that of lizards (Gans, 1961; Carroll, 1988; Zug et al., 2001; Kley,

2001), which supports a hypothesis of a common ancestor between scolecophidia and alethinophidia that had an alethinophidian jaw suspension, rather than a lizard-like mechanism.

Contrary to those morphological and ecological studies, (Vidal & Hedges, 2004), placed snakes as sister to the Iguania (a clade containing iguanas, chameleons, and agamids), based upon Bayesian analysis of two nuclear gene sequences, oocyte maturation factor (C-mos) and recombination-activating gene 1 (RAG1). Vidal and

Hedges claimed that their Bayesian analysis refuted the marine origin hypothesis for snakes, because the clade to which snakes are most closely related consists of primarily terrestrial groups. Yet, the marine origin hypothesis to which Vidal and Hedges refer was based primarily upon the phylogenetic position of fossil taxa (Caldwell & Lee, 1997; Lee et al., 1999; Scanlon & Lee, 2000; Rage & Escuillie, 2000), for which molecular evidence is unavailable. The position of those fossil taxa is in dispute, to be sure

(Rieppel, 1988; Lee, 1997; Rieppel & Zaher, 2000; Tchernov et al., 2000; Zaher &

Rieppel, 2000; Lee et al., 2000; Lee, 2000; Scanlon & Lee, 2000; Zaher & Rieppel, 2002;

Rieppel & Kearney, 2002; Rieppel et al., 2003; Kearney, 2003), but their position can not be determined using molecular data without also using morphology. Hedges (Hedges &

Maxson, 1996), however, opposes the “total evidence” approach to phylogenetic systematics. Instead, he prefers to assign morphological, behavioral, and ecological character states to taxa after generating a phylogeny based exclusively upon molecular data. One serious flaw with that approach, however, is that all fossil taxa are excluded from such an analysis.

The data presented in Vidal and Hedges (2004), by the most generous interpretation, merely indicate that varanid lizards and snakes are not sister taxa. By removing varanid lizards, and mosasaurs by association, from the sister position to snakes, Vidal and Hedges (2004) believe that they have refuted the marine origin hypothesis for snakes. In my opinion, the nodes of interest are those that indicate the positions of the pachyophiid and madstoiid snakes, not varanids and mosasaurs. To refute the marine origin hypothesis, Vidal and Hedges would have to demonstrate that pachyophiid and madstoiid snakes either form a monophyletic group within a clade of terrestrial snakes (Fig 1.3A), or that they represent at least two separate groups, each separated from the other by at least one node representing a clade of terrestrial species

(Fig. 1.3B). None of the studies of those fossil snakes determined the monophyly either of pachyophiid or madstoiid snakes. Thus, while they did produce an interesting rearrangement of squamate taxa, Vidal and Hedges provided no evidence relevant to the ecological origin of snakes (Fig. 1.3C). Moreover, their Bayesian consensus tree showed

little support for the relationship between snakes and any other squamate group. Indeed, in their own summary tree, snakes form a polytomy with other squamates. Thus, the conflict among those differing phylogenies remains unresolved.

Another molecular phylogeny (Vidal & David, 2004), incorporating a subset of the same data as Vidal and Hedges (2004), but with fewer taxa, found that species with macrostomatan gape characteristics were distributed throughout their consensus tree. This is significant because Tchernov et al. (2000) claimed that fossil marine snakes with legs were advanced snakes, based upon their macrostomatan traits. This led them to hypothesize that the fossil marine snakes had re-elaborated hind limbs, perhaps via activation of dormant genetic pathways. Vidal and David’s (2004) phylogeny placed some macrostomatan snakes at the basal end of the tree, rather than in apical groups.

They concluded, therefore, that there is no need to evoke a secondary evolution of hind limbs to explain their presence in fossil taxa, which significantly weakens the scenerio proposed by Tchernov et al. (2000). In doing so, Vidal and David (2004) eliminated the chief evidence against a marine origin, yet they reiterate “that a marine origin of snakes can be safely ruled out,” erroneously based upon the findings of Vidal and Hedges

(2004).

THE SIGNIFICANCE OF SNAKE EYES

[T]he ophidian eye exhibits not one solitary structural feature which would enable a comparative ophthalmologist, handed a microscopic preparation of it, to place its owner in the Sauropsida at all (Walls, 1940).

Image-forming eyes evolved independently in a minimum of 40 different animal lineages (Salvini-Plawen & Mayr, 1977), although the developmental pathways of all

metazoan eyes may share many of the same genetic components (Gehring & Ikeo, 1999).

Nilsson and Pelger (1994) used a computer simulation to demonstrate that an image- forming eye, similar to the vertebrate lens-bearing eye, could evolve from a single photoreceptive integumentary cell in a little less than 400,000 generations. For many animals, that equates to between one-half million and one million years. While many vertebrates have reduced eyes, there is substantial variation in the structure of vertebrate eyes. That great variation implies that eyes are extremely important to the survival and reproduction of individual vertebrates. Thus, any substantial differences in ophthalmic structures between closely related taxa require an extrordinary explanation.

Walls (1940, 1942) argued for a burrowing origin of snakes based upon the differences between their eyes and those of other squamate reptiles. Walls proposed that snake eyes degenerated in response to an ancestry in a low-light environment, specifically underground, followed by a period of nocturnality on the terrestrial surface.

Underwood (1970) supported a modification of Walls’ (1940) hypothesis, in which the low-light environment was not specified, but nocturnality seemed likely. Both attributed the current morphological state of snake eyes to the evolution of functional novelties from a simpler condition than that even of extant basal reptiles.

The eyes of turtles, tuataras, most lizards, and most birds all share a suite of ophthalmic features, including: cartilaginous and/or bony elements embedded in the sclera, a vascular protrusion of tissue, the conus papiliaris, into the body of the vitreous, an equatorial thickening of the lens epithelium, the annular pad or ringwülst, and a number of photoreceptor organelles, including large oil droplets in their distal inner segments, paraboloids, and contractile myoids (Fig 1.4). Both the paraboloids and oil

droplets may act as refractile elements, funnelling light to the photoreceptor outer segements. Crocodilians lack scleral ossicles, have a reduced annular pad, no conus papiliaris, and their cone oil droplets, when present, are colorless. Crocodilians, like many turtles, are amphibious, but also mostly active at night. Walls (1942) interpreted the differences between crocodilian eyes and those of other reptiles as a result of that nocturnal, amphibious lifestyle.

Snake eyes resemble the eyes of eutherian mammals more than those of other reptiles. Snakes and eutherian mammals both have retinal blood vessels and a collagenous sclera lacking skeletal elements. They both also lack paraboloids, myoid contractility, and large oil droplets. Moreover, diurnal species of both snakes and eutherian mammals evolved yellow lens pigments (Walls, 1942). Most researchers agree that eutherian mammals arose from nocturnal ancestors. Walls attributed those evolutionary losses of photoreceptor oil droplets and scleral skeletal features to that nocturnal ancestry. Only the ciliary muscles, present in the eyes of many mammals, are lacking in snake eyes. Those mammals that lack ciliary muscles include insectivores, nocturnal rodents, baleen whales, and sirenians. But Walls (1940, 1942) attributed the differences between snake eyes and those of lizards to a burrowing, rather than a nocturnal ancestry.

Central to his burrowing origin hypothesis was Walls’ (1940) contention that the iris muscles of modern snakes were evolutionarily derived from remnants of a reduced ciliary muscle. During development, typical squamate iris muscles derive from the same neurectodermal precursors that give rise to the iris pigment epithelium and stroma, while the ciliary muscles condense in the surrounding mesenchyme, presumeably from

mesodermal tissue (Underwood, 1970). Walls (1942) reported that the iris muscles of snakes are derived from mesoderm, rather than ectoderm as in lizards, but it is unclear what observations prompted that assertion. The only methods available prior to Walls’ investigation, and indeed for many years after were those of classical embryology, which are unable to determine the stem cells from which the intraocular muscles derive. New methods have uncovered the genetic regulation of eye development in several model organisms (Chow & Lang, 2001; Oakley, 2003), and these discoveries provide the means by which to investigate the development of snake eyes.

Birds’ eyes are derived from the same ancestral precursors as lizard, turtle, and crocodilian eyes, and appear much the same as in those other taxa. Two recent studies

(Volpe et al., 1993; Barrio-Asensio et al., 1999) on the development of the iris muscles in chickens may explain Walls’ (1942) observations in snakes. Chicken iris muscles are composed of both smooth muscle and striated muscle, but at different times during development. Cells expressing smooth muscle myosin heavy chain protein first appear at the pupillary margin of the iris during development, and later at the ciliary margin. The same cells then express striated muscle proteins, beginning at the ciliary margin and ending at the pupillary margin, where smooth muscle cells may remain in adults(Volpe et al., 1993). Both that transdifferentiation and a similar process in the ciliary muscle of chicks are initiated by the inhibition of a transcription factor, activin, by a second transcription factor, follistatin (Link & Nishi, 1998). Moreover, skeletal muscle in in both the iris and ciliary body have multiple developmental precursors, including the epithelium of the optic cup, neural crest, and mesenchyme (Barrio-Asensio et al., 1999;

Barrio-Asensio et al., 2002). Perhaps Walls (1942) misinterpreted the pattern of

differentiation of snake iris muscles as the iris coopting evolutionary remnants of the ciliary muscle.

Snakes provide an interesting model for studying the genetic regulation of eye development because they differ strikingly from their lizard relatives. But, snakes have yet to be evaluated for the expression of regulatory genes discovered in other animals. In particular, the absence of numerous features typical of other reptiles from the eyes of snakes may be explained by changes in the expression, or timing thereof, of regulatory genes. It is intriguing that some of the evolutionary losses of features from the eyes of snakes are shared with placental mammals, such as the scleral skeletal elements, photoreceptor mobility, paraboloids, and large oil droplets. Those shared deficiencies suggest that snake eye development may provide direct insight to the evolution of mammalian eyes.

Retinal patterns were an important source of data for Underwood’s (1967) classification of snakes. Indeed, nearly every combination of visual cells found in other vertebrates are also found in snakes (Walls, 1942). Fig. 1.5 is a partial survey of visual cells among snakes. There are three main types of photoreceptor among snakes: single cones, double cones, and rods. Each of these three types, in turn, varies in shape and size among species. The scolecophidian snakes, Typhlops and Leptotyphlops have only small rods (Fig. 1.5 H). Viperid snakes, possess all three types of photoreceptor, in four morphologically distinct classes: large and small single cones, double cones, and small rods, all of which have short myoids, such that the ellipsoids are adjacent to the limitans

(Fig. 1.5 A, B, C, and H). Underwood (1970) reported that this pattern is typical for many alethinophidian snakes. Some of the exceptions include many diurnal colubrids, which do

not have rods, crotaline snakes, which do not have small single cones, boas and pythons, which have no double cones (Sillman et al., 1999; Sillman et al., 2001), and some nocturnal colubrids, which have neither double cones nor small single cones (Rasmussen

1990). The cones of nocturnal snaile-eating snake (Sibon) have long slender myoids (Fig.

1-5 E and F), and the single cones of cat and herald snakes (Boiga, Telescopus, and

Crotaphopeltis) have myoids (Fig. 1-5 D) that are intermediate between the viperid morphology and that of Sibon (Underwood, 1970; Rasmussen, 1990).

The apparent contradiction between the function of vision in the natural history of snakes and the perception of snakes as having poor eyesight deserves investigation. That perception, for the most part, is based upon Walls’ (1942) comparative study of vertebrate eye morphology. Yet, when Walls examined snake eyes, he restricted his comparisons to within squamate reptiles and did not include other vertebrate groups, despite having considerable data at hand. It is interesting that many of the features absent from snake eyes also are absent from the eyes of eutherian mammals. In mammals, the evolutionary losses of scleral skeletal elements, photoreceptor oil droplets, myoid contractility, and paraboloids are all attributed to nocturnal ancestry. But, their absence in snakes is attributed to burrowing ancestry. Two other peculiarities of snake eyes are the method of focussing by moving a rigid, spherical lens toward the cornea, and the fusion of their eyelids to form a transparent covering, the spectacle or brille. Spectacles are present in some nocturnal squamates, as well as some burrowing species, but also are common among primitively aquatic animals. While many tetrapods, including most nocturnal and burrowing squamates deform their soft, flattened lens to focus, primitively and secondarily aquatic vertebrates focus by movement of a spherical lens. This

mechanism is necessary because of the loss of corneal refractive power underwater. A spherical lens compensates for the loss of corneal refraction, but deforming it along its optic axis distorts the image, particularly at its edges (Sivak, 1975). Those similarities justify the evaluation of alternative evolutionary explanations for the unique combination of features that comprise snake eyes.

Figure 1.1. Skull of Python molurus, illustrating cranial kinesis and major components of the jaw suspension. A) Articulated skull, showing the position of the jaw elements with the mouth closed. B) Skull with the jaws disarticulated. Each element articulates with a moveable joint. C) Articulated skull, showing the position of the jaws during a strike. Abbreviations: art – articular, col – columella, den – dentary, ect – ectopterygoid, fro – frontal, max – maxilla, pal – palatine, par – parietal, prm – premaxilla, por – postorbital, prf – prefrontal, pte – pterygoid, qua – quadrate, sup – supratemporal.

Figure 1.2. The distribution of snakes around the world. Yellow areas represent terrestrial, amphibious, and aquatic species. Red areas indicate the seasnakes, seakraits, and acrochordid snakes.

Scolecophidia Scolecophidia Terrestrial snakes A Pachyophiid snakes Madtsoiid snakes Terrestrial snakes B Scolecophidia Terrestrial snakes A Pachyophiid snakes Madtsoiid snakes Terrestrial snakes B Scolecophidia Terrestrial snakes A Pachyophiid snakes Madtsoiid snakes Terrestrial snakes B

Figure 1.3. Three possible phylogenies given Vidal and Hedges (2004) findings. See the text for an explanation. (A) The first of two phylogenies that refute a marine origin hypothesis for snakes. (B) Second of two phylogenies that refute a marine origin hypothesis for snakes. (C) The only hypothesis supported by Vidal and Hedges (2004) data.

Figure 1.4. Photoreceptors of a basal reptile, the red-eared slider, Trachemys scripta. (A) A single cone, (B) a double cone, and (C) a rod. Abbreviations: el – ellipsoid, fp – foot piece, lim – limitans, my – myoid, nu – nucleus, od – oil droplet, os – outer segment, pa – paraboloid. The arrow indicates the direction of light as it passes through the retina.

Figure 1.5. Representative morphology of snake photoreceptors: (A) double cone, (B) large single cone, (C) small single cone, (D) single cone with a long myoid, (E) small single cone with an extremely long myoid, (F) double cone with an extremely long myoid, (G) long rod, (H) short rod. Abbreviations: el – ellipsoid, fp – foot plate, lim – limitans, my – myoid, nu – nucleus, os – outer segment. The arrow indicates the direction of light as it passes through the retina.

CHAPTER 2

COMPARATIVE OPHTHALMIC ANATOMY OF TWO ECOLOGICALLY DISTINCT COLUBRID SNAKES, THE NORTHERN PINE SNAKE (PITUOPHIS MELANOLEUCUS), AND THE BROWN TREESNAKE, (BOIGA IRREGULARIS).

Animals that are active both in bright and dim light must have the visual sensitivity to see an image under dim illumination, but also the ability to reduce the quantity of light on the retina when the ambient light is intense. Animals achieve maximum sensitivity by having many low-threshold photoreceptors (rods), whose collective output is summed over many fewer ganglion cells (Peterson, 1992). Such low- threshold photoreceptors, however, are overwhelmed by intense light. Vertebrates with sensitive retinas can reduce the intensity of light reaching their retinas by one of two mechanisms: either by contracting the pupil, or by adjusting the position of the photoreceptors relative to the retinal pigment epithelium. Many lizards possess contractile filaments in their photoreceptor myoids. When expanded, the photoreceptor outer segment disks are surrounded by processes of the pigment cells. The myoids withdraw the outer segments from the pigment epithelium when they contract. Snakes do not have contractile myoids (Walls, 1942) and must rely on their pupils alone to adjust the intensity of light reaching the retina.

There is a trade-off between sensitivity and acuity in visual systems. To create a bright image in dim light, the size of the image on the retina must be small.

Consequently, the image is projected on only a few photoreceptors. Groups of tens to hundreds of receptors converge on only a few higher neurons in sensitive retinas.

Summation of this magnitude reduces the resolution of the image. To maintain visual resolution during scotopic acuity, nocturnal animals often have very slim photoreceptors, approaching the diffraction limit. Eyes that maximize acuity, on the other hand, spread the image over a broad array of receptors, whose outputs remain independent. This strategy will produce a relatively dim image. Consequently, the adaptations to increase sensitivity degrade acuity and vice versa.

Northern pine snakes are diurnal, non-venomous constrictors, which may become crepuscular or nocturnal during the hotter months of the year (Conant & Collins, 1998).

Females excavate tunnels in sandy loam in which to lay their eggs, and both sexes occupy the burrows of gopher tortoises or rodents. The adult diet consists mostly of rodents. Prey capture may occur on the surface or in burrows (Rudolph et al., 2002). Consequently, northern pine snakes experience a broad range of ambient light throughout their lives.

Pituophis is most closely related to the genera Elaphe (rat snakes) and Lampropeltis

(king snakes and milk snakes) (Highton et al., 2002). Rat snakes, milk snakes, and king snakes have simplex, all-cone retinae, in which small, single cones have a rod-like morphology while the large, single and double cones possess broader and longer outer segments than in strictly diurnal genera such as Coluber (the racers). Those specializations found in Elaphe and Lampropeltis increase sensitivity in dim light, probably allowing some scotopic visual ability (Walls, 1942; Underwood, 1970).

All snakes have small eyes relative to their body mass, due mainly to their having small heads. As a consequence, their pupils are relatively large and admit light from a wide range of incident angles (Walls, 1942). Light arriving at a photoreceptor from an oblique angle is less likely to be captured than light arriving parallel to the longitudinal axis of the outer segment (Stiles & Crawford, 1933). Wong (1989) and Bossomaier et al.

(1989) found a retinal mechanism for overcoming the Stiles-Crawford effect in garter snakes. Wong (1989) found microdroplets in the peripheral region of cone ellipsoids in eastern garter snakes (Thamnophis sirtalis) that acted as light guides, analogous to the large oil droplets in the photoreceptors of other squamates (Bossomaier et al., 1989).

These microdroplets are concentrated at the periphery of the organelle, such that light that arrived at the ellipsoids of garter snake cones was strongly refracted around the central mitochondria, and was funnelled to the cone outer segments. Consequently, light from oblique angles is captured by garter snake cone outer segments that, in the absence of these droplets, would not be absorbed. While northern pine snakes are larger than eastern garter snakes, and have larger eyes, a similar retinal mechanism may improve their scotopic vision.

All the species in the genus Boiga, Old-World cat snakes, are semi-arboreal to arboreal, with enlarged posterior maxillary teeth and large DuVernoy’s (venom) glands

(Greene, 1989). Brown treesnakes, B. irregularis, are nocturnal specialists whose native range includes northern Australia and western Indonesia, east of Wallace’s line (Rodda et al., 1999). These snakes are best known for their role in the extinction of native birds

(Savidge, 1987; Mccoid, 1991; Reichel et al., 1992; Fritts et al., 1998; Wiles et al., 2003) and lizards (Rodda & Fritts, 1992a; Rodda & Fritts, 1992b) after their accidental

introduction to the island of Guam. While there are nearly 150 published investigations of brown treesnake control on Guam, information on their general biology is more limited.

Some of those studies included reproductive biology (Whittier & Limpus, 1996; Bull et al., 1997; Greene & Mason, 1998; Greene & Mason, 2000; Greene et al., 2001; Greene &

Mason, 2003), home-range activity (Santana-Bendix & Maughan, 1992; Bull & Whittier,

1996; Tobin et al., 1999), foraging behavior (Rodda, 1992; Rochelle & Kardong, 1992;

Rochelle & Kardong, 1993), and venom characteristics (Vest et al., 1991; Zalisko &

Kardong, 1992; Hayes et al., 1993; Weinstein & Kardong, 1994; Broaders & Ryan, 1997;

Kardong et al., 1999). Their visual ecology, however, is known only anecdotally.

Brown treesnakes have large eyes with vertically elliptical pupils, typical of nocturnal animals, and visually orient under extremely low light levels both in the lab

(personal observation), and in the field (Rodda et al., 1999). Because scotopic vision requires considerable summation across visual receptors, resolution in a scotopic eye is limited not by the density of the photoreceptors themselves, but by the density of more complex units consisting of groups of photoreceptors and the ganglion cells that recieve the output of each group (Walls, 1942). Rasmussen (1985) studied the retinas of mangrove snakes, Boiga dendrophila, as part of an investigation of the systematics of opisthoglyphous snakes, and found that mangrove snakes have a duplex retina with densely-packed, long, rod photoreceptors, with a few cones interspersed throughout.

Retinal morphology seems to be conserved within snake genera (Walls, 1942;

Underwood, 1970), so other Boiga should have retinas similar to that of B. dendrophila.

Herein, I compare the eyes of pine snakes, a primarily diurnal, terrestrial species, that

nonetheless needs some scotopic ability, with those of brown treesnakes a primarily nocturnal, arboreal species that is rarely active under photopic conditions.

MATERIALS AND METHODS

I obtained the pine snakes used in this investigation from the Ophidian Research

Colony at the University of Texas, Tyler, TX. All Boiga irregularis used in this study were collected on the Pacific island of Guam. All the individuals investigated for this study had been euthanised following their use in other experiments (Shulse, 1997). To ensure the freshness of specimens for light and electron microscopy, I removed their eyes immediately following euthanasia, punctured them through the cornea with a dissection needle, and placed them in a paraformaldehyde-glutaraldehyde fixitive for 24 hours. I embedded those eyes in JB-4 medium (Polysciences, Inc.), after dehydration in an ethanol series, and sectioned the blocks on a rotary microtome using a stainless steel knife for thick section light microscopy. I stained those sections using toluidine blue.

I prepared some specimens for detailed analysis of the retina, first by removing the cornea and lens, and then cutting radial, triangular sections of the sclera and retina, approximately 3 mm across the base and 4 mm long. I post-fixed those specimens in 4% osmium tetroxide (OsO4), dehydrated them through an ethanol series followed by propylene oxide, and then embedded them in Spurr medium. I sectioned those blocks on an ultramicrotome using a glass knife for thin section light microscopy. I stained those sections using methylene blue. I took additonal sections for electron microscopy from those blocks using a diamond knife. I placed those sections on copper grids, and treated them with uranyl acetate and lead citrate, followed by washes in an ethanol-methanol

solution, and finally distilled water. I collected images of those tissue samples using a

Philips CM 12 transmission electron microscope.

I dissected the spectacle, eyeball, and lens from 6 pine snakes and 4 (10) brown treesnakes previously fixed in formalin and stored in 70% ethanol. I measured, using digital calipers (Mitutoyo, model CM-6”) the width and depth of the spectacle and the axial and equatorial diameters of the eyeball and lens. I approximated the volume of the lens and the eye for each individual, assuming each was spherical. I assumed the refractive indices of the ocular media, including the lens and spectacle to be the same as for other species of snakes (Sivak, 1977). I calculated the radius of curvature, in meters,

2 of the spectacle as rSpec = (y /2S) + S/2, where y = ½ the sagittal chord of the spectacle, S

= depth of that chord. I estimated the radius of curvature of the lens, rLens, as ½ the diameter of the lens along the optic axis. Using constants derived from the refractive indices mentioned above, I then calculated the refractive power (in diopters) of the spectacle and the lens using the thin lens equation, which simplifies to FSpec =

0.3355/rSpec, and FLens = 0.1579/rLens, respectively. RESULTS

The eyeball of pine snakes is approximately spherical. The pupil forms a wide verticle ellipse on contraction. Following euthanasia, the dilated pupil is approximately

30% of the equatorial diameter of the eyeball. The lens is spherical and amber in color, both before and after fixation. By contrast, the brown treesnake pupil forms a very narrow ellipse when contracted, and occupies approximately 40% of the diameter of the equatorial diameter of the eyeball when dilated. The lens, while also spherical, has much less of the yellow pigment found in pine snakes. Instead, it is a translucent white with a 26

yellow-tinted core. Calculated metrics, based upon the gross measurements of the spectacle, eyeball, and lens are provided in Table 2.1.

In both species, the sclera is approximately 50 µm thick and loosely connected to the choroid, which contains numerous, broad venous sinuses. The cornea is approximately 400 µm thick at its base, and contains a circular lymphatic sinus (the canal of Schlemm), centered between its outer and inner margins. The choroid folds inward at the uvea, forming the stroma of the iris. The iris has an inner layer of dark pigment that is continuous with the pigment of the choroid (Fig. 2.1A). The ciliary body consists of a columnar epithelium (Fig. 2.1A & B), with zonular fibers joining the epithelial cells to the lens capsule. The base of this epithelium is heavily pigmented and that layer of pigment is continous with the retinal pigment layer. The iridial muscle of accommodation is circular in cross-section, and circumscribes the perimeter of the iris (Fig 2.1C). The remaining iris muscles are arranged in both radial and circular diffuse bundles throughout the stroma.

The retinas of these two species, like the lenses, are dramatically different. Pine snakes have large single cones, small single cones, and double cones, all of which are quite stout, with tapering outer segments, that are approximately 8 µm across their base and 10 µm long (Fig. 2.2). The outer segments of both the principle and accessory members of the double cones are immediately adjacent to one another. The ellipsoids of the large single cones, small single cones, and the principle members of double cones are located outside of the limitans and stain only lightly with toluidine blue, but intensely with methylene blue around their periphery. Their central regions have a brownish tint when stained with methylene blue. The ellipsoids of the accessory cells of the double

cones are inside the limitans and also stain more intensely with methylene blue than toluidine blue. I observed no rods in pine snake retinas. Brown treesnake retinas consist primarily of densely-packed rods, with extremely long outer segments (about 110 µm), and only a few cones (Fig. 2.3). The cone ellipsoids are outside the level of the rod ellipsoids, approximately 14 µm tall and 7 µm wide. Their cone outer segements are 15

µm long and 4 µm wide at their base, and do not reach the pigment epithelium. The ellipsoids of both rods and cones stain poorly with toluidine blue, but more intensely with methylene blue.

In pine snakes, the photoreceptor nuclei form a single row, immediately internal to the limitans, with a few, scattered nuclei in a second row. There are appoximately eight to nine inner nuclear rows and a single row of tightly-packed ganglion cell nuclei. There is an approximately 2:1 ratio of photoreceptor to ganglion cell nuclei (Fig. 2.4A). Brown treesnakes have approximately six rows of photoreceptor nuclei, four inner nuclear rows, and one row of widely-spaced ganglion cell nuclei. There is a 38:1 ratio of photoreceptor to ganglion cell nuclei (Fig. 2.4B).

Light transmitted through a tangential section of the pine snake retina bends around the ellipsoids of large and small single cones, and the principle members of double cones (Fig. 2.5). The light appears to be focused on the base of the outer segments, which are surrounded by melanin. The inner segments of the accessory cells of double cones appear as small points of light adjacent to the ellipsoids of the remaining cells, and are not surrounded by melanin. I observed no such phenomenon in the retinas of brown treesnakes. Transmission electron microscopy of pine snake retinas (Fig. 2.6) reveals spherical droplets, between 100 nm and 200 nm in diameter, in the peripheral

region of the cone ellipsoids. The core of the ellipsoid contains densely-packed mitochondria. The peripheral droplets are more electron dense than the mitochondria, and are uniformly stained. They do not appear to be membrane-bound, and are present in all the cone ellipsoids, save those of the accessory cells of the double cones. Electron micrographs of brown treesnake retinas confirm the identity of the rods (Fig. 2.7). The rod ellipsoids are long and narrow, and contain numerous, dense mitochondria. Stacks of independent membranous disks surrounded by a separate, continuous membrane are apparent at the base, middle, and distal rod outer segments. As seen in the light micrographs, the distal ends of the rods contact, but do not penetrate the pigment epithelium. The pigmented epithelium is intensely-stained and contains dense pigment granules between 300 and 900 nm in diameter. Surprisingly, brown treesnake rods also contain ellipsoidal microdroplets, between 100 and 250 nm in diameter (Fig. 2.8). These spherules are not bound by membrane and appear less electron dense than the mitochondria, in contrast to the cone microdroplets seen in pine snakes.

DISCUSSION

Ophthalmic characteristics in common between northern pine snakes (Pituophis m. melanoleucus) and brown treesnakes (Boiga irregularis) are shared by all described advanced snakes (Alethinophidia). These features include striated intraocular muscles, a large peripheral iridial muscle of accommodation, a simple columnar ciliary epithelium, and corneal placement of the scleral-venous sinus (Fig. 2.1A-C), as well as spherical lenses, retinal blood vessels (Fig. 2.4A), and a fascicular optic nerve. The differences between the eyes of these two species, however, reflect their different ecologies, and

meet expectations for animals that are specialized for particular light environments, but retain some ability to function under the opposite conditions.

Refraction. Brown treesnake lenses are significantly larger than northern pine snake lenses for eyes of similar size (Mann-Whitney test, p = 0.042). That difference, and the difference in lens pigmentation exemplifies the contrasting diurnal and nocturnal activity of these species. The size difference in lenses, relative to the eyeballs, corresponds to a difference in refraction, with northern pine snakes achieving 67% more refractive power in their lenses and 43% more from their spectacles than brown treesnakes. Because of the large lens, the distance from the lens to the retina in brown treesnakes is much shorter than that for northern pine snakes. As a consequence, the image is approximately 29% smaller and, consequently 8% brighter for brown treesnakes than for nothern pine snakes. This is expected, given the typical scotopic activity of brown treesnakes. Additionally, the spectacle of the brown treesnake contributes considerably more to the eye’s refractive power than the lens, while the lens and spectacle of pine snakes have roughly equal contributions (Fig. 2.9).

Photoreceptors. Pine snake cone ellipsoids have densely-concentrated mitochondria centrally, surrounded by microdroplets in their periphery (Fig. 2.6). The microdroplets in eastern garter snakes (Bossomaier, 1989; Wong, 1989) are lighter than the mitochondria, and are not uniformly colored. The apparent difference between eastern garter snake and northern pine snake microdroplets may be due to differences in preparation of the tissue and quality of the sections. On the other hand, northern pine snake microdroplets may have a different composition than those of eastern garter snakes. Northern pine snake microdroplets, like those of eastern garter snakes are

refractile in fixed and stained specimens, as evident from transmission of light through tangential retinal sections (Fig. 2.5). Thus, northern pine snakes appear to employ the same mechanism as eastern garter snakes to avoid the consequences of the Stiles-

Crawford effect.

The electron micrographs of the retina of brown treesnakes reveal microdroplets in the rod ellipsoids. Such droplets thus far have been found only in cones, where their function as light guides was supported by Wong (1989), Bossomaier et al. (1989), and my results. These droplets may serve the same function in the retinas of brown treesnakes, by increasing the likelihood of photon capture at the outer segment. Walls

(1942) hypothesized that snake rods were evolutionarily transformed cones. The discovery of rhodopsin in some cone photoreceptors of Anolis carolinensis (McDevitt et al., 1993), and cone opsins in rod photoreceptors in geckos (Kojima et al., 1992; Loew,

1994; Loew et al., 1996) supported Walls’ (1942) transformation hypothesis, and indicate that such transformations represent recurrent evolutionary phenomena. The presence of microdroplets in rod ellipsoids, similar to the droplets found in cones, suggests homology between these two types of photoreceptors, and lends further support to Walls’ transformation hypothesis. As with the cone microdroplets, the composition of the rod microdroplets is unknown. No tangential sections were available to assess the light guide properties of these intracellular inclusions.

Sensitivity. The differences between numbers of inner, outer, and ganglion cell nuclear rows indicate a difference in summation of signals from the photoreceptors, which also is consistant with the difference in visual ecology between these species.

Specifically, on average each ganglion cell collects the output of nearly 1100

photoreceptors in brown treesnakes, whereas each ganglion cell in northern pine snakes sums seven photoreceptors, on average. Brown treesnake have an order of magnitude more receptors per unit area than in northern pine snakes, and their photoreceptor outer segments are 11 times longer. The molecular mechanism of phototransduction in typical

(human) rods provides a one hundred-fold increase in sensitivity over cones (Barlow,

1982). But in human rods, the outer segments are about the same length as the cones. If the difference in sensitivity between rods and cones is a function of the difference between the lengths of outer segments, then a brown treesnake’s rod will be approximately 1100 times more sensitive than the cones of northern pine snakes. Those differences provide brown treesnakes with substantially greater sensitivity than pine snakes.

Acuity. From these data, it is clear that brown treesnake eyes are much more sensitive than northern pine snake eyes, but it is more difficult to assess the difference in acuity between these two species. Without mapping the receptive fields of the retinal ganglion cells, or performing psychophysical tests, the best these data can provide is an approximate range within which the true visual acuity of each species lies. The lowest estimate of visual acuity for both species is calculated by assuming that there is no overlap between ganglion cell receptive fields. For a brown treesnake, in a thin section there are 38 photoreceptors for each ganglion cell. Assuming that the spacing between the centers of adjacent outer segments is approximately 2.5 µm, the spacing between receptive fields is 95 µm. A retinal image, 100 µm in height corresponds to an object height of 236 mm at 10-m distance. Thus, given the assumptions above, that is the narrowest separation of two points that a brown treesnake could resolve at 10 m. If the

receptive fields of ganglion cells in brown treesnake retinas overlap considerably, which is typical for most animals (Peterson, 1992), then the ability to resolve two points at a given distance will be much greater. If we assume a receptive field overlap of 75%, then the minimum separation for two points at 10 m that a brown treesnake can resolve is 59 mm. The values for northern pine snakes, based upon a receptive field diameter of approximately 20 µm, are 60 mm for non-overlapping fields and 16 mm for 75% overlap.

The angle of separation of two points in both species is approximately 20′ of arc. But, the values for brown treesnakes are under scotopic conditions while the values for northern pine snakes are under photopic conditions. Thus, both species may have similar visual acuities under the conditions in which they are most active.

Conclusions. The small eye to body size in snakes requires them to have a relatively large pupil, which consequently requires them to suffer a large Stiles-Crawford effect as in garter snakes (Bossomaier et al., 1989). The presence of microdroplets in the cone ellipsoids (Fig. 2.6) of northern pine snakes may compensate for the loss of light entering the eye through oblique angles by funneling photons to the outer segment disks.

Wong (1989) and Bossomaier et al. (1989) hypothesized this to be the case in garter snakes. Light transmitted through tangential retinal sections supports that hypothesis, revealing bright, circular areas corresponding to the peripheral ellipsoids and bright points surrounded by dense pigment, corresponding to the bases of cone outer segments

(Fig. 2.5). This light guide effect of the cone microdroplets may also increase the light captured by the cones, allowing pine snakes some scotopic vision during crepuscular or nocturnal activity, or when foraging in rodent burrows. The microdroplets of brown treesnake rod photoreceptors are sparse by comparison, although a light guide effect

cannot be ruled out. Such an effect would be useful to an animal that consistantly experiences scotopic conditions. My results indicate that brown treesnake rods provide this species with considerable scotopic sensitivity, yet they also appear to have an acuity under scotopic conditions similar to that of the diurnal acuity of northern pine snakes.

This is consistent with observations of nocturnal behavior in brown treesnakes, and their ability to navigate in complex nocturnal habitats and feed on small, quick, nocturnal lizards.

Northern pine Feature Brown treesnakes snakes Snout-vent length (cm) 106 ± 10 115 ± 20 Eyeball equatorial diameter (mm) 5.38 ± 0.54 5.59 ± 0.30 Lens axial diameter (mm) 2.43 ± 0.43 3.59 ± 0.20 Spectacle refractive power (diopters) 164 ± 20 115 ± 10 Lens refractive power (diopters) 154 ± 22 92 ± 5 Outer segment length (µm) 10 110 Outer segment density (receptors/mm2) 7.8 x 103 8.7 x 104 Photoreceptor nuclear layers 1½ 6 Inner nuclear layers 8-9 4 Ganglion cell layers 1 1 Pupil shape (contracted) broad ellipse narrow ellipse Lens color yellow – amber clear – pale yellow Photoreceptors lsc, ssc, dc rods, lsc Photoreceptor cells : Ganglion cells 2 : 1 38 : 1

Table 2.1. Comparison of ophthalmic features between northern pine snakes and brown treesnakes. Measurements are means ± standard error of the mean. Abbreviations: dc − double cones, lsc − large single cones, ssc − small single cones.

Figure 2.1. (A) Light micrograph of a radial section (methacrylate 10 µm, toluidine blue, 100x) through the anterior portion of the eye of a pine snake, Pituophis melanoleucus. Bar = 100 µm. (B) Radial section (methacrylate10 µm, toluidine blue, 1000x) through the ciliary body of a brown treesnake, Boiga irregularis. Bar = 1 µm.(C) Circumferential section (methacrylate10 µm, toluidine blue, 400x) through the ciliary body and iris of a brown treesnake. Bar = 5 µm. Abbreviations: co – cornea, cb – ciliary body, cs – scleral-venous sinus (canal of Schlemm), ir – iris, ma – muscle of accommodation, re – retina, sc – sclera.

Figure 2.2. (A) Retina of a pine snake, Pituophis melanoleucus, with a large single cones visible at the center and a small single cone to its left (methacrylate 12-µm section, toluidine blue, 1000x). Bar = 10 µm. (B) Thin section (Spurr resin 1-µm section, methylene blue, 1000x) showing a double cone. Bar = 10 µm. Abbreviations: ael – accessory cell ellipsoid, anu – accessory cell nucleus, aos – accessory cell outer segment, el – ellipsoid layer, lim – limitans, lsc – large single cone, onu – outer nuclear layer, os – outer segment layer, pel – principle cell ellipsoid, pnu – principle cell nucleus, pos – principle cell outer segment, ssc – small single cone. The arrow indicates the direction of light as it passes through the retina.

Figure 2.3. Radial sections through the retina of a Brown treesnake, Boiga irregularis. (A) A 10-µm section (methacrylate, toluidine blue, 400x) showing the densely packed rods with long, thin outer segments, and two cones, whose large ellipsoids are at the base of the rod outer segment layer. (B) A 1- µm section (Spurr resin, methylene blue, 400x) showing the rod nuclei, ellipsoids, outer segments, and a single, large cone, with a large ellipsoid and long, tapering outer segment. Bars = 10 µm. Abbreviations: cel – cone ellipsoid, co – cone, cos – cone outer segment, lim – limitans, nu – nucleus, os – outer segment layer, pe – pigmented epithelium. The arrow indicates the direction of light as it passes through the retina.

Figure 2.4. Radial retinal sections of (A) pine snake, Pituophis melanoleucus and (B) brown treesnake, Boiga irregularis. Both sections: Spurr resin, 1-µm section, methylene blue, 400x, Bars = 20 µm. Abbreviations: gcl – ganglion cell layer, inl – inner nuclear layer, onl – outer nuclear layer, rel – rod ellipsoids, ros – rod outer segments, rv – retinal vessel. The arrow indicates the direction of light as it passes through the retina.

Figure 2.5. Tangential section (methacrylate 12 µm, toluidine blue, 400x) through the ellipsoid region of the retina of a pine snake, Pituophis melanoleucus. This section passes through the base of the outer segments in the upper half and through the middle of the ellipsoids in the lower half. Light transmitted through the retina funnels through the periphery of the ellipsoids (el) of large and small single cones, and the principle members of double cones, and is focused at the base of the outer segments (os). The accessory cells (ac) of double cones also are indicated. Bar = 50 µm.

Figure 2.6. Electron micrographs (6300x) of a large single cone from a pine snake, Pituophis melanoleucus. A. Outersegment (os) and ellipsoid (el) containing microdroplets. B. Enlargment of the ellipsoid in (A), providing a closer look at the microdroplets. C. Distal portion of the ellipsoid of the principle member of a double cone, showing the mitochondria (mt) and microdroplets. Bar = 1 µm.

Figure 2.7. Transmission electron micrographs of brown treesnake, Boiga irregularis, retinas showing characteristics of the rods. (A) Three rods showing irregular mitochondria in their ellipsoids. Bar = 5 µm. (B) A section of a rod outer segment showing membranous disks within and independent of the plasma membrane. Bar = 2 µm. (C) The distal ends of rod outer segments are adjacent to, but not embedded in the pigmented epithelium. Bar = 2 µm.

Figure 2.8. (A) Transmission electron micrograph of a brown treesnake, Boiga irregularis, rod at the junction of the inner and outer segments. Bar = 2 µm. (B) An enlargement of the rod ellipsoid between the mitochondria, showing small droplets. Bar = 500 nm.

200 p = 0.48 175 p = 0.0002 150

125

100

Refractive Power (Diopters) Power Refractive 50

0 BTS Spectacle BTS Lens NPS Spectacle NPS Lens

Figure 2.9. Comparison of refractive powers of the spectacle and lens of brown treesnakes (BTS) and northern pine snakes (NPS). The p-values are based upon paired t- tests, 1-tailed.

CHAPTER 3

GEOMETRIC OPTICS AND VISUAL ECOLOGY AMONG SNAKES.

Ophthalmic morphology and paremeters of visual fields usually correspond with light environment, habitat, and lifestyle (Walls, 1942). In snakes, Kahmann (1934) found the degree of binocular overlap to vary from about 20 to 46 degrees, with terrestrial species at the low end and arboreal species at the high end of the range. Henderson and

Binder (1980) proposed that an extremely elongated head, combined with deep lateral indentations between the eyes and the tip of the snout may provide a particularly high degree of binocular overlap in arboreal diurnal specialists, such as vine snakes. Marx and

Rabb (1972) noted that among snakes, arboreal species had larger eyes than terrestrial species, and nocturnal species had larger eyes than diurnal species.

In contrast, the shapes of the eyeballs and lenses of snakes appear to be conserved across species (Walls, 1942; Sivak, 1977). That stability suggests that their refractive powers likewise should be conserved, varying only with the size of their eyes. Walls found the eyeballs of most snakes to be spherical or slightly elongated in the optic axis.

Sivak (1977), on the other hand, noted that the eyes of three colubrid species, yellow rat snakes (Elaphe quadravittatta), corn snakes (Elaphe guttatta), and southern black racers

(Coluber coluber priapus) were noticeably subspherical, with the spectacle and the cornea beneath it less curved than the sclera in each specimen. He verified that the 45

spectacle is a powerful refractive element, as predicted by Walls (1942), providing roughly half the eye’s refractive power. The nearly spherical lens contributed the remaining refractive power.

Sivak’s (1977) data indicated that two species, Elaphe guttatta and E. quadravittatta, had nearly double the refractive power of Coluber constrictor priapus.

Moreover, the relative contribution of spectacle and lens was reversed between the two genera, with the spectacle being slightly more powerful in Coluber while the lens was more powerful in Elaphe. All of his specimens were approximately the same size, but

Elaphe had substantially smaller eyes than Coluber. That suggests that the difference in total refractive power between the two genera was due entirely to eye size (Fig. 3.1). This makes sense because the smaller the eye, the stronger its curvature, which results in a ray of light having a greater angle of incidence with the refractive surface, and consequently greater refraction.

In this investigation, I built upon Walls’ (1942) comparative work by examining members of two unrelated families: the polyphyletic Colubridae, and the monophyletic

Crotalidae, or pit vipers. I chose those two taxonomic groups because of their global distributions, diverse ecologies, and distinct morphological differences. Additionally, colubrid genera have distinct photic specialists and generalists. For example, the colubrid brown treesnake (Boiga irregularis) is strictly nocturnal, foraging in trees and shrubs at night and sheltering in tree hollows, detritus piles, and leaf axils through the day. During the day, when disturbed, brown treesnakes are slow to rouse, despite a normally pugnacious nature (personal observation). The photic ecology of pit vipers is less clear.

Pit vipers may move extensively during the night, but are found in ambush postures

during the day or night (Greene, 1997). The extent to which vision plays a role in either activity in pit vipers is unknown. Rods outnumber the cones in the retinas of northern copperheads, Agkistrodon contortrix mokeson, with ratios similar to those in humans

(Walls, 1942). That suggests that copperheads, at least, are photic generalists, able to see during the day, but also having limited night vision.

In this chapter, I quantified orbital area, binocular overlap, lens refractive power, and spectacle refractive power among colubrid and crotalid snakes. First, I assessed the ecological correlates of binocular overlap and orbital area noted by Marx and Rabb

(1972) and Henderson and Binder (1980). Secondly, I measured the shape of the eyeballs and lenses to determine whether Walls’ (1942) or Sivak’s (1977) description of eyeball shape better characterizes snakes in general. Lastly, I attempted to determine if the relative refractive contributions of the spectacle and lens found by Sivak (1977) apply generally to snakes, or if they are confounded by phylogeny, ecology, and body size.

MATERIALS AND METHODS

I studied specimens from three herpetological collections: the California

Academy of Sciences, the National Museum of Natural History, and the Ohio State

University Museum of Biological Diversity (Appendix A). I collected data on 925 specimens in two families, 16 species of colubrids and 35 species of crotalids (pit vipers).

For each species, I recorded their typical habitat, light environment, and distribution based upon descriptions in Coburn (1991) and Greene (1997). Using digital calipers

(Mitutoyo Model CM-6”) I measured to the nearest 10-µm the length of the head (HL), the length of the snout (LS), the width of the base of the snout (SBW), the width across

the nares (NW), and the height (OrH) and width (OrW) of the orbit of every specimen

(Fig. 3.2). I used the following formula to calculate the angle of binocular overlap:

Binoc = 2 x (arctangent(((SBW-NW)/2)/SL)).

Additionally, I calculated the area of the orbit as:

OrA = ((OrH+OrW)/4)2 x π).

To reduce the effect of size on my analyses of orbital area, I divided OrA by head length

(HL). I subjected the size-adjusted OrA and binocular overlap to one-way ANOVAs with habitat, light environment, and genus as factors. I also performed post-hoc multiple comparisons using Bonferroni tests in all of these analyses. Because it is not clear under what lighting conditions crotalid snakes rely on vision, I treated all crotalid species as photic generalists.

I used a subset of those specimens, all from the Ohio State University Museum of

Biodiversity, representing ten species, eight colubrids and two crotalids, to estimate the refractive powers of the spectacle and lens. I measured the snout-vent length (SVL) of each specimen by tracing a string along the spinal column and comparing that length obtained to a meter-stick. I dissected one spectacle and the underlying eyeball from each specimen. I next measured the width of the spectacle (its chord) and its depth, and the diameters of the eyeball along its equatorial and optical axes. Next, I opened the eyeball using a scalpel and removed the lens. I subsequently measured each lens along its equatorial and optic axes to obtain the diameters. I used those measurements, and the formulae described in Chapter 2, to calculate the refractive powers of the spectacle and lens. I performed all statistical analyses using SPSSTM on an IBM PC-compatable computer, running Microsoft Windows XPTM.

RESULTS

I found a significant, but weak correlation between size-adjusted orbital area

(sOrA) and angle of binocular overlap for the colubrid snakes (Pearson correlation = -

0.117, p = 0.004), but no such correlation for pit vipers (Pearson correlation = 0.006, p =

0.919). I subsequently tested for correlations between those two variables independently for each colubrid species. Three of the colubrid species showed significant negative correlations between sOrA and the angle of binocular overlap: rough green snakes

(Opheodrys aestivus, Pearson correlation = -0.580, p < 0.001), northern black racers

(Coluber constrictor, Pearson correlation = -0.665, p < 0.001), and black rat snakes

(Elaphe obsoleta, Pearson correlation = -0.499, p < 0.001). I removed those three colubrid species from further analyses, and treated sOrA and the angle of binocular overlap separately for the remaining species.

Among colubrid snakes, I found significantly greater average sOrA in arboreal species than in aquatic (p < 0.001) or terrestrial (p < 0.001) species, while aquatic and terrestrial species did not differ significantly (Fig. 3.3A). Among pit vipers, the average sOrA was significantly greater in aquatic species when compared to arboreal (p < 0.001) or terrestrial (p < 0.001) species. Arboreal pit vipers had a significantly (p = 0.005) greater average sOrA than terrestrial species (Fig. 3.3B). Diurnal colubrids had a significantly (p = 0.042) greater average sOrA than those active in mixed lighting conditions and a significantly (p < 0.001) smaller average sOrA than nocturnal species.

Colubrids that are active in mixed lighting also had significantly (p < 0.001) smaller sOrA than nocturnal species (Fig. 3.4).

Arboreal colubrids had a significantly (p = 0.012) narrower average angle of binocular overlap than terrestrial species (Fig. 3.5A). In contrast, arboreal pit vipers had significantly (p < 0.001) wider average angle of binocular overlap than terrestrial species

(Fig. 3.5B). Neither arboreal nor terrestrial species of either colubrids or pit vipers differed significantly in the angle of binocular overlap from aquatic species. Diurnal colubrids had a significantly narrower average angle of binocular overlap than either nocturnal specialists (p < 0.011) or generalists (p < 0.035), while those generalist and nocturnal species did not differ significantly (Fig. 3.6).

I subdivided the colubrid species into groups based upon their combined habitat and light environment and performed another one-way ANOVA. Those results are presented in table 3.1. Based upon those results, colubrid snakes can be arranged in order of decreasing sOrA as follows: arboreal nocturnal specialists, terrestrial diurnal specialists, terrestrial nocturnal specialists, arboreal diurnal specialists, aquatic diurnal specialists, and terrestrial generalists (Fig. 3.7). Alternatively, those species also may be arranged in decreasing order of average angle of binocular overlap: terrestrial nocturnal specialists, aquatic diurnal specialists, terrestrial generalists, terrestrial diurnal specialists, arboreal nocturnal specialists, and arboreal diurnal specialists (Fig. 3.7).

I performed paired sample t-tests between axial and equatorial diameters for the eyeballs and lenses for each species (Fig. 3.8 A-B). I used one-tailed tests for the eyeballs of all species because they were consistently wider in the equatorial dimension than the axial dimension, both within and among species. Alternatively, I used two-tailed tests for the lenses because the differences in dimensions were less clear and not consistent among species. The eyeballs were significantly subspherical for nine species, with brown

treesnakes (Boiga irregularis) being the only exception. The lenses of five species differed significantly from spherical. The lenses of eastern hognose snakes (Heterodon platyrhinos) and northern black racers (Coluber c. constrictor) were elongated significantly in the optic axis, while those of eastern cottonmouths (Agkistrodon piscivorous), eastern milk snakes (Lampropeltis triangulum), and northern water snakes

(Nerodia sipedon) were significantly subspherical.

The lenses had higher average refractive powers than the spectacles for eight species (Fig. 3.9). The difference in refractive power for the lens and spectacle was not significant for northern copperheads. For brown treesnakes, on the other hand, the mean refractive power of the spectacle was significantly greater than that of the lens (about

27%). Snout-vent length and the refractive power of the lens were correlated significantly and negatively for all ten species. The refractive power of the spectacle also was correlated significantly and negatively with SVL for eight of the ten species, with brown treesnakes and eastern garter snakes (Thamnophis sirtalis) as the two exceptions (Table

3.2). That corresponds to a strong positive correlation between SVL and the sizes of the eyes and lens for all ten species and the spectacle for all but brown treesnakes. The spectacle was correlated significantly and positively with SVL for eastern gartersnakes, but that correlation was weaker than those for the other species. Thus, larger snakes generally had larger eyes and correpondingly weaker refractive elements than smaller species.

DISCUSSION

Both orbital area and binocular overlap varied with habitat and photic ecology.

While SVL-adjusted orbital area varied among colubrid snakes as expected binocular 51

overlap did not. Nocturnal arboreal specialists had the largest eyes among colubrid snakes, but arboreal diurnal species had the smallest average degree of binocular overlap, contrary to expectations (Marx & Rabb, 1972; Henderson & Binder, 1980). I believe this is due to the length of their snouts, which is an obstacle to vision across the medial plane.

The elongated, narrow heads of these snakes provides them with a large gape and large number of teeth, due to their long mandibular, palatine, pterygoid and maxillary bones.

Vine snakes typically consume lizards (Henderson and Binder, 1980), although fish are also eaten by South Asian vine snakes, Ahaetulla fronticincta (R. Lucas, personal communication), and Central American vine snakes, Oxybelis aeneus (T. Hetherington, in press). Both lizards and fish tend to be elongate, slippery, and sometimes armored animals. The long jaws and many teeth of vine snakes enhance their ability to maintain a grip on these prey (Greene, 1997). At the same time, the elongated head enhances the camouflage of vine snakes, by limiting the distinction between the head and body as well as giving the illusion that the anterior body recedes into the distance, unlike the offset head and abrupt snout of most snakes. This represents a trade-off between selection for camouflage and binocular vision. Instead of broad binocular overlap in the frontal plane, the narrow head of vine snakes provides them with wide lateral and posterior views, allowing them to detect approaching predators, while they rely on stealth and camouflage to ambush prey as they approach closely to the snake (Henderson & Binder, 1980).

My results illuminated distinct differences between the eyes of pit vipers and colubrids. Indeed, pit vipers have a visual morphology opposite that of colubrids. The extremely wide head and short snout of pit vipers provides them with exceptionally wide binocular overlap, unlike that in colubrid snakes. On the other hand, with the exception of

eastern cottonmouths (Agkistrodon piscivorous), they have much smaller eyes than colubrids of the same size. That is consistent with a hypothesis that terrestrial and arboreal pit vipers rely on camouflage to hide from predators and prey, venom for defense and digestion, and thermoreceptors (i.e., the pit organ) for detecting threats and prey (Greene, 1997). If eye size is a valid indicator, then vision in terrestrial and arboreal pitvipers appears to be less important than in colubrids of the same size. The wide binocular overlap, rather than being indicative of selection for binocular vision, may be a consequence of their wide heads. Their wide heads accommodate enlarged, temporally- located, venom glands and long quadrate bones, both of which enable pit vipers to subdue, consume, and digest prey many times their own girth (Greene, 1997). Moreover, afferent pathways from the pit organs converge on the optic tectum in rattlesnakes and pythons, providing a thermal overlay on their visual perception (Newman & Hartline,

1982). These infrared-sensitive pit organs guide the feeding strike in pit vipers and pythons, and operate under dim illumination. It is unclear to what extent they function under bright illumination. Nonetheless, the overlap between the right and left pit organs may require a similar degree of overlap of the visual fields to prevent a mismatch between the sensory modalities, akin to vertigo.

The large eyes of eastern cottonmouths, an aquatic pit viper, may represent a convergence with nocturnal colubrids. Terrestrial and arboreal pit vipers mostly consume endotherms and terrestrial ectotherms, which stand out against background thermal radiation, permitting them to use their thermosensitive pits. Eastern cottonmouths have a catholic diet that includes both endotherms, particularly wading birds, and aquatic ectotherms, primarily fish, but also frogs, salamanders, juvenile alligators and other

snakes (Conant & Collins, 1991; Greene, 1997). Most aquatic ectotherms are isothermic with their environment. Thus cottonmouths may rely on vision when foraging in water, rather than their pits, resulting in larger than expected eyes for their body size.

For the subset of specimens I investigated for refractive power, the lens was stronger than the spectacle in every species except brown treesnakes, for which the spectacle was significantly stronger than the lens, and in eastern garter snakes, in which there was no significant difference (Fig. 3.10). Brown treesnakes are nocturnal, while northern black racers, eastern garter snakes, northern water snakes, and rough green snakes are active diurnally and black rat snakes (Elaphe obsoleta), eastern cottonmouths, eastern milk snakes, eastern hognose snakes, and northern copperheads are active in many light environments. The lens of brown treesnakes is much larger than that of a similar-sized eye in a diurnal animal (Fig. 3.11). The larger lens is a direct result of the larger aperture (pupil) of the eye in nocturnal species (Fig. 3.12). Much of the light entering the pupil would not pass through the lens were it to remain proportional between nocturnal and diurnal species. Consequently, light passing through the brown treesnake’s large lens is not refracted as strongly as it is through the lenses of other species with similar-sized eyeballs. I believe that examination of other nocturnal species will reveal the same pattern.

Lens shape varied among the ten species I studied (Fig. 3.8B). The two pit vipers had markedly subspherical lenses, while those of Lampropeltis triangulum and Nerodia sipedon were less so. Four species of colubrids with different photic and habitat ecologies: Boiga irregularis, Elaphe obsoleta, Opheodrys aestivus, and Thamnophis sirtalis all had roughly spherical lenses. Coluber constrictor and Heterodon platyrhinos

both had lenses that were slightly elongated in the optic axis. Those differences in lens shape may be due to differences in the thickness of the anterior epithelium, the “anterior pad” (Walls, 1942).

My data differed from those of Sivak (1977), who described the power of the spectacle as nearly identical to that of the lens in each specimen of southern black racer and in one corn snake, while the lenses of four yellow rat snakes were consistently more powerful, although only slightly so, than the spectacles. I found much greater differences between the spectacle and lens refractive powers for all ten species that I sampled. Sivak used eyes from freshly killed specimens, froze those eyes immediatly upon dissection, and then measured frozen sections cut on a cryomicrotome, while those I studied were stored in 70% ethanol storage. Either method may have distorted the thin tissue of the spectacle, or the shape of the eyeball, accounting for our different results.

The power of both the lens and spectacle were negatively correlated with body size (SVL) for all nine species I examined. Snout-vent length and lens size are correlated positively for nocturnal specialists (Pearson correlation = 0.919, p < 0.001), diurnal specialists (Pearson correlation = 0.659, p < 0.001), and generalists (Pearson correlation

= 0.971, p < 0.001). I employed multiple regression analysis to determine if the slopes and intercepts of the fit lines for the three photic categories differed. Based on Tukey post-hoc tests the fit line for the nocturnal specialist (brown treesnakes) had a significantly larger intercept (p < 0.001) than both diurnal specialists and photic generalists, while the intercept did not differ between diurnal specialists and photic generalists (p = 0.222),. Thus, while larger snakes reliably have larger lenses, the lens of

brown treesnakes is substantially larger for any SVL than that of diurnal specialists and photic generalists of the same size.

In conclusion, a broad sampling of species reveals that morphological patterns previously observed do not occur universally among snakes. In particular, while eyeball shape was similar across species, lens shapes varied from subspherical to elongate in the optic axis. Orbital area varied as expected among species, but binocular overlap did not, particularly for colubrids. The ecological correlations with binocular overlap and orbital area differed between pit vipers and colubrids, perhaps reflecting a constraint upon eye position due to prominent venom glands in pit vipers.

Coluber constrictor priapus 250.00 Elaphe guttata Elaphe quadrivittata

200.00

150.00 Total refractive power (diopters)

100.00

3.00 4.00 5.00 Eyeball axial radius (mm)

Figure 3.1. A scatterplot of the total refractive power as a function of the radius of the eyeball for three species of snakes from a study done by Sivak (1977). The fit line was graphed for the combined data and has a Pearson correlation of -0.97.

Figure 3.2. Measurements obtained from snake heads used to calculate the angle of binocular overlap in the frontal plane, illustrated using the head of an urutu, Bothrops alternatus, a South American pit viper Abbreviations: HL – head length, NW – width between the nares, ORH – height of the orbit, ORV – width of the orbit, SBW – snout base width, SL – snout length, θ – the angle of binocular overlap. 58

A ground

arboreal

aquatic

0.20 0.40 0.60 0.80 1.00 SVL-adjusted OrA

B ground

arboreal

aquatic

0.40 0.50 0.60 SVL-adjusted OrA

Figure 3.3 Plot of the size-adjusted OrA (mm) for colubrid snakes (A) and pit vipers (B), grouped by typical habitat. Filled boxes are means. Whiskers are one standard error of the mean in length.

nocturnal

mixed

diurnal

0.20 0.40 0.60 0.80 1.00 1.20 SVL-adjusted OrA

Figure 3.4. Plot of the size-adjusted OrA (mm) for colubrid snakes, grouped by typical light environment. Filled boxes are means. Whiskers are one standard error of the mean in length.

ground

arboreal

aquatic

18.0 20.0 22.0

ground

arboreal

aquatic

44.00 46.00 48.00 50.00

Figure 3.5. Plots of the angle of binocular overlap (degrees) for colubrid snakes (A) and crotalid snakes (B), grouped by typical habitat. Boxes are means. Whiskers are one standard error of the mean in length.

nocturnal

mixed

diurnal

18.00 19.00 20.00 21.00 22.00

Figure 3.6. Plot of the angle of binocular overlap (degrees) in colubrid snakes, grouped by light environment. Boxes are means. Whiskers are one standard error of the mean in length.

Arboreal Arboreal Aquatic Terrestrial Terrestrial Terrestrial

diurnal nocturnal diurnal diurnal generalist nocturnal Arboreal – *-0.57 0.14 -0.05 *0.15 -0.01 diurnal Arboreal 1.76 – *0.71 *0.22 *0.72 *0.56 nocturnal Aquatic 4.55 2.80 – -0.18 0.01 -0.15 diurnal Terrestrial 1.76 2.01 -2.79 – *0.20 0.04 diurnal Terrestrial ‡3.76 2.00 -0.79 ◊3.36 – *-0.16 generalist Terrestrial *9.60 *7.84 5.05 *7.83 †5.84 – nocturnal

Table 3.1. Mean differences among combined habitat and light categories in sOrA (upper cells) and angle of binocular overlap (lower cells) for colubrid snakes. In each cell, the value indicates the mean for the category in the row heading minus the mean for the category in the column heading. Significant differences are indicated by symbols as follows: * – p < 0.001, † – p = 0.001, ‡ – p = 0.012.

Figure 3.7. Comparison of the size-adjusted orbital area and binocular overlap among ecological groups of snakes, based upon the data in Table 3.1.

Figure 3.8. Boxplots of comparing axial diameters (clear boxes) and equatorial diameters (filled boxes) of the eyeballs (A) and lenses (B) for eight species of colubrid snakes and two pit viper species. P-values are based upon paired t-tests. Abbreviations: Ag_co – Agkistrodon contortrix, Ag_pi – A. piscivorous, Bo_ir – Boiga irregularis, Co_co – Coluber constrictor, El_ob – Elaphe obsoleta, He_pl – Heterodon platyrhinos, La_tr – Lampropeltis triangulum, Ne_si – Nerodia sipedon, Op_ae – Opheodrys aestivus, Th_si – Thamnophis sirtalis.

Figure 3.9. Boxplots of refractive powers (diopters) of the spectacles (open boxes) and lenses (closed boxes) for eight species of colubrid snakes and two species of pit vipers. P- values are based on paired t-tests. Abbreviations: Ag_co – Agkistrodon contortrix, Ag_pi – A. piscivorous, Bo_ir – Boiga irregularis, Co_co – Coluber constrictor, El_ob – Elaphe obsoleta, He_pl – Heterodon platyrhinos, La_tr – Lampropeltis triangulum, Ne_si – Nerodia sipedon, Op_ae – Opheodrys aestivus, Th_si – Thamnophis sirtalis.

Species n Spectacle Lens Black rat snake -0.946 -0.977 10 * * Elaphe obsoleta p < 0.001 p < 0.001 Brown treesnake -0.639 -0.948 10 * Boiga irregularis p = 0.088 p < 0.001 Eastern cottonmouth -0.855 -0.960 8 * * Agkistrodon piscivorous p = 0.007 p < 0.001 Eastern garter snake -0.698 -0.902 10 * Thamnophis sirtalis p = 0.054 p = 0.002 Eastern hognose snake -0.943 -0.941 10 * * Heterodon platyrhinos p < 0.001 p < 0.001 Eastern milk snake -0.895 -0.937 10 * * Lampropeltis triangulum p < 0.001 p < 0.001 Northern copperhead -0.657 -0.919 10 * * Agkistrodon contortrix p = 0.039 p < 0.001 Northern water snake -0.781 -0.935 10 * * Nerodia sipedon p = 0.008 p < 0.001 Northern black racer -0.913 -0.947 10 * * Coluber constrictor p = 0.001 p < 0.001 Rough green snake -0.743 -0.893 10 * * Opheodrys aestivus p = 0.014 p = 0.001

Table 3.2. Correlations between snout-vent length (mm) and refractive power (diopters) for eight species of colubrid snakes and two species of pit vipers. Values are Pearson correlations followed by p-values (two-tailed). Significant correlations are indicated by an asterisk (*).

4.00 Diurnal Mixed Nocturnal

3.00

2.00 Lens axial diameter (mm) 1.00

0.00 2.00 4.00 6.00 8.00 Eyeball axial diameter (mm)

Figure 3.10. Scatter plot of axial diameters of the lenses and eyes of ten species of colubroid snakes, sorted by the typical light level during which those species are active. The solid black circles represent the only nocturnal species in the sample, brown treesnakes. The fit lines have similar slopes but the intercept is greater for brown treesnakes.

4.0 Diurnal Mixed Nocturnal 3.0

2.0

1.0 Axial diameter of the lens (mm) lens the of diameter Axial

0 500 1000 1500 SVL (mm)

Figure 3.11. Scatterplot of lens size and body size for nine colubrid species, grouped by light environment. The solid black circles represent the only nocturnal species in the sample, brown treesnakes.

Figure 3.12. Illustration of the paths of light emanating from infinity and refracted through the eye of a snake. In a nocturnal species, the large pupil allows more light into the eye, but requires a large lens (dotted border) to focus the image on the retina. A diurnal snake’s lens (solid border) is too small, and would result in a blurred image because peripheral rays pass the lens unrefracted and focus behind the retina.

CHAPTER 4

THE ORIGIN OF SNAKES (SERPENTES) FROM THE PERSPECTIVE OF VERTEBRATE EYE ANATOMY.

Limbless, snake-like bodies evolved independently among numerous squamate lineages, many of which exhibit terrestrial and fossorial or semifossorial ecologies

(Wiens & Slingluff, 2002). One might infer from these observations that similar conditions gave rise to snakes, but recent fossil discoveries (Caldwell & Lee, 1997; Lee et al., 1999; Tchernov et al., 2000; Rage & Escuillie, 2000) revived an alternative hypothesis that early snakes lived in marine environments (Nopsca, 1923). Specifically,

Cretaceous marine snakes with hind limbs, hypothesized to be transitional between lizard and snake body plans, provided the impetus for new phylogenetic analyses of snakes and their relatives. Some studies found these limbed marine snakes to be basal snakes, supporting the marine origin of snakes (Caldwell & Lee, 1997; Lee et al., 1999; Scanlon

& Lee, 2000; Rage & Escuillie, 2000). Others concluded they were not primitive snakes but were advanced (macrostomatan) snakes which had re-evolved legs (Zaher, 1998).

These analyses placed the extant, burrowing blindsnakes and anilioids as the most basal snakes, thus reaffirming the traditional view of a burrowing origin of snakes (Tchernov et al., 2000).

Other evidence relevant to question of snake origins comes from comparative vertebrate ophthalmology (Fig. 4.1). Specifically, the extreme structural and functional differences between the eyes of lizards (Fig. 4.1A and B) and of snakes (Fig. 4.1C and D) implies that snake eyes were dramatically altered during their origin from lizard ancestors

(Walls, 1940). The most substantial differences involve the structures directly associated with focusing an image onto the retina. Snakes focus by applying pressure to the vitreous body via enlarged peripheral iris muscles, thus forcing a rigid, spherical lens forward within the eyeball. Relaxation of those muscles results in passive retraction of the lens

(Michel, 1933; Sivak, 1977). That contrasts with the lizard mechanism in which robust ciliary muscles embedded in the choroid and anchored to bony elements in the sclera squeeze a thick annular pad bounding the soft, flattened lens (Walls, 1942). Given that lizards are ancestral to snakes, and that many superficially snake-like lizards are burrowers, one explanation for these ophthalmic observations was that snakes went through a burrowing phase in their origin, and in that low-light environment lost the typical lizard mechanism for precise image focusing. After the functional and structural degeneration of their eyes, snakes re-colonized light-rich environments and evolved a totally different focusing mechanism. That explanation is widely cited, with some subsequent modifications (Bellairs & Underwood, 1951; Underwood, 1970) as compelling evidence that snake ancestors had greatly reduced eyes and were burrowers

(Greene, 1997; Zaher & Rieppel, 2000; Coates & Ruta, 2000). The functional degeneration of snake eyes during their early evolution also is consistent with the similar fusion or loss of neural layers in the optic tectum observed in snakes and squamate species with reduced visual function (Senn & Northcutt, 1973).

Snake eyes, however, also bear many intriguing similarities to the eyes of aquatic vertebrates. Primitively aquatic animals, such as fishes and amphibians, have a rigid spherical lens that focuses by movement, usually toward the cornea, accomplished using one or more subsets of iridial musculature directly attached to the lens epithelium (Walls,

1942; Fernald, 1990). Secondarily aquatic animals, including mammals and birds, also have more spherical lenses than their terrestrial relatives (Walls, 1942; Sivak, 1975).

Among aquatic mammals, whales accommodate by forward lens movement (Supin et al.,

2001), and a similar mechanism was proposed for pinnepeds (West et al., 1991). Aquatic turtles and birds, such as cormorants use robust iris sphincter muscles to squeeze the anterior surface of the lens to focus underwater. In proposing similarities between the eyes of snakes and burrowing reptiles, Walls (Walls, 1940) did not compare the eyes of snakes to those of the only extant marine lizard, the Galapagos marine iguana

Amblyrhynchus cristatus Bell, whose eyes remain unstudied, nor did he compare them to other aquatic vertebrates. Similarly, aquatic species in general and the marine iguana, in particular, have yet to be investigated with regard to visual centers in the brain. Thus, the marine origin hypothesis has never been evaluated using comparative ophthalmic data.

Here, we apply parsimony and phenetic clustering methods to ophthalmic and orbital data across a wide array of vertebrate taxa to investigate the probable ecological conditions responsible for snake eye anatomy.

MATERIALS AND METHODS

I constructed a matrix containing 69 ophthalmic and orbital characters (Table 4.1,

Appendix B) across 53 vertebrate taxa compiled from the extensive literature

(Walls1942; Rochon-Duvigneaud, 1943; Duke-Elder, 1958; Underwood, 1970; Sillman, 73

1973; Sivak, 1977; Fite & Lister, 1981; Fernald, 1990; Murphy et al., 1990; Schmid et al., 1992; Pardue et al., 1993; Supin et al., 2001). These were subjected to parsimony analyses (which unites taxa based on derived similarity) and phenetic analyses (which groups taxa based on overall similarity).

In the parsimony analyses I treated 11 multi-state characters as ordered (according to morphoclines), and the remaining multi-state characters as unordered. Additionally, I performed analyses with all multi-state characters treated as ordered and with all multi- state characters treated as unordered. Prior to our analyses, I identified and removed seven cladistically uninformative characters. Multiple searches on the resulting matrix were performed using the Parsimony Ratchet (Nixon, 2000) in the tree-searching program NONA (Goloboff, 1999). In Winclada (Nixon, 1999), I scanned the trees produced by the Parsimony Ratchet for unsupported nodes (which were collapsed) and created a strict consensus of the remaining trees. I expected snakes to align either with varanoid lizards, if eye characters reflected mainly shared ancestry, or unrelated fossorial or aquatic taxa, if eye characters reflected mainly convergent adaptation. To determine which characters were responsible for each cluster in the consensus tree, the distribution of each of the 62 informative characters were analyzed separately under accelerated and delayed optimization strategies. Next, I entered a generally-accepted vertebrate phylogeny assembled from other studies (Sibley & Ahlquist, 1990; Helfman et al., 1997;

Lee, 2000; Zug et al., 2001; Murphy et al., 2001; Scally et al., 2001) and performed character optimization using our ophthalmic data to identify lineages at the end of long branches, i.e. which had undergone extensive eye evolution.

Finally, I converted the matrix to binary data for each character state and constructed a distance matrix from those data in SPSS using the pattern algorithm for calculation of dissimilarity. The distance matrix was inspected directly, rather than forced into a cluster diagram (phenogram), because such clustering usually distorts phenetic distances. I subdivided taxa into four ecological categories: aquatic, amphibious, terrestrial, and fossorial, and then ranked distances between each taxon and separately for scolecophidians (blindsnakes) and alethinophidians (advanced snakes). I also calculated the median rank for the distances within each ecological category as a score of overall similarity between that category and either blindsnakes or advanced snakes.

RESULTS

In the consensus tree from the analysis with selected characters ordered (Fig.

4.2A), taxa tend to align with each other based upon similar ecologies rather than accepted phylogenetic relationships. In particular, mammals and reptiles with reduced eyes emerge at the base of the tree. Snakes, which might be expected to align either with lizards, or with eye-reduced forms, instead cluster with aquatic forms, nesting deeply within fishes and caecilian amphibians.

Three characters that unite snakes with primitively aquatic taxa (fishes and caecilians) are a flattened cornea, a thickened corneal margin, and a spherical lens. These characters are shared by all primitively aquatic animals, and also expressed in secondarily aquatic animals to a greater degree than in their terrestrial relatives (Walls, 1942; Sivak,

1975). They thus appear to be associated with aquatic habits. Another character uniting snakes with the aquatic clade is the presence of blood vessels on the surface of the retina.

These vessels on the inner surface of the retina nourish the vitreous humor and retina. In 75

snakes, they likely represent the arrest of a developmental program (Jokl, 1923; Walls,

1942), but as they are not also expressed in secondarily aquatic forms, the correlation of this character with aquatic habits is less compelling. Three other characters uniting snakes with the aquatic clade are losses of (in snakes) various land vertebrate synapomorphies: the lachrymal gland, nictitans, and retractor bulbi muscles. Lachrymal glands, which usually secrete an aqueous fluid, were lost by numerous tetrapod lineages including some whales, murid rodents, penguins, some owls, Sphenodon, some geckos, pygopodids (legless geckoes), and snakes. The nictitans, or nictitating membrane also was lost by numerous taxa of varying ecologies: whales, echidnas, opossums, marsupial moles, largely burrowing squamates such as pygopodids, amphisbaenians, and dibamids, chameleons, and snakes. Retractor bulbi muscles are also synapomorphic for tetrapods and were subsequently lost by snakes and birds, and co-opted by caecilian amphibians to manipulate their tentacles. The broad ecological distribution of these three characters makes them ambiguous regarding the ecological origin of snakes.

The eye data were mapped onto a phylogeny depicting widely (but not universally) accepted relationships among extant vertebrates, synthesized from a number of other studies using different data (Helfman et al., 1997). Branch lengths were calculated for those data optimized only for unambiguous changes. The longest branch

(length = 27) leads to snakes (Scolecophidia plus Alethinophidia). The next three longest branches include those for dibamid and amphisbaenian squamates (length = 17), marsupial moles (length = 13), and talpid moles (length = 10). Both delayed and accelerated optimization identified the same long branches. Thus, snakes have indeed undergone substantial ophthalmic change, as have some burrowing forms. However, the

previous results show that the actual nature of the changes is very different. Note that many of the nodes in Fig. 4.2B are unsupported by any synapomorphies in ophthalmic characters, further demonstrating the lack of correlation between eye anatomy and phylogeny.

In the phenetic analysis, the eyes of blindsnakes and advanced snakes were, as expected, most similar to one another. When compared to other taxa, the eyes of advanced snakes shared the greatest similarity with mainly aquatic taxa: sharks, whales, sirenians, gars and lampreys, but also caecilians, shrews, mice, and opossums (Table 4.2).

Blind snake eyes were most similar to those of caecilians, shrews, mice, lungfishes, echidnas, lampreys, hagfishes, dibamids, and sirenians (Table 4.2). The median ranks

(Table 4.3) for distances between both advanced snakes and blindsnakes with the remaining taxa subdivided into ecological categories were least (nearest) for aquatic animals, indicating greatest overall similarity to aquatic taxa. The next greater median between blindsnakes and the remaining taxa was for fossorial groups, followed by amphibious taxa, and lastly terrestrial forms. For advanced snakes, the next greatest medians were for fossorial and terrestrial groups (tied), and followed by amphibious taxa.

Thus, the eyes of both groups of snakes are most similar to the eyes of aquatic rather than fossorial taxa, though the eyes of blindsnakes are also somewhat similar to those of fossorial species, as is expected given other convergent features (Lee, 1998).

DISCUSSION

It is not surprising that previous workers (Walls, 1940; Bellairs & Underwood,

1951; Underwood, 1970) considered the highly modified snake eye as evidence of a fossorial or sheltering ancestor, given that ophthalmic and orbital characters show the 77

greatest degree of change among burrowing taxa and snakes (Fig. 4.2B). However, the exact nature of the changes in snakes, and in burrowing taxa, are very different (Fig.

4.2A). Previous studies only compared snake eyes with those of lizards, and thus overlooked the striking ocular similarities between snakes and a variety of primarily aquatic vertebrates.

The placement of caecilians with snakes in our parsimony consensus tree reflects the ambiguity that has plagued attempts to understand the ecological forces that molded the unique snake body plan (Coates & Ruta, 2000). Caecilians contain species that are either burrowing or aquatic, and this cluster thus fits predictions of either an aquatic or burrowing origin of snakes. Indeed, these hypotheses are not mutually exclusive (Nopsca,

1923; McDowell, 1972; Rieppel, 1988). However, the nesting of the snake-caecilian

"clade" within a plexus of aquatic vertebrates (fish) strongly supports the aquatic hypothesis for snake origins. None of the characters supporting the fish-caecilian-snake cluster are shared with exclusively burrowing taxa. In contrast, three are shared with exclusively aquatic or amphibious taxa: the flattened cornea, thickened corneal margin, and spherical lens. Moreover, the loss of the retractor bulbi muscles in snakes, resulting in a condition convergent with primitively aquatic animals, does not necessarily imply visual reduction, since a similar loss has occurred in large-eyed forms with great visual acuity (birds). These ophthalmic characters thus constitute strong evidence that snakes had aquatic ancestors.

Figure 4.1. Functional anatomy of lizard (A) and snake (B) eyes, illustrating major differences between the two general types. C. Lizards focus by contracting large ciliary muscles ( bm, cm) anchored to scleral ossicles (so) thereby applying pressure to the lateral surface of the lens (ln) via the annular pad (ap). D. Snakes focus by moving their lens forward via increased pressure on the vitreous (vi) due to peripheral iris muscle (im) contraction. Abbreviations: an, anterior pad; bm, Brücke’s ciliary muscle; cb, ciliary body; ch, choroid; cm, Crompton’s ciliary muscle; cn, conus papilliaris; co, cornea; el, eye lid; fv, fovea; id, iris dilator muscle; is, iris sphincter muscle; ln, lens; re, retina; rv, retinal blood vessels; sc, scleral cartilage; sl, sclera; sp, spectacle; vi, vitreous; zf, zonular fibers.

Characters Taxa 0 1 2 3 4 5 6 7 8 9 10 11 Myxiniformes 0 0 0 - - - 0 0 0 0 0 0 Petromyzontiformes 0 0 0 - - - 0 1 1 1 1 1 Chondrichthyes 0 0 0 - 0 0 1 1 1 1 1 1 Acipenseriformes 0 0 0 - - - 0 1 1 1 1 1 Semionotiformes 0 0 0 - - - 0 1 1 1 1 1 Latimeria 0 0 0 - - - 0 1 1 1 1 1 Dipnoi 0 0 0 - - - 0 1 1 1 1 1 Teleostei [02] 0 0 - 0 2 0 1 [01] 1 [01] [01] Anura 0 0 [01] 1 [01] 0 2 1 1 1 1 1 Caudata 0 1 1 1 [012] 0 [02] 1 1 1 1 1 Gymnophiona 0 0 2 ? - - 0 1 1 1 1 2 Ornithorhyncus 0 1 2 2 0 0 2 1 1 1 1 1 Tachyglossa 0 1 1 2 0 0 0 1 1 1 1 1 Didelphidae 0 1 1 2 0 0 0 1 1 1 1 1 Macropodidae 0 1 1 2 0 0 2 1 1 1 1 1 Noteryctidae 0 ? ? ? 2 0 0 0 0 0 0 0 Soricidae 0 2 1 2 1 0 3 1 1 1 1 1 Talpidae 0 2 1 2 2 0 3 0 0 0 0 0 Primata 0 2 1 2 1 0 3 1 1 1 1 1 Felidae 0 2 1 2 1 0 2 1 1 1 1 1 Mustellidae 0 2 1 2 1 0 2 1 1 1 1 1 Sirenia 0 0 2 0 2 0 2 1 1 1 1 1 Pinnipedia 0 1 2 0 1 0 2 1 1 1 1 1 Cetacea 0 [01] 2 0 [12] 0 0 1 1 1 1 1 Sciuridae 0 2 1 2 1 0 3 1 1 1 1 1 Muridae 0 0 2 2 1 0 3 1 1 1 1 1 Cheloniidae 0 2 2 2 1 0 1 1 1 1 1 1 Chelydridae 0 1 2 2 1 0 1 1 1 1 1 1 Dermochelyidae 0 2 2 2 1 0 1 1 1 1 1 1 Emydidae 0 1 2 2 1 0 1 1 1 1 1 1 Testudinidae 0 1 2 2 1 0 1 1 1 1 1 1 Trionychidae 0 1 2 2 1 0 1 1 1 1 1 1 Sphenodontidae 0 0 2 2 1 0 1 1 1 1 1 1 Scincidae 0 1 2 2 [13] [012] [01] 1 1 1 1 1 Aniella 0 1 2 2 0 0 ? 1 1 1 1 1 Lacertidae 0 2 2 2 1 [01] 1 1 1 1 1 1 Dibamidae 0 ? ? ? - - 0 0 0 0 0 0 Amphisbaenia 0 0 2 2 - - 0 0 0 0 0 0 Iguanidae 0 2 2 2 1 [01] 1 1 1 1 1 1 Agamidae 0 2 2 2 1 [01] 1 1 1 1 1 1 Chamaeleonidae 0 2 2 2 2 0 0 1 1 1 1 1 Pygopodidae 0 0 2 2 3 2 0 1 1 1 1 1 Gekkonidae 0 [01] 2 2 [123] [012] [01] 1 1 1 1 1 Lanthanotidae 0 2 2 2 1 1 1 1 1 1 1 1 Varanidae 0 2 2 2 1 0 1 1 1 1 1 1 Scolecophidia 0 0 2 2 3 2 0 1 1 1 1 1 Alethinophidia 0 0 2 2 3 2 0 1 1 1 1 1 Crocodylia 0 1 2 2 1 0 1 1 1 1 1 1 Passeriniformes 0 2 1 2 0 0 1 1 1 1 1 1 Strigiformes 2 [01] 2 2 0 0 1 1 1 1 1 1 Falconiformes 0 2 2 2 0 0 1 1 1 1 1 1 Phalacrocoracidae 0 1 2 2 0 0 1 1 1 1 1 1 Sphenisciformes 0 0 2 2 0 0 1 1 1 1 1 1

Continued

Table 4.1 Character by taxon data matrix.

Table 4.1 continued

Characters Taxa 12 13 14 15 16 17 18 19 20 21 22 23 Myxiniformes 0 0 0 0 0 0 ? 0 1 - - - Petromyzontiformes 1 0 0 0 0 0 1 1 0 1 0 1 Chondrichthyes 1 0 0 0 1 0 0 1 0 1 0 1 Acipenseriformes 1 0 0 0 1 0 0 1 0 1 0 1 Semionotiformes 1 0 0 0 1 0 0 1 0 1 0 1 Latimeria 1 0 0 0 0 0 0 1 0 0 ? ? Dipnoi 1 0 0 0 1 0 2 1 1 0 0 1 Teleostei [01] 0 0 0 1 1 [23] 1 0 0 0 1 Anura 1 1 1 0 1 0 [01] 1 1 1 0 1 Caudata 1 1 1 0 1 0 [01] 1 [01] 1 0 1 Gymnophiona 1 2 2 0 0 0 1 1 1 0 0 1 Ornithorhyncus 1 1 0 0 0 0 0 1 1 1 0 1 Tachyglossa 1 1 0 0 0 0 0 1 1 0 1 1 Didelphidae 1 1 0 0 0 0 0 1 2 0 0 1 Macropodidae 1 1 0 0 0 0 0 1 2 0 0 1 Noteryctidae 0 0 0 0 0 0 0 ? ? ? ? ? Soricidae 1 1 0 0 0 0 0 1 1 0 0 1 Talpidae 0 0 0 0 0 0 0 1 1 0 0 0 Primata 1 1 0 0 0 0 0 1 2 0 0 1 Felidae 1 1 0 0 0 0 0 1 2 0 0 1 Mustellidae 1 1 0 0 0 0 0 1 2 0 0 1 Sirenia 1 1 0 0 0 0 0 1 1 0 1 1 Pinnipedia 1 1 0 0 0 0 0 1 0 0 1 1 Cetacea 1 1 0 0 0 0 0 1 0 1 1 1 Sciuridae 1 1 0 0 0 0 0 1 2 0 0 1 Muridae 1 1 0 0 0 0 0 1 2 0 0 1 Cheloniidae 1 1 1 0 1 0 0 1 1 0 0 1 Chelydridae 1 1 1 0 1 0 0 1 1 0 0 1 Dermochelyidae 1 1 1 0 1 0 0 1 1 0 0 1 Emydidae 1 1 1 0 1 0 0 1 1 0 0 1 Testudinidae 1 1 1 0 1 0 0 1 2 0 0 1 Trionychidae 1 1 1 0 1 0 0 1 1 0 0 1 Sphenodontidae 1 1 1 1 1 0 0 1 2 0 0 1 Scincidae 1 1 1 [01] 1 0 [03] 1 2 0 [12] 1 Aniella 1 1 1 1 1 0 0 1 2 0 0 0 Lacertidae 1 1 1 1 1 0 0 1 2 0 [12] 0 Dibamidae 0 0 0 0 0 0 3 0 1 0 0 0 Amphisbaenia 0 0 0 0 0 0 3 1 1 0 0 0 Iguanidae 1 1 1 1 1 0 0 1 2 0 [12] 0 Agamidae 1 1 1 1 1 0 0 1 2 0 [12] 0 Chamaeleonidae 1 1 0 0 1 0 0 1 2 0 [12] 0 Pygopodidae 1 1 0 0 1 0 3 1 1 0 0 0 Gekkonidae 1 1 1 [01] 1 0 [03] 1 [12] 0 [12] 0 Lanthanotidae 1 1 1 1 1 0 0 1 2 0 [12] 0 Varanidae 1 1 1 1 1 0 0 1 2 0 [12] 0 Scolecophidia 1 0 0 0 0 0 3 0 1 1 0 1 Alethinophidia 1 0 0 0 0 0 3 0 0 1 0 1 Crocodylia 1 1 ? ? 0 0 0 1 1 0 0 1 Passeriniformes 1 0 0 0 0 0 0 1 2 0 0 1 Strigiformes 1 0 0 0 0 0 0 1 1 0 0 1 Falconiformes 1 0 0 0 0 0 0 1 2 0 0 1 Phalacrocoracidae 1 0 0 0 0 0 0 1 1 0 0 1 Sphenisciformes 1 0 0 0 0 0 0 1 0 0 0 1

Continued

Table 4.1 Continued

Characters Taxa 24 25 26 27 28 29 30 31 32 33 34 35 Myxiniformes - - - 0 - - 0 0 0 0 - - Petromyzontiformes 0 0 0 1 0 0 0 0 0 0 0 0 Chondrichthyes 1 0 [01] 0 1 0 2 0 1 [013] 1 1 Acipenseriformes 1 0 0 1 1 0 2 0 0 0 1 0 Semionotiformes 1 1 0 1 1 0 2 0 0 0 1 0 Latimeria ? ? 1 ? 1 0 2 1 0 0 0 0 Dipnoi 1 0 0 0 1 0 1 0 0 1 0 0 Teleostei 1 1 [01] 1 1 0 [12] [012] [01] [013] 1 [012] Anura 1 0 0 0 1 2 [12] [01] 1 1 1 [01] Caudata 1 0 0 0 1 1 [012] 0 1 1 1 0 Gymnophiona 1 0 0 0 ? ? 0 0 0 0 0 1 Ornithorhyncus 1 0 0 0 1 2 2 0 1 ? 1 1 Tachyglossa 1 0 0 0 1 2 2 0 1 ? 1 2 Didelphidae 1 0 0 0 1 2 0 0 1 ? 3 0 Macropodidae 1 0 0 0 1 2 0 0 1 ? 3 2 Noteryctidae ? ? 0 0 ? ? 1 0 0 0 - - Soricidae 1 0 0 0 1 2 0 0 1 3 1 1 Talpidae 0 0 0 0 1 2 0 0 1 3 1 1 Primata 1 0 0 0 1 2 0 0 1 3 1 2 Felidae 1 0 0 0 1 2 0 0 1 3 1 2 Mustellidae 1 0 0 0 1 2 0 0 1 3 1 1 Sirenia 1 0 0 0 1 2 0 0 1 3 1 2 Pinnipedia 1 0 0 1 1 2 0 0 1 3 1 0 Cetacea 1 0 0 0 1 2 0 0 1 3 1 0 Sciuridae 1 0 0 0 1 2 0 0 1 3 1 2 Muridae 1 0 0 0 1 2 0 0 1 3 1 1 Cheloniidae 1 0 0 0 1 2 2 2 2 0 3 0 Chelydridae 1 0 0 0 1 2 2 2 2 0 3 0 Dermochelyidae 1 0 0 0 1 2 2 2 2 0 3 0 Emydidae 1 0 0 0 1 2 2 2 2 0 3 1 Testudinidae 1 0 0 0 1 2 2 2 2 0 3 2 Trionychidae 1 0 0 0 1 2 2 2 2 0 3 0 Sphenodontidae 1 0 0 0 1 2 2 2 2 3 2 1 Scincidae 1 0 0 1 1 2 2 2 2 3 2 2 Aniella 1 0 0 1 1 2 2 2 2 3 2 1 Lacertidae 1 0 0 1 1 2 2 2 2 3 2 2 Dibamidae 1 0 0 0 1 2 0 0 0 0 - - Amphisbaenia 1 0 0 0 1 2 [12] [01] 0 0 - - Iguanidae 1 0 0 1 1 2 2 2 2 3 2 2 Agamidae 1 0 0 1 1 2 2 2 2 3 2 2 Chamaeleonidae 1 0 0 1 1 2 1 2 2 3 2 2 Pygopodidae 1 0 0 1 1 2 2 2 2 3 [03] 0 Gekkonidae 1 0 0 1 1 2 [012] [12] 2 3 2 2 Lanthanotidae 1 0 0 1 1 2 2 2 2 3 2 2 Varanidae 1 0 0 1 1 2 2 2 2 3 2 2 Scolecophidia 1 0 0 0 1 1 0 0 2 0 4 0 Alethinophidia 1 0 0 0 1 [12] 0 0 2 [023] 4 [01] Crocodylia 1 0 0 0 1 2 2 0 2 2 3 1 Passeriniformes 1 0 0 0 1 2 2 2 2 3 2 2 Strigiformes 1 0 0 0 1 2 2 2 2 3 2 1 Falconiformes 1 0 0 0 1 2 2 2 2 3 2 2 Phalacrocoracidae 1 0 0 0 1 2 2 2 2 3 2 1 Sphenisciformes 1 0 0 0 1 2 2 2 2 3 2 1

Continued

Table 4.1 Continued

Characters Taxa 36 37 38 39 40 41 42 43 44 45 46 47 Myxiniformes - - 0 - 0 0 0 0 0 - - - Petromyzontiformes 1 0 2 1 0 1 1 0 0 0 0 0 Chondrichthyes [01] 1 0 - 0 1 1 0 0 0 0 0 Acipenseriformes 0 0 1 [01] 0 1 1 [01] 0 ? 1 1 Semionotiformes 0 1 2 1 0 1 1 1 0 1 1 1 Latimeria 0 1 0 ? 0 1 1 0 0 ? 1 1 Dipnoi 0 1 2 ? 0 1 [01] [01] 0 ? [01] 1 Teleostei [01] 1 [012] [01] [012] 1 1 1 0 1 0 1 Anura 0 1 2 1 0 1 1 1 0 1 [01] 1 Caudata ? 1 2 1 0 1 1 1 0 1 0 1 Gymnophiona 0 1 2 0 0 1 [01] 0 0 0 0 0 Ornithorhyncus 0 1 2 0 0 1 1 1 0 0 1 0 Tachyglossa 0 1 2 0 0 1 0 0 0 0 0 0 Didelphidae 0 1 2 ? 0 1 1 1 0 ? 1 0 Macropodidae 0 1 2 ? 0 1 1 1 0 ? 1 0 Noteryctidae - - ? ? 0 0 0 0 0 - - - Soricidae 0 1 1 0 0 1 [01] 0 0 0 0 0 Talpidae 0 1 1 0 0 1 1 0 0 0 0 0 Primata [01] 1 1 0 [012] 1 1 0 0 0 0 0 Felidae 0 1 1 0 0 1 1 0 0 0 0 0 Mustellidae 0 1 1 0 0 1 1 0 0 0 0 0 Sirenia 0 1 1 0 0 1 1 0 0 0 0 0 Pinnipedia 0 1 1 0 0 1 1 0 0 0 0 0 Cetacea 0 1 1 0 0 1 1 0 0 0 0 0 Sciuridae 1 1 1 0 0 [01] 1 0 0 0 0 0 Muridae 0 1 1 0 0 1 1 0 0 0 0 0 Cheloniidae 0 0 2 1 0 1 1 1 0 1 1 1 Chelydridae 0 0 2 1 0 1 1 1 0 1 1 1 Dermochelyidae 0 0 2 1 0 1 1 1 0 1 1 1 Emydidae 0 0 2 1 [01] [01] 1 1 0 1 1 1 Testudinidae 0 0 2 1 0 1 1 1 0 1 1 1 Trionychidae 0 0 2 1 1 1 1 1 0 1 1 1 Sphenodontidae 0 0 2 ? 1 1 1 0 1 ? 1 1 Scincidae 0 0 2 1 [01] 0 1 1 0 1 1 1 Aniella 0 0 2 1 0 0 1 1 0 1 1 1 Lacertidae 0 0 2 1 [01] 0 1 1 0 1 1 1 Dibamidae - - 1 ? 0 0 0 0 0 - - - Amphisbaenia - - 2 ? 0 0 1 0 0 0 [01] [01] Iguanidae 0 0 2 1 [01] 0 1 1 0 1 1 1 Agamidae 0 0 2 1 [01] 0 1 1 0 1 1 1 Chamaeleonidae 0 0 2 1 2 0 1 1 0 1 1 1 Pygopodidae 0 0 2 1 0 0 1 1 0 1 0 1 Gekkonidae [01] 0 2 1 0 [01] 1 1 1 1 1 1 Lanthanotidae 0 0 2 1 1 0 1 1 0 1 1 1 Varanidae 0 0 2 1 1 0 1 1 0 1 1 1 Scolecophidia 0 1 2 0 0 1 [01] 0 0 0 0 0 Alethinophidia [01] 1 2 0 0 [01] [01] [01] 0 0 0 0 Crocodylia 0 ? 2 0 0 1 1 1 0 1 0 1 Passeriniformes 0 ? 2 1 2 1 1 1 0 1 1 1 Strigiformes 0 ? 2 1 0 1 1 1 0 1 1 1 Falconiformes 0 ? 2 1 2 1 1 1 0 1 1 1 Phalacrocoracidae 0 ? 2 1 2 1 1 1 0 1 1 1 Sphenisciformes 0 ? 2 1 2 1 1 1 0 1 1 1

Continued

Table 4.1 Continued

Characters Taxa 48 49 50 51 52 53 54 55 56 57 58 59 Myxiniformes ? 2 ? ? 0 - - 0 0 0 0 0 Petromyzontiformes 1 2 1 0 0 0 0 0 0 0 0 0 Chondrichthyes 1 [012] 1 0 0 0 1 [02] 0 [012] [01] 1 Acipenseriformes 1 2 1 [01] 0 0 1 0 0 1 0 0 Semionotiformes 1 2 1 1 0 0 2 0 0 1 0 0 Latimeria 1 2 1 1 0 0 1 0 0 0 0 ? Dipnoi 1 1 1 0 0 0 1 0 0 0 0 0 Teleostei 1 1 1 1 0 0 [12] 0 [01] [02] [01] 1 Anura 1 2 1 1 0 0 0 0 0 [012] 0 1 Caudata 1 1 1 0 0 [01] 0 0 0 0 0 1 Gymnophiona ? 0 1 0 0 0 0 0 0 0 0 0 Ornithorhyncus ? 1 1 0 0 0 ? 0 0 0 0 2 Tachyglossa ? 1 1 0 0 0 ? 0 0 0 0 2 Didelphidae ? 1 1 1 0 0 2 0 0 0 0 1 Macropodidae ? 1 1 1 0 0 ? 0 0 0 0 1 Noteryctidae - ? ? ? 0 ? 0 0 0 0 0 0 Soricidae 1 1 1 1 0 0 0 0 0 0 0 1 Talpidae 1 1 1 1 0 0 0 0 0 0 0 1 Primata 1 1 1 1 0 0 [01] [02] 0 0 0 1 Felidae 1 1 1 1 0 0 1 [02] 0 [01] 0 1 Mustellidae 1 1 1 1 0 0 1 0 0 [02] 0 1 Sirenia 1 1 1 1 0 0 1 0 0 2 0 2 Pinnipedia 1 1 1 1 0 0 1 [02] 0 [02] 0 2 Cetacea 1 1 1 1 0 0 1 0 0 2 [01] 2 Sciuridae [01] 1 1 1 0 0 0 0 0 0 0 1 Muridae 1 1 1 1 0 0 0 0 0 0 0 1 Cheloniidae 1 1 1 0 0 0 0 1 0 0 0 2 Chelydridae 1 1 1 0 0 0 0 1 0 0 0 2 Dermochelyidae 1 1 1 0 0 0 0 1 0 0 0 2 Emydidae 1 1 1 0 0 0 0 1 0 0 0 2 Testudinidae 1 1 1 0 0 0 0 0 0 0 0 2 Trionychidae 1 1 1 0 0 0 0 1 0 0 0 2 Sphenodontidae 1 0 1 0 0 0 0 0 0 0 0 1 Scincidae 0 2 1 0 1 0 0 0 [01] 0 0 1 Aniella 0 2 1 0 1 0 0 0 0 0 0 1 Lacertidae 0 2 1 0 2 0 0 0 0 0 0 1 Dibamidae - 2 1 0 0 0 0 0 0 0 0 0 Amphisbaenia 0 2 1 0 0 0 0 0 0 0 0 0 Iguanidae 0 2 1 0 2 0 0 0 [01] 0 0 1 Agamidae 0 2 1 0 2 0 0 0 0 0 0 1 Chamaeleonidae 0 2 1 0 2 0 0 0 0 0 0 1 Pygopodidae 0 2 1 0 1 0 0 0 0 0 0 0 Gekkonidae [01] 2 1 0 1 0 0 [02] 1 [01] [01] 1 Lanthanotidae 0 2 1 0 2 0 0 ? 0 0 0 1 Varanidae 0 2 1 0 2 0 0 0 0 0 0 1 Scolecophidia 1 0 1 1 0 1 0 0 0 0 0 0 Alethinophidia 1 0 1 1 [01] 1 0 [012] 1 [012] 0 [12] Crocodylia 1 0 1 0 0 1 2 0 0 0 0 1 Passeriniformes 1 1 1 0 2 0 0 0 [012] 0 0 1 Strigiformes 1 1 1 0 2 0 0 0 1 0 0 1 Falconiformes 1 1 1 0 2 0 0 [02] 1 0 0 1 Phalacrocoracidae 1 1 1 0 2 0 0 1 0 0 0 2 Sphenisciformes 1 1 1 0 2 0 0 0 0 0 0 2

Continued

Table 4.1 Continued

Characters Taxa 60 61 62 63 64 65 66 67 68 Myxiniformes 0 0 0 0 0 0 0 0 0 Petromyzontiformes 0 0 0 2 0 1 0 0 0 Chondrichthyes 1 [012] 0 1 2 0 0 0 0 Acipenseriformes 0 [01] 0 0 0 0 0 0 0 Semionotiformes 0 0 0 1 2 0 0 0 0 Latimeria ? 0 0 0 0 0 0 0 0 Dipnoi 0 [02] 0 0 0 0 0 0 0 Teleostei 1 [012] 0 2 1 0 1 0 0 Anura 1 [012] 0 1 2 0 1 0 0 Caudata 1 0 0 2 2 0 0 0 0 Gymnophiona 0 0 0 0 0 0 0 0 0 Ornithorhyncus 0 0 0 0 0 0 0 0 0 Tachyglossa 0 0 0 0 0 0 0 0 0 Didelphidae 0 1 ? 0 0 0 1 0 0 Macropodidae 0 2 ? 0 0 0 1 0 0 Noteryctidae 0 0 - - - 0 1 0 0 Soricidae 0 0 0 0 0 0 0 0 0 Talpidae 0 0 0 0 0 0 0 0 0 Primata 1 0 2 0 0 0 1 0 1 Felidae 1 [01] 2 0 0 0 1 0 0 Mustellidae 1 [012] 2 0 0 0 1 0 0 Sirenia 1 2 0 0 0 0 0 0 0 Pinnipedia 1 1 2 0 [02] 0 1 0 1 Cetacea 1 2 [02] 0 [02] 0 [01] 0 [01] Sciuridae 1 0 2 0 0 0 1 0 0 Muridae 0 0 0 0 0 0 1 0 0 Cheloniidae 0 0 1 0 0 0 2 0 0 Chelydridae 0 0 1 0 0 0 2 0 0 Dermochelyidae 0 0 1 0 0 0 2 0 0 Emydidae 0 0 1 0 0 0 2 0 0 Testudinidae 0 0 1 0 0 0 2 0 0 Trionychidae 0 0 1 0 0 0 2 0 0 Sphenodontidae 1 1 1 0 0 0 2 0 0 Scincidae 1 0 [01] 0 0 0 2 2 0 Aniella 1 1 1 0 0 0 2 2 0 Lacertidae 1 0 1 0 0 [02] 2 2 0 Dibamidae 0 0 0 0 0 0 0 0 0 Amphisbaenia 0 0 0 0 0 0 0 0 0 Iguanidae 1 0 1 0 0 [02] 2 2 0 Agamidae 1 0 1 0 0 [02] 2 2 0 Chamaeleonidae 1 0 1 0 0 [02] 2 2 0 Pygopodidae 0 1 1 0 0 0 [02] [02] 0 Gekkonidae 1 [01] [01] 0 0 0 [02] [02] 0 Lanthanotidae 1 0 1 0 0 0 2 2 0 Varanidae 1 0 1 0 0 0 2 2 0 Scolecophidia 0 0 0 0 0 0 0 0 0 Alethinophidia 1 [012] 0 1 2 0 0 0 0 Crocodylia 1 1 1 0 0 0 2 0 0 Passeriniformes 1 0 1 0 0 2 2 2 0 Strigiformes 1 0 1 0 0 2 2 2 0 Falconiformes 1 0 1 0 0 2 2 2 0 Phalacrocoracidae 1 0 1 0 0 2 2 0 0 Sphenisciformes 1 0 1 0 0 2 2 0 0

Figure 4.2. (A) The strict consensus of 12 most parsimonious trees from the matrix of 69 ophthalmic and orbital characters, each with length = 295, consistency index = 0.35, retention index = 0.77, shows how ophthalmic characters reflect common ecology, rather than common ancestry, among vertebrates. (B) A traditional vertebrate phylogeny synthesized from other studies and data (Sibley & Ahlquist, 1990; Helfman et al., 1997; Lee, 2000; Zug et al., 2001; Murphy et al., 2001; Scally et al., 2001): length = 405, consistency index = 0.25, retention index = 0.64. In both trees, the two groups of snakes are at the top. Yellow boxes show the distribution and relationships within the squamate clade for both trees. Numbers above each branch length indicate that length for optimised ophthalmic and orbital characters. Branch lengths of zero indicate nodes unsupported by ophthalmic characters. 86

Taxa Alethinophidia Scolecophidia Taxa Alethinophidia Scolecophidia Alethinophidia 0.000 0.004 Dibamidae 0.026 0.012 Scolecophidia 0.004 0.000 Sphenisciformes 0.028 0.024 Chondrichthyes 0.015 0.023 Talpidae 0.030 0.017 Gymnophiona 0.017 0.006 Sphenodontidae 0.030 0.024 Muridae 0.017 0.010 Pygopodidae 0.030 0.019 Soricidae 0.018 0.009 Strigiformes 0.030 0.025 Cetacea 0.018 0.015 Amphisbaenia 0.031 0.015 Mustellidae 0.019 0.017 Phalacrocoracidae 0.031 0.024 Sirenia 0.019 0.014 Falconiformes 0.032 0.029 Petromyzontiformes 0.021 0.011 Teleostei 0.034 0.038 Semionotiformes 0.021 0.016 Cheloniidae 0.034 0.020 Didelphidae 0.021 0.013 Chelydridae 0.034 0.020 Felidae 0.021 0.017 Dermochelyidae 0.034 0.020 Dipnoi 0.022 0.010 Emydidae 0.034 0.024 Tachyglossa 0.022 0.010 Testudinidae 0.036 0.022 Pinnipedia 0.022 0.020 Trionychidae 0.036 0.022 Sciuridae 0.022 0.016 Passeriniformes 0.037 0.030 Anura 0.023 0.027 Gekkonidae 0.039 0.039 Crocodylia 0.023 0.017 Scincidae 0.041 0.036 Latimeria 0.024 0.014 Aniella 0.041 0.039 Ornithorhyncus 0.024 0.016 Chamaeleonidae 0.048 0.039 Macropodidae 0.024 0.015 Varanidae 0.053 0.044 Primata 0.024 0.018 Lanthanotidae 0.054 0.045 Myxiniformes 0.025 0.011 Lacertidae 0.055 0.045 Acipenseriformes 0.025 0.014 Iguanidae 0.055 0.047 Caudata 0.026 0.021 Agamidae 0.056 0.046 Noteryctidae 0.026 0.016

Table 4.2. Pairwise distances comparing Alethinophidia (advanced snakes) and Scolecophidia (blindsnakes) to other vertebrate taxa. Distances are shown in ascending order (i.e. decreasing similarity) for advanced snakes.

Aquatic Amphibious Terrestrial Fossorial Alethinophidia 11.500 32.500 29.500 29.500 Scolecophidia 11.000 30.000 38.000 17.500

Table 4.3. Medians of ranked pairwise distances from blindsnakes and advanced snakes for vertebrate taxa grouped by ecology

APPENDIX A. SPECIMENS

Abbreviations: COL – Colubridae, CRO – Crotalidae.

California Academy of Sciences, Herpetology Collections

FAM Genus Accession Number COL Ahaetulla fronticincta 212162, 212163, 212164, 212165, 212166, 212167, 212168, 212169, 212170, 212171, 212172, 212173, 212174, 212175, 212176, 212177, 212178, 212179, 212180, 212181, 212182, 212183, 212184, 212185, 212186, 212187, 212188, 212189, 212190, 212191, 212192, 212193, 212195, 212196, 212197, 212198, 212199, 212200, 212201, 212202, 212203, 212204, 212205, 212206, 212207, 212208, 212209, 212210, 212211, 212213, 212214, 212215, 212216, 212217, 212218, 212219, 212220, 212221, 212222, 212223, 212224, 212225, 212226, 212294

COL Chrysopelea ornata 12384, 12385, 12386, 12387, 12388, 125206, 13097, 13098, 13100, 13101, 13102, 136751, 16707, 17256, 17257, 179160, 18444, 18446, 18790, 204852, 208433, 210708, 212084, 39528, 55139, 55140, 8483, 8484, 8485, 8486, 8487, 8536, 8866, 8867, 8868

COL Chrysopelea paradisi 125172, 125331, 125332, 128032, 128982, 129207, 131698, 132942, 133711, 135656, 135657, 137180, 140230, 140234, 15330, 15331, 15332, 15810, 18572, 20691, 26612, 27311, 27313, 27314, 27471, 28003, 28147, 60470, 60574, 60942, 62427, 62491

COL Dispholidus typus 103144, 111830, 122310, 122311, 131170, 141751, 147998, 148037, 152790, 153360, 159919, 168906, 168907, 173815, 66029, 86011

COL Imantodes cenchoa 119916, 140968, 140986, 144534, 145214, 163738, 163820, 163864, 163905, 175934, 66940, 66944, 66945, 79004, 79005, 79006, 79007, 79008, 84180, 93204

Continued

Appendix A Continued

Abbreviations: COL – Colubridae, CRO – Crotalidae.

California Academy of Sciences, Herpetology Collections

FAM Genus Accession Number COL Leptodeira annulata 114048, 134468, 134469, 134470, 135701, 139843, 140989, 140993, 140996, 140997, 140999, 141007, 141008, 141009, 142472, 142473, 142474, 142475, 142476, 142477, 142478, 142479, 142586, 143152, 156685, 156686, 169522, 169533, 169554, 169590

COL Oxybelis aneus 113723, 113724, 113725, 113726, 116161, 116162, 116163, 116164, 116173, 116174, 116175, 116176, 116177, 116206, 116207, 116289, 12476, 12495, 14547, 4080, 66939, 71344, 71432, 71433, 74398, 79009, 79010, 79011, 94623, 94624

COL Psammophis schokari 100029, 100030, 120498, 120499, 120500, 120538, 120710, 120711, 120712, 120713, 120987, 13243, 134163, 135150, 135529, 135530, 135564, 135979, 135980, 136066, 136475, 136543, 136553, 138748, 138996, 139519, 139751, 140416, 140417, 140418, 140419, 140420, 140421, 140493, 140499, 148594, 148610, 149451, 86375

COL Trimorphodon biscutatus 121081, 122445, 132167, 132168, 132170, 132171, 13219, 135254, 140948, 142645, 143411, 144559, 15384, 169623, 169632, 24052, 34048, 4087, 97110

Continued

Appendix A Continued

Abbreviations: COL – colubridae, CRO – crotalidae. National Museum of Natural History FAM Species Accession Numbers COL Boiga irregularis 22357, 22358, 22359, 22362, 22367, 22377, 22383, 22389, 22220, 22221, 22222, 22223, 22224, 22225, 22226, 22227, 22228, 22229, 22230, 22231, 22256, 22282, 22283, 22284, 22285, 22287, 22288, 22289, 22290, 22291, 22292, 22293, 22294, 22295, 22296, 22297, 22298, 22299, 22300, 22301, 22302, 22303, 22304, 22305, 22306, 22307, 22308, 22309, 22310, 22311, 22312, 22313, 22314, 22315, 22316, 22317, 22318, 22319, 22320, 22321, 22322, 22323, 22324, 22325, 22326, 22327, 22328, 22329, 22330, 22331, 22333, 22334, 22335, 22336, 22337, 22338, 22339, 22340, 22341, 22342, 22343, 22344, 22346, 22347, 22348, 22349, 22350, 22351, 22352, 22353, 22354, 22355, 22356, 22357, 22358, 22359, 22360, 22361, 22362, 22363, 22364, 22365, 22366, 22367, 22368, 22369, 22370, 22371, 22372, 22373, 22374, 22375, 22376, 22377, 22378, 22379, 22380, 22381, 22382, 22383, 22384, 22385, 22386, 22387, 22388, 22389, 22390, 22391, 22392, 22393

CRO Agkistrodon bilineatus 30492, 523659, 82180, 84055, 85093, 85094

CRO Agkistrodon blomhoffi 15421, 15423, 23436, 31866, 34038, 34039, 56200, 67027, 67031, 67032

CRO Agkistrodon intermedius 136575, 203374, 203376, 203377, 203378, 203380, 203381, 203382, 81917

CRO Agkistrodon strauchi 107665, 66632, 66633, 67813, 81979, 81980, 81981

CRO Atropoides nummifer 110426, 110428, 123709, 123712, 129426, 61994, 6747, 86863

CRO Bothriechis bicolor 127973, 46511

CRO Bothriechis lateralis 339805

CRO Bothriechis marchi 319942, 337488, 337489

CRO Bothriechis nigroviridis 32580, 68856

CRO Bothriechis schlegelii 13558, 14040, 14044, 19522, 19740, 25169, 29855, 32599, 32600, 32602, 32603, 32604, 32605, 32606, 32607, 32608, 72360

Continued

Appendix A Continued

Abbreviations: COL – colubridae, CRO – crotalidae.

National Museum of Natural History

FAM Species Accession Numbers CRO Bothriopsis albocarinata 165183, 165184, 165185, 165187, 165189, 165190, 165193

CRO Bothriopsis bilineatus 165262, 165263, 165265, 165268, 165270, 165271, 165274, 165275, 165276, 165277, 165279, 165280, 538552

CRO Bothriopsis punctata 124258, 165286, 20629, 232521, 72355

CRO Bothriopsis taeniata 165231, 165282, 165283, 165284, 165285, 167625, 232513, 232516

CRO Bothrops alternatus 100706, 100722, 165459, 165467, 165468, 165469, 38182, 69288, 69291, 69292, 69294, 69295, 71135, 71136, 71137, 76321, 76322

CRO Bothrops asper 123496, 123497, 148515, 165195, 165196, 20693, 20789, 32149, 323227, 333104, 333105, 338263, 5886, 82156

CRO Bothrops atrox 11188, 135234, 165336, 165337, 17731, 17732, 17733, 17734, 232510, 306172, 401, 477, 531703, 531704, 531705, 6177

CRO Bothrops jararaca 1000000, 56176, 69319, 69320, 69322, 69324, 69325, 69326, 69327, 69328, 69330, 69331, 69332, 71139, 76313, 98628, 98629, 98631, 98632

CRO Bothrops jararacusa 165509, 165510, 165511, 165512, 207676, 208138, 217841, 253589, 253890, 253891, 39054, 69300, 69301, 69302

CRO Bothrops moojeni 165485, 253143, 253144, 342425, 342426, 38181

CRO Bothrops neuweidi 165494, 165495, 347915, 69296, 69297, 69299, 71141, 73420, 76318, 76319

CRO Deinagkistrodon acutus 145561, 157010, 203364, 64024, 73139

CRO Hypnale hypnale 203372, 254536, 254537, 254730, 267792, 267794, 531216

CRO Lacheis muta 165008, 165009, 165010, 165966, 192284

CRO Ophryacus undulatus 304849, 46435, 46436, 46466 Continued 92

Appendix A Continued

Abbreviations: COL – colubridae, CRO – crotalidae.

National Museum of Natural History

FAM Species Accession Numbers CRO Porthidium dunni 110419, 110421, 110423, 110425, 196292, 30265, 30266, 30269, 46422

CRO Porthidium nasutum 110415, 151711, 151712, 165317, 165319, 165320, 165321, 20625, 22442, 232520, 338629, 536001, 536002

CRO Trimeresurus albolabrus 102949, 134074, 53441, 70342, 72076, 72077, 72078, 84834, 90369

CRO Trimeresurus flavomaculatus 266613, 266614, 266615, 266635, 266636, 319043, 319044, 319045, 319046, 319047, 319050, 326683, 34709, 36111, 37872, 37873, 56014

CRO Trimeresurus flavoviridis 123312, 123313, 123483, 123485, 141693, 31818

CRO Trimeresurus jerdonii 76246, 85158, 93860, 94015, 94016, 94092

CRO Trimeresurus 140817, 140818, 140820, 142534, 63417, 82652, 84634 mucrosquamatus CRO Trimeresurus trigonocephalus 254789, 254966, 267797, 267799

CRO Tropidolaemus 103526, 26526, 267803, 267806, 33161, 33162, 33163, 33180, 51647

Continued

Appendix A Continued

Abbreviations: COL – colubridae, CRO – crotalidae.

The Ohio State University, Museum of Biological Diversity, Division of Higher Vertebrates FAM Species Accession Numbers COL Coluber constrictor 128, 129, 139, 140, 143, 594, 1752, 1773, 2191, 128, 129, 131, 132, 133, 134, 137, 138, 139, 140, 141, 142, 143, 1432, 1432, 144, 1455, 1456, 1479, 1544, 1546, 1547, 1578, 1578, 1598, 1655, 1677, 1677, 1677, 1677, 1773, 1794, 1794, 18, 18, 1923, 2166, 2191, 558

COL Elaphe obsoleta 82, 90, 97, 104, 105, 573, 1023, 1578, 1595, 1690, 2214, 100, 102, 1023, 1093, 1365, 1550, 1575, 1690a, 1690b, 1691, 1717, 2194, 2214,

2216, 2228, 2294, 329, 608, 82, 90, 92, 95a, 95b, 95c

COL Heterodon platyrhinos 5, 200, 203, 204, 210, 216, 1089, 1826, 2091, 207.2

COL Lampropeltis triangulum 48, 49, 56, 61, 63, 66, 71, 1543, 1545, 1836, 1085, 1086, 1087, 1238, 1239, 1357, 1433, 1434, 1437, 1443, 1549, 41, 42, 43, 44, 45, 46,

464, 47, 48, 49, 50, 51a, 51b, 52, 53, 56, 58, 60, 61, 62, 65, 66, 67, 68, 69, 69, 70a, 70b, 70c, 72

COL Nerodia sipedon 461, 1327, 1355, 1375, 1463, 1640, 1731, 1841, 2193, 2267

COL Opheodrys aestivus 113, 115, 117, 121, 1487, 1611, 1637, 1641, 1761, 1774, 111, 112, 113, 114, 115, 116a, 116b, 116c, 117, 118, 119, 120, 121, 122, 123, 1232, 124, 1487, 1568, 1611, 1630, 1637, 1641, 1702, 1774

COL Thamnophis sirtalis 249, 251, 253, 1359, 1425, 1435, 2218, 2222

CRO Agkistrodon contortrix 19, 23, 36, 37, 38, 1051, 1270, 1325, 1760, 2096

CRO Agkistrodon piscivorous 682, 683, 1187, 1482.1, 1482.2, 1555, 2127, 2245

APPENDIX B: CHARACTER STATE DESCRIPTIONS

Numbers correspond to the character numbers in Table 4.1. Numbers in parentheses represent character states. The * indicates characters coded as ordered. All remaining characters were unordered. The † indicates characters found to be uninformative for our analysis.

1† Eyeball shape: hemispherical (0), flattened tubular (1). 2* Lachrymal gland: absent (0), small (1), large (2) 3* Harderian gland: absent (0), small (1), large (2) 4 Orbital drainage: none (0), soft tissue canal (1), bony canal (2) 5 Eyelid mobility: both mobile (0), reduced mobility in one lid (1), reduced mobility in both (2), lids fused (3) 6 Eyelid transparency: both opaque (0), one eyelid transparent (1), both eyelids transparent (2) 7 Nictitans: none (0), actively mobile (1), passively mobile (2), immobile (3) 8 Inferior oblique: absent (0), present (1) 9 Superior oblique: absent (0), present (1) 10 Inferior rectus: absent (0), present (1) 11 Superior rectus: absent (0), present (1) 12 Medial rectus: absent (0), present (1) 13 Lateral rectus: absent (0), present (1) 14 Retractor bulbi: absent (0), present (1), co opted (2) 15 Levator bulbi: absent (0), present (1), co opted (2) 16 Bursalis: absent (0), present (1), co opted (2) 17 Protractor lentis: (transversalis) absent (0), present (1) 18† Retractor lentis: absent (0), present (1) 19 Spectacle: none (0), primary (1), secondary (2), tertiary (3) 20 Choroid and sclera: fused (0), separate (1) 21 Corneal curvature: flattened (0), convex but little or no sulcus (1), extremely convex with distinct sulcus (2) 22 Corneal margin: as thick as center (0), thicker than center (1) 23 Corneal epithelium: stratified squamous (0), cornified stratified squamous (1) 24 Descemet's lamina and mesothelium: absent (0), present (1) 25 Corneal substantia propria: absent (0), present (1) 26 Autochthonous layer of cornea: absent (0), present (1) 27† Corneal pigment: none (0), present (1) 95

28 Pectinate or annular ligament: absent (0), present (1) 29† Aqueous production: diffusion through cornea (0), secreted internally (1) 30 Aqueous drainage: diffusion (0), general lymphatic (1), canal of Schlemm (2) 31* Scleral cartilage: absent (0), reduced (1), robust (2) 32* Scleral ossicles: absent (0), reduced (1), robust (2) 33 Iris muscle fibers: absent (0), smooth (1), striated (2) 34 Pupillary contraction: none (0), slow dilation, slow contraction (1), slow dilation, fast contraction (2), fast dilation and contraction (3) 35 Lens attachment: none (0), zonular fibers only (1), distinct ringwulst (2), reduced ringwulst (3), anterior pad (4) 36* Lens shape: spherical (0), slightly lenticular (1), markedly lenticular (2) subspherical along the equatorial axis (3) 37 Lens pigment: none (0), yellow (1) 38 Lens sutures: absent (0), present (1) 39* Pigmented epithelium: unpigmented (0), sparse (1), heavy (2) 40 Photomechanical response of pigment epithelium: absent (0), present (1) 41* Central fovea: absent (0), shallow (1), deep central (2) 42 Rods: absent (0), present (1) 43 Cone: absent (0), present (1) 44 Double cones: absent (0), present (1) 45 Double rods: absent (0), present (1) 46 Myoid contractility: none (0), present (1) 47 Oil droplets: absent (0), present (1) 48 Parabaloids: absent (0), present (1) 49 Rhodopsin or porphyropsin: absent (0), present (1) 50* Optic nerve condition: fascicular (0), some septa (1), no septa (2) 51† Choroidal vasculature: none (0), present (1) 52 Retinal vasculature: none (0), present (1) 53* Conus papilliaris or pecten absent (0), small or simple (1), large or elaborate (2) 54 Corneal blood vessels: none (0), present (1) 55 Tapetum: absent (0), choroidal (1), retinal (2) 56 Accessory focus: absent (0), iris sphincter deforms lens (1), stenopaic pupil (2) 57* Temporal fovea: absent (0), shallow (1), deep (2) 58 Pupil shape when dilated: round (0), vertical ellipse (1) horizontal ellipse (2) 59† Pupillary operculum: absent (0), present (1) 60* Iris sphincter: absent (0), normal (1), robust (2) 61 Iris dilator: absent (0), present (1) 62 Pupil shape when contracted: round (0), vertical slit (1), horizontal slit (2) 63 Lens deformation: none (0), squeezed by ciliary contraction (1), elastic recoil during ciliary contraction (2) 64 Lens movement rearward: none (0), passive (1), active (2) 65 Lens movement forward: none (0), passive (1), active (2) 66 Deformation of eyeball: none (0), external muscles (1), internal muscles (2) 67 Brücke’s ciliary muscle: absent (0), smooth (1), striated (2) 68 Crompton’s ciliary muscle: absent (0), smooth (1), striated (2)

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