University of California, San Diego

UNIVERSITY OF CALIFORNIA, SAN DIEGO

Heterochrony and the evolution of an aggressive display of the Sarcastic Fringehead

(Blenniiformes: Neoclinus blanchardi)

A Thesis submitted in partial satisfaction of the requirements for the degree Master of Science

Marine Biology

Watcharapong Hongjamrassilp

Committee in charge:

Professor Philip A. Hastings, Chair Professor Gregory W. Rouse Professor Jennifer R. Taylor

2016

The Thesis of Watcharapong Hongjamrassilp is approved and it is acceptable in quality and form for publication on microfilm and electronically:

Chair

University of California, San Diego

2016

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DEDICATION

This thesis is dedicated to my family members and Fondue who always support and encourage me to pursue my dream to be a scientist.

This thesis is also dedicated to my first ethology teacher, Associate Professor

Usanee Yodyingyuad, Ph. D., who introduced me to the study of animal behavior, and

Dr. Chitchai Chantangsi, who always generously gives me valuable suggestions and encouragement while I have problems with my graduate student life and teaches me how to be a good scientist.

TABLE OF CONTENTS

Signature Page……………………………………………………………………………iii

Dedication Page…………………………………………………………………………..iv

Table of Contents………………………………………………………………………….v

List of Figures…………………………………………………………………..………...vi

List of Tables……………………………………………………………………...……...ix

Acknowledgements…………………………………………………………...…………...x

Abstract of the Thesis…………………………………………..……………….……….xii

Introduction………………………………………………………………………………..1

Materials and Methods………………………………………………………..………...... 5

Results……………………………………………………………….…………………...10

Discussion……………………………………………………………….……………….17

Figures………………………………………………………………………….………...22

Tables……………………………………………………………….……..……….….…40

References……………………………………………………………….…………….…44

LIST OF FIGURES

Figure 1 Phylogenetic tree of Neoclinus based on morphological characters with M. sandae as an outgroup.The yellow lines show the posterior extent of the maxilla. .... 22

Figure 2 Genital papilla used for sex identification of Neoclinus spp. (Left) Urogenital area of female without pointed genital papilla. (Right) Urogenital area of male with pointed genital papilla shown in the dashed circle...... 23

Figure 3 Homologous landmarks of three species of Neoclinus fishes shown in upper case letters...... 23

Figure 4 Method of digitizing 36 pseudo-landmarks on the maxilla from a cleared-and- stained specimen of N. stephensae (SIO 86-60). The first mark (red dots in upper figure) was made at similar points on the maxilla of all three species. The second and third marks are digitized at the midpoint between the first and second marks, respectively...... 24

Figure 5 N. blanchardi exhibiting a typical guarding display by projecting the head out from its shelter (shell of wavy turban snail, Megastraea turbanica). This display is common in most hole-dwelling blennies...... 25

Figure 6 N. blanchardi performing the gaping display to an intruder by opening the mouth and lateral flaring of the maxillae...... 26

Figure 7 Lateral view of N. blanchardi performing the gaping display. The red arrow points to the head-bending gesture which is unusual among fishes...... 27

Figure 8 Kinematic diagram showing a flowchart of transitions between behaviors observed in N. blanchardi (N=32)...... 28

Figure 9 Reflectance property of the buccopalatal membrane under (A) normal light and (B) UV light...... 28

Figure 10 Fluorescence in the buccopalatal membrane of N. blanchardi. (A) shows the margin of the mouth under normal visible light. (B) shows the margin of the mouth under 470 nm. Green color indicates the fluorescence property of that area...... 29

Figure 11 Frontal view of N. blanchardi while performing a gaping display. This display not only allows the species to show the adductor mandibulae complex but also the well- armed oral dentition...... 29

Figure 12 Buccopalatal membrane of N. blanchardi (SIO 14-194) with an open mouth (left) and closed mouth (right). The enlargement of this membrane permits the unusual flaring of the maxillae during the gaping display...... 30

Figure 13 Cross section of buccopalatal membrane of N. blanchardi under 400X magnification...... 30

Figure 14 Cleared-and-stained maxilla of three species of Neoclinus fishes (N. blanchardi = SIO 60-67-61A; N. uninotatus = SIO uncataloged; N. stephensae = SIO 86- 60)...... 31

Figure 15 Dissected specimen of 210 mm SL male N. blanchardi (SIO 64-95-61A) showing muscles and bones involved in the gaping display...... 32

Figure 16 Drawing illustrating bones and adductor mandibulae muscles in N. blanchardi. (A) Innermost layer of skull; (B) inner layer of muscles; (C) outermost layer of muscles...... 33

Figure 17 Scatter plot of log-transformed maxilla length against PC1 score for three Neoclinus species. Regression line of each species is shown in the graph with the value of slope, y-intercept and correlation in the table...... 34

Figure 18 Scatter plot of log-transformed calcified maxilla length against PC1 score for three Neoclinus species. Regression line of each species is shown in the graph with the value of slope, y-intercept and correlation in the table...... 35

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Figure 19 Scatter plot of log-transformed uncalcified maxilla length against PC1 score for N. blanchardi juveniles, males, and females...... 36

Figure 20 Thin plate spline of maxilla deformation from N. stephensae to N. uninotatus after procrustes transformation. Red and blue gradient shows expansion and contraction of each area, respectively. In this deformation, expansion is greatest in the posterior part of maxilla, while some parts in the anterior part show contraction...... 37

Figure 21 Thin plate spline of maxilla deformation from N. stephensae to N. blanchardi after procrustes transformation. Red and blue gradient shows expansion and contraction of each area, respectively. In this deformation, expansion is also greatest in the posterior part of the maxilla, while most contraction is in the anterior part of the maxilla...... 38

Figure 22 Thin plate spline of maxilla deformation from N. uninotatus to N. blanchardi after procrustes transformation. Red and blue gradient shows expansion and contraction of each area, respectively. As in the other deformation comparisons, expansion is greatest in the posterior part of the maxilla, while contraction occurs in the anterior part...... 39

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LIST OF TABLES

Table 1 List of specimens used in this study from the Marine Vertebrate Collection at Scripps Institution of Oceanography, UCSD...... 40

Table 2 Descriptive ethogram of territorial behaviors in N. blanchardi...... 42

Table 3 Percentage of maxilla length (ML) per standard length (SL) from the biggest available specimen of three species of Neoclinus...... 42

Table 4 PC1 scores of each studied characters from 55 specimens of Neoclinus…….....43

ACKNOWLEDGEMENTS

I would like to express my deep gratitude to my advisor, Professor Philip A.

Hastings for giving me an invaluable opportunity to become his student here at SIO.

Having a chance to study here not only opens my eyes into another world of science but also gives me an inestimable experience. Thank you for allowing me to conduct my research under your kindly and helpful advice, teaching me to think scientifically and academically, and always having an unlimited time for me when I have problems. Most importantly, thank you for your understanding of our cultural differences and helping me to improve my English skills with your unlimited patience.

My sincere thanks are also due to Professor Greg Rouse for giving me critical comments on my thesis and also teaching me wonderful classes on marine invertebrates and phylogenetics which really strengthen my skills in evolutionary biology. I would also like to thank Associate Professor Jennifer Taylor for her suggestions about graduate student life and introducing me to the biomechanics field of study. Her teaching is amazing and has changed my perspective about physics to become the interesting topics as same as biology. Also, thank you Professor Nicholas Holland for teaching me to do histological techniques with his patience.

Many thank are also due to my first office mate, H.J. Walker, who always brings me back while I feel blue with his sense of humor and also makes me feel excited with a thousand of his extraordinary experiences especially the most excited one “Oarfish head” which I will never forget until my dying day. Moreover, I would like to thank Cynthia

Klepadlo and Noelle Bowlin for being good friends and always supporting me with their amazing suggestions.

My life as an international student in SIO would not be complete without these following people. I would like to thank my SIO friends especially Angela Zoumplis, Jack

B. Pan, Jennifer Le, Arturo Ramírez Valdez, Kaitlyn Lowder, Garfield Kwan, Steven

Kelley, Sumaetee Por Tangwancharoen for their friendship and for creating unforgettable moments during my stay in San Diego. Very special thanks are due to Varoth

Lilascharoen for helping me with everything since I first arrived in San Diego until now.

Last in sequence but not the least important, I owe more than thanks to my family members which includes my parents and grandparents, my elder brother and sister, and

Fondue for their support and encouragement throughout my life and for helping me pursue my dream for being a scientist. I promise to bring these priceless experiences back and improve our beloved country and if possible to the world.

ABSTRACT OF THE THESIS

Heterochrony and the evolution of an aggressive display in the Sarcastic Fringehead

(Blenniiformes: Neoclinus blanchardi)

Watcharapong Hongjamrassilp

Master of Science in Marine Biology

University of California, San Diego, 2016

Professor Philip A. Hastings, Chair

Signals are evolved to help animals communicate and thereby maximize their inclusive fitness. To increase the efficiently of communication, selection favors animals that are able to increase the effectiveness or amplify their signals over evolutionary time.

Relatively few studies of signal amplification have been conducted on fishes although fishes include many species whose behavior is well-known. The Sarcastic Fringehead

(Neoclinus blanchardi, Teleostei) exhibits an extreme version of a common aggressive display, the “gaping display” in which an open mouth is presented toward an opponent.

This species has extremely long maxillae that are flared laterally during the display. In

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this study, display behaviors of three live specimens and 14 videos of N. blanchardi were observed, described, and analyzed. The comparative anatomy of N. blanchardi and the related species N. uninotatus and N. stephensae, were examined to document the structures related to the unusual gaping display. Fluorescence of the buccopalatal membrane was investigated. Finally, geometry morphometry, including the truss network system and thin-plate spline, and PCA were used to study the evolution of the maxilla under the heterochrony framework. The results show that the unusual gaping display is used for intraspecific territorial defense. Three main morphological modifications related to amplification of this display are enlargement of the buccopalatal membrane, enlargement of the adductor mandibulae muscle complex, and lengthening of the maxilla.

The elongate maxilla of N. blanchardi evolved via acceleration (faster growth compared to outgroups) and hypermorphosis (continued growth to a larger body size), both forms of peramorphosis. All of these characters play a role in the amplification of the gaping display in N. blanchardi.

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Introduction

In animal communication systems, signals are among the most important elements used to convey information between individuals. Animal signals are defined as structures or behaviors that when displayed can change another organism's behavior

(Searcy and Nowicki, 2005; Smith and Harper, 2003). They can appear in many different forms including electrical, visual, tactile, chemical, and acoustical displays depending on the sensory system of the receiver (Laidre & Johnstone, 2013). Moreover, signals are used in many contexts such as mating, socialization, and territory defense. Having reliable signaling is important for survival and increased reproductive success, and as a consequence they are often under strong selection (Dall et al., 2005; Wilgers & Hebets,

2011).

The reliability of signals (Számadó, 2011) can vary depending on how the signals evolved. One hypothesis is if a signaler and receiver share a common interest in the outcome, a signal will have greater reliability (Smith and Harper, 1995). For example, in aposematic warning coloration (Sherratt and Beatty, 2003), both signaler, such as venomous insects, and receiver, such as a bird, can benefit from a reliable signal because the venomous insect will survive and the bird does not ingest toxins from the insect. This type of signal is recognized as an evolutionarily stable signal (Bergstrom and Lachmann,

1997; Johnstone and Grafen, 1992; Rodríguez-Gironés et al, 1996) and comes along with the stability of the signaling system (Donath, 2007).

However, some animals are able to cheat by showing unreliable or dishonest signals that mimic existing reliable signals (Smith et al, 1988; Wilson, 2007). An example of dishonest signals is Batesian mimicry (Dawkins and Guilford, 1991; Marek and Bond, 2009) in which harmless animals mimic an extant warning signal from a harmful species to deceive a predator. Once an unreliable signal exists, it can disturb the dynamic of a signaling system because receivers may ignore a reliable signal. In this case, both signalers who produce reliable and unreliable signals will lose benefit leading to an unstable dynamic of the signaling system (Donath, 2007). Nevertheless, unreliable signals can still exist if they are in low frequency (Dawkins and Guilford, 1991)

The evolutionary process of signal diversification in animals is generally poorly understood. Display behaviors have been hypothesized to drive the diversification of morphological traits related to those displays (Bateson, 2004; Duckworth, 2009; Huey et al., 2003; Smith and Harper, 2003) as well as features that increase the effectiveness or amplify those displays (Hasson, 1990, 1997). Teleost fishes are the most diverse vertebrate lineage (Helfman et al, 2009) and exhibit a wide variation in morphology and behaviors making them a good model for studying the role of behaviors in signal and morphological diversification.

One common territorial behavior which can be found in many fishes and other vertebrates is the so-called "gaping display" in which an open mouth is presented toward an intruder (Horback et al, 2010; Sibley, 1955; Lappin and Huska, 2005; Ritte, 2008).

The purpose of the gaping display in agonistic encounters is to advertise the signaler’s size, the size of a weapon (jaws and teeth), or a structure that infers a weapon's

3 performance (Lappin et al, 2006). For example, mandrills gape to advertise their large and prominent teeth (Bout and Thierry, 2005), and the gaping display of male collared lizards (Crotaphytus sp.), reveals muscles of the adductor mandibular complex (AMC) that are a reliable signal indicating their bite force (Lappin et al, 2006).

Fishes in the blenniiform genus Neoclinus are known to show the gaping display for territorial defense (Lindquist, 1975). Neoclinus includes eleven species of benthic shallow-water fishes, three in the eastern Pacific, distributed from northern California to

Baja California (N. blanchardi, N. uninotatus, and N. stephensae), and eight in the western Pacific, known from Japan and South Korea to northern Taiwan (Fukao, 1987;

Murase, 2010; Love, 2011). Phylogenetic relationships based on morphological characters (unpublished data) (Figure 1) reveals that N. blanchardi is a sister taxon to N. uninotatus and both are sister taxa to N. stephensae. The western Pacific species are hypothesized to share a common ancestor, and are sister to this clade, with

Mccoskericthys sandae as the sister group to the entire genus (Lin and Hastings, 2011,

2013). Neoclinus fishes have been hypothesized to originate in the eastern Pacific

(Hastings, 2009), while some members dispersed into western Pacific, probably through the Aleutian Islands during an interglacial period (Hubbs, 1953; Stephens 1961)

While gaping displays can be found among other Neoclinus species, the gaping display of the Sarcastic Fringehead (Neoclinus blanchardi) is exceptional in that it includes lateral flaring of the extremely long maxillae. In addition, this display is made more conspicuous (or amplified in the sense of Hasson, 1990) by a bright color inside the oral cavity and a yellow margin of the jaws.

To decipher the pattern of visual signal amplification in N. blanchardi, basic information about their biology is needed. However, the basic behavioral and ecological information about members of Neoclinus are poorly known, especially that of N. blanchardi. Thus, in this study, I investigated (1) the general behavior including the unusual gaping display in N. blanchardi; (2) the morphological characters related to the extreme gaping display in N. blanchardi; and (3) the role of heterochrony in the evolution of the maxilla in Neoclinus using the truss network based on homologous landmarks and geometric morphometry.

Materials and Methods

1) Samples

In this study, preserved specimens of the three eastern Pacific (EP) species (N. blanchardi, N. uninotatus, and N. stephensae), as well as specimens of three Japanese species were obtained from the Marine Vertebrate Collection (MVC) at Scripps

Institution of Oceanography. The specimens were first identified to sex based on the morphology of the genital papilla located at the posterior end of the urogenital opening

(Figure 2). Males have a pointed papilla while females have a rounded papilla. Some juvenile, male, and female specimens were selected for gonadal histology to confirm the reliability of this gender identification method. A list of the specimens used in this study is shown in Table 1.

2) Behavioral observation of N. blanchardi

The general behaviors of N. blanchardi, including the gaping display, were recorded for live specimens and from videos available on the internet. Three live

N. blanchardi (2 ♂ 1 ♀) were collected by SCUBA at 30 m depth from the La Jolla

Canyon and held in aquaria for behavioral observations under the approval of IACUC

(protocol number S00080). The fishes were first acclimated in 95-liter aquaria for one month before quantifying their behaviors. In addition, 14 video clips of N. blanchardi from anonymous divers were obtained from youtube.com. Thirty-two intraspecific interactions between 25 individuals (3 live specimens and 22 from 14 video clips) were observed. A mirror was presented to the three live specimens to test their reaction to their

6 own image. Display behaviors were recorded by hand-held video camera (Sony FDR

AX333) and described. An ethogram was constructed to describe the actions performed in territorial behaviors. A kinematic diagram was used to show the sequence of various territorial behaviors.

3) Description of morphologies related to the gaping display in N. blanchardi

The gaping display in N. blanchardi is different from that of other Neoclinus as far as known in that the maxillae are flared outward. This extreme display is made possible by enlargement of membranes between the jaw elements and by muscles of the adductor mandibularis complex. From our observations, this display occurs frequently in male-male encounters, thus I examined the morphology of males in preserved specimens of N. blanchardi, N. uninotatus, and N. stephensae. The muscles and membranes were identified according to the definitions of Datovo and Vari (2013). Some specimens were selected for double straining of cartilage and bone following the protocol of Datovo and

Bockmann (2010) to facilitate study of the attachment of muscles with bones. This included a destaining step in which specimens were soaked in distilled water for 3 days

(changed every day) to remove color from the muscle. Specimens were then moved to

50 % isopropanol for 2 weeks (changed once per week). The complete destained specimens had little stain in the muscle while the bone and cartilage were red and blue, respectively. For the osteology study of these fishes, clearing and double staining followed the protocol of Taylor and van Dyke (1985). Drawings and photographs were made of all relevant anatomical features.

The fluorescence properties of the inside of the mouth cavity were examined from a freshly dead specimen of N. blanchardi using an epifluorescence stereoscope (Nikon

SMZ1500 with 100 W mercury lamp and filter cube with excitation at 450–490 nm and longpass emission barrier >500 nm; Melville, NY, USA). The pictures were taken by

QImage Retiga 2000R color digital camera.

4) Heterochrony study of the maxilla in Neoclinus species

The maxilla of N. blanchardi is extremely long, extending posterior to the head in large males. To study the evolutionary development of this maxilla in N. blanchardi, the heterochrony framework was used to describe and compare the pattern of growth of the maxilla in three closely related species of the genus. Ideally heterochrony compares character size or shape in developmental time (McKinney, 2013). However, the age of each preserved specimen could not be determined, therefore, body size was used as a proxy for developmental time. A series of undistorted specimens from the MVC was selected for inclusion in the study: 21 specimens of N. blanchardi (12 ♂, 9 ♀: SL 27.19 -

245 mm), 13 specimens of N. uninotatus (6 ♂, 7 ♀: SL 52.51-180 mm), and 17 specimens of N. stephensae (9 ♂, 8 ♀: SL 47.42 – 82.99 mm). Single specimens of four

Japanese species were also examined. Each specimen was photographed in lateral view with an included scale bar, and traditional truss distances (Strauss and Bookstein, 1982) were measured from the photographs. These included the following seven homologous landmarks (Figure 3): (1) anterior tip of nasal bone, (2) insertion of first dorsal-fin spine,

(3) insertion of first of dorsal-fin ray, (4) upper tip of dorsal hypural bone, (5) lower tip of ventral hypural bone, (6) insertion of first anal-fin spine, and (7) base of pelvic-fin spine.

These landmarks were indicated by insect pins inserted in a silicone plate on the left side of each specimen prior to photographing it with a digital camera (Nikon D3200).

Landmarks in the images were digitized in a computer and the coordinate points were obtained using tpsDig 2.0 (Rohlf, 2004). Coordinate points for all landmarks were used to generate 13 truss distances using PAST 3.11(Hammer et al, 2001). In addition to these 13 truss distances, the following nine characters were measured directly from specimens with a Pro-Max Electronic Caliper: (1) head length, (2) upper jaw length, (3) eye diameter, (4) distance from base of pelvic fin to base of anal fin, (5) body depth at anal area, (6) predorsal distance, (7) standard length, (8) first anal soft fin ray length, and (9)

5th dorsal fin ray length. All data from 13 truss distances along with nine mensural characters were log-transformed and analyzed by principal component analysis (PCA) under the covariance matrix (Bookstein et al, 1985) in PAST 3.11. PC1 score, which is representative of body size, was plotted against log-transformed maxilla length, calcified maxilla length, and uncalcified maxilla length (see below). The results were analyzed using ANCOVA in PAST 3.11 to compare the differences of slope and/or y-intercept among the three study species. Ratio of maxilla length per standard length was determined from all specimens of the three species and compared using ANOVA in

PAST 3.11.

5) Shape deformation of the maxilla in Neoclinus species

The maxilla of N. blanchardi is not only larger than that of related fishes, but also differs in shape. The deformation direction of the maxilla in these three species of

Neoclinus was investigated using thin-plate spline (TPS) deformation (Thompson, 1917;

Bookstein, 1989) to quantify the shape changes in N. blanchardi relative to the other species. This method can visualize the change from one shape to another shape by generating a deformation grid using homologous landmarks from two different shapes.

The difference between the homologous landmarks will show as “bending energy” on the grid. The maxilla was dissected from the largest available adult male specimen for each species. Each maxilla was photographed using a digital camera to obtain a 2D shape image. While homologous landmarks of this structure are unclear, 36 pseudo-landmarks were used instead. The maxilla images were digitized (Figure 4) using tpsDig 2.0 to obtain coordinate points. The coordinate points of pseudo-landmarks from the three species were procrustes-transformed for uniform scaling purpose before the data were analyzed by thin plate spline in PAST 3.11 (Hammer and Harper, 2008). A deformation grid was used to demonstrate the changes in maxilla shape. In addition, the maxilla of the two largest species was processed for standard paraffin histology and slides were stained with hematoxylin and eosin to examine the cellular structure of the posterior portion in

N. blanchardi and N. uninotatus.

Results

1) General description of the behavior of N. blanchardi including the gaping display

From my observations, the gaping display in N. blanchardi is different from that of other Neoclinus in that the head is raised while the lower jaw is depressed, the premaxillae are projected only slightly, while the maxillae are flared outward at approximately a 90 degree angle from the body axis (Figure 6, Figure 7). Male N. blanchardi normally live in holes of available objects underwater such as vacant shells of the wavy turban snail (Megastraea turbanica), crevices in rocky substrates, and discarded items such as cans and bottles. These serve as refuges and as egg depositions sites

(Lindquist, 1981; Hastings and Petersen, 2010) and are vigorously defended from conspecifics, especially by males.

Regarding territorial behavior, when confronted with other fish species, Sarcastic

Fringeheads interact by burst swimming and biting their intruder or sometimes retreating inside the shelter depending on the size of the intruder. In contrast, if approached by other individuals of the same species, they typically perform a gaping display. The gaping display starts by opening the mouth, flaring the maxilla outward, and bending head upward (Figure 6, Figure 7). While the mouth is open, each individual uses the mouth to push the opponent. The smaller individual generally retreats from the area without fighting, but if individuals are similar in size, a fight with biting may ensue.

Sometimes, gaping behavior occurs when no other fish is around their shelter and resembles a “yawn” as seen in numerous other fishes. When presented with a mirror

11 image, the fish perform the gaping display (N=3) indicating that they can recognize individuals of the same species but have no self-recognition. Habituation was also observed in this study with males declining to gape after several presentations of the mirror. No gaping display was observed in live specimens in females in this study, either when presented with a mirror or approached by another conspecific. However, the occurrence and frequency of this display by females in the field is unknown.

All observed territorial behaviors in N. blanchardi are described in an ethogram

(Table 2) and kinematic diagram that explains the sequence of territorial behaviors

(Figure 8). No gaping display was shown to other animal species during observations suggesting that this display is used only for intraspecific communication. When a conspecific intruder swam near to its shelter, male N. blanchardi performed a gaping display in 69% of the encounters, but performed no gaping display in 31% of the encounters. In the latter case, the intruder was smaller and left the area quickly. This implies that additional external morphological characters serve as an index of size when opponents differ greatly in size. These may be overall head size, an enlarged, brightly colored maxilla, or the bright spot on the dorsal fin of males.

2) Morphological traits related to the gaping display in N. blanchardi

Three morphological characters in the cranial area of N. blanchardi are related to the unusual gaping display of this species. These are (1) the buccopalatal membrane

(projugal laminar and inferior labial membrane), (2) the maxilla and anterior maxillary joint, and (3) the adductor mandibulae muscles.

2.1) Buccopalatal membrane

The buccopalatal membrane generally comprises dense irregular connective tissue located between the maxilla (upper jaw), and the dentary (lower jaw). This membrane is dramatically larger in N. blanchardi compared to the other members of the genus

Neoclinus as well as most other fishes (Figure 12). The enlargement of this membrane allows the jaws to open wide and permits significant lateral movement of the maxillae. It may also help to return the maxillae into the resting position when the mouth is closed.

In live male and female N. blanchardi, this membrane is brown in color with a thick, yellow edge starting from the posterior end of the maxilla extending along the infralabial ligament (Figure 9A). This yellow membrane is visible even with the mouth closed. Under 470 nm light, this membrane is fluorescent (Figure 10) appearing greenish.

Under UV light, most areas of the membrane reflect UV light making the membrane appear purple (Figure 9B). Results from histological study showed that buccopalatal membrane consists of two type of connective tissue: dense regular connective tissue and dense irregular connective tissue (Figure 13). The dense regular connective layer is located on the external side of the membrane while dense irregular connective layer is located on the internal side.

2.2) Maxilla

The maxilla of male N. blanchardi shows an extreme enlargement in size and shape compared to females and both sexes of the other two species. The ratio of maxilla length and standard length is significantly different among three species (P<0.001;

ANOVA) and the percentage of maxilla length per standard length of the biggest specimens of the three species is shown in Table 3 indicating that these three species differ in relative maxilla length. In large adult N. blanchardi, the maxilla can extend posteriorly well past the posterior margin of the head. Histological investigation of the maxilla shows that entire of maxilla consists of acellular bone. In cleared-and-stained specimens of N. blanchardi and N. uninotatus, the posterior portion of this bone does not retain Alizarin red, a stain specific to calcium, while in N. stephensae, the western Pacific species of Neoclinus, and M. sandae the entire maxilla retains the red stain. This implies that the posterior portion of the elongate maxilla of N. blanchardi and N. uninotatus is uncalcified bone, while the maxilla of other Neoclinus species is fully calcified to its posterior tip (Figure 14).

2.3) Adductor mandibulae muscles

As in most other fishes, the adductor mandibulae muscles of Neoclinus species consist of three main parts: the par rictalis, par malaris, and par stegalis (Datovo & Vari

2013). These muscles were identified in Neoclinus based on the bones to which they attach. The par rictalis is a muscle bundle which attaches to the preoperculum, quadrate, and lower jaw, the par malaris attaches on the hyomandibular, dorsal portion of the preopercle and the ectopterygoid, and the par stegalis is covered by the par malaris and par rictaris and attaches on the metapterygoid (Figure 15, Figure 16). These muscles in all three eastern Pacific species are the same in attachment site but apparently differ in relative size. In N. blanchardi, ligaments from the anterior part of the par stegalis connect to the external process of the maxilla. This muscle may be one factor that helps to control

14 the movement of maxilla. When N. blanchardi shows the gaping display, a portion of the adductor manibulae complex (AMC) can be observed inside the oral cavity (Figure 11).

3) Heterochrony study of maxilla in Neoclinus fishes

PC1 from the PCA accounts for 94.56 % of the variation in the 13 truss distances and nine external measurable characters. Loading factors of PC1 shown in Table 4 are all positive. Therefore, PC1 score is used to represent overall body size. In most cases, body size has a direct correlation with developmental time, so, in this study, body size is used as a proxy for developmental time. Love (1991) reported similar maximum ages of N. blanchardi (6 years) and N. uninotatus (7 years) indicating that at least these two species have similar developmental times.

The relationships between maxilla length and PC1 score (body size) are shown in

Figure17 for three EP species of Neoclinus, and the value of the y-intercept, slope, and correlation are shown in the table in Figure 17. All three regression lines show positive correlation between maxilla length and PC1 score. The rate of growth of the maxilla as inferred by the slope of this regression line is similar in N. blanchardi and N. uninotatus

(P =0.699). However, the rate of maxilla growth in both species is significantly higher than in N. stephensae (P < 0.000; ANCOVA). The relative length of the maxilla in N. blanchardi is larger than N. uninotatus. and N. uninotatus is larger than N. stephensae. In

Neoclinus species from the western Pacific, as well as that of the outgroup species,

Mccoskericthys sandae, the relative length of the maxilla is equal to or less than that of N. stephensae. Given the hypothesized phylogenetic relationships, the simplest explanation for the enlargement of the maxilla in N. blanchardi is that it evolved via a combination of

15 two types of peramorphosis, acceleration and hypermorphosis. In acceleration the development of the feature occurs at a faster rate, i.e., the slope of the regression line is steeper, while in hypermorphosis the feature develops for a longer period of time, i.e., as reflected by a larger body size under the assumption that body size is a proxy for developmental time (McKinney and McNamara, 1991; McNamara, 2012; McKinney,

2013).

Comparing the length of the calcified and uncalcified portions of the maxilla separately with body size (PC1) reveals that the calcified portion has a similar slope in N. blanchardi and N. uninotatus (P = 0.604) and both have a steeper slope compared to N. stephensae (P < 0.0001; Figure 18). This implies that the increase in length of the maxilla of N. blanchardi and N. uninotatus occurs in the uncalcified portion. All three regression lines for PC1 score and the calcified maxillary length in three species show positive correlation. Thus, the calcified maxilla of N. blanchardi and N. uninotatus show peramorphosis via acceleration compared to N. stephensae. Although N. blanchardi and

N. uninotatus have a similar rate of growth in the maxilla, the maxilla in N. blanchardi has delayed offset of growth compared to N. uninotatus which is peramorphosis via hypermorphosis.

The relative length of the maxilla is sexually dimorphic in N. blanchardi, but insufficient data are available to confidently test for dimorphism N. uninotatus and N. stephensae. Comparison of growth trajectories of male and female N. blanchardi (Figure

19) indicates that the relatively larger maxilla of males is the result of acceleration (faster rate of growth) and hypermorphosis (males grow to a larger size) in the uncalcified

16 posterior portion of the maxilla. The increase in growth rate of the maxilla in males begins around the time of sexual maturation of N. blanchardi. After maturation, the slope of the growth is significant greater in males compared to females (P = 0.047).

Finally, the results from the thin-plate spline analysis show comparative deformation of the maxilla in the three study species (Figure 20, Figure 21, Figure 22).

Comparing the shape change from N. stephensae to N. uninotatus, most of the area of the maxilla of N. uninotatus expands posteriorly, with the greatest expansion in the uncalcified part, while the anterior area shows contraction (Figure 20). A similar pattern is seen comparing N. stephensae to N. blanchardi and N. uninotatus to N. blanchardi except in these comparisons, the anterior part is more strongly condensed (Figure 21,

Figure 22). In both N. blanchardi and N. uninotatus the greatest expansion occurs in the posterior, uncalcified portion. These results are consistent with the heterochrony analysis implicating peramorphosis in the enlargement of maxilla in N. uninotatus and especially in N. blanchardi.

Discussion

In an attempt to explain the evolution of signal amplification of the gaping display in N. blanchardi, information about basic behavior, comparative morphology, and patterns of heterochrony for jaw morphology were examined. Results show that male N. blanchardi display the unusual gaping display for territorial defense and this display evolved for intraspecific communication. The context of this gaping display is similar to gaping display in other members of Neoclinus and also the closely related group, chaenopsid blennies (Lindquist, 1975). However, the mechanisms differ in that the mouth is more widely opened and the maxillae are flared outward making an especially large gaping display in N. blanchardi. The display exposes the well-armed dentition on the dentary, premaxilla, vomer and palatine bones, as well as the anterior portion of the adductor mandibulae muscles (Figure 11).

The ability to show this unusual gaping display in N. blanchardi is determined by having unique morphological modifications in three characters, the buccopalatal membrane, the adductor mandibulae muscles, and the maxilla. The buccopalatal membrane in N. blanchardi is dramatically enlarged compared to other fishes.

Histological study reveals that this membrane consists of both dense regular and irregular connective tissue while other fishes consist of just dense irregular connective tissue

(Datovo and Bockmann, 2010). Having dense regular connective tissue may help to facilitate the gaping display because fibers in the regular connective tissue are oriented in a single direction that would increase support for a unidirectional pull (Cormack, 2001).

This membrane allows the jaws to be opened widely and the maxilla to flare laterally

18 during the gaping display. Moreover, the area inside this membrane has a fluorescence property on the yellow edge of the mouth, as well as a UV reflectance property at the center of membrane. Because N. blanchardi can be found between 3-73 meters depth

(Love, 2011), there is a possibility that the yellow color, fluorescence, and UV reflectance property improve the signaling, but whether or not this species has the appropriate receptors and thus the importance of these visual signals in the environment, have not been demonstrated. The light yellow margin apparently functions as an amplifier (Hasson, 1989, 1990, 1991, 1997), providing information about the body size of the displaying male and thus his competitive ability in territorial defense.

Regarding the adductor mandibulae muscles, they normally are involved in mouth opening and closing in fishes (Winterbottom, 1974; Gosline, 1971). However, in N. blanchardi, these muscles are especially large and thick and can be seen inside their mouth during the display (Figure 11). This situation is similar to the male collared lizard

(Crotaphytus sp.) in which the adductor mandibulae complex (AMC) serves as an honest signal inferring biting performance (Lappin et al, 2006). However, the relationship of

AMC size and biting force has not been tested for N. blanchardi.

The last morphological character related to the gaping display is the maxilla. My results show that N. blanchardi has an extremely long maxilla with an uncalcified posterior part. This novel morphological character seen in males of this species and to a lesser extent in males of N. uninotatus evolved via acceleration, an increase in growth rate, and hypermorphosis, continued growth in this feature associated with the increase in body size seen in these two species compared to other species in the genus. In N.

19 blanchardi males, the accelerated rate of growth begins at approximately the size of attainment of sexual maturity and is seen primarily in the uncalcified posterior portion of the maxilla. This accelerated growth is likely associated with increased levels of hormones related to sexual maturity as shown for secondary sex characters in male fishes

(Pandey, 1969; Turner, 1960).

Phylogenetic analysis based on morphological characters (unpublished data) suggests that N. blanchardi is a sister taxon to N. uninotatus, and together both species are sister to the third eastern Pacific species, N. stephensae. This clade appears to the sister to the western Pacific members of the genus, and the tropical eastern Pacific species, Mccoskerichthys sandae, is sister to the entire genus Neoclinus. The relative maxilla length is similar in M. sandae (Rosenblatt and Stephens, 1987), the western

Pacific clade [N. okazaki, N. chihiro, N. bryope (Fukao, 1987); N. nudus (Fukao, 1990);

N. monogrammus and N. nudiceps (Murase et al,2010)] and N. stephensae. In all of these species, the maxilla extends posteriorly to the level of posterior orbit or slightly beyond it. The maxilla of N. uninotatus is relatively longer, extending well past the orbit to just before the posterior margin of the preopercle, while that of N. blanchardi is even longer, extending well-past the posterior margin of the head. This pattern of increase in relative size of the maxilla is similar to the increase in their maximum body size (Table 3) within

Neoclinus. Although not examined in the present study, the size of the buccopalatal membrane and the adductor mandibulae muscles may also increase in relative size in these fishes via similar patterns of heterochrony.

Among the 11 species of Neoclinus and over 80 species the related chaenopsid blennies, N. blanchardi is the largest species. As all members of this clade are hole- dwelling (Hastings and Springer, 1994) and these shelters serve both as refuges and as egg-deposition sites, making them key resources for male reproductive success (Hastings,

1986; Hastings and Petersen, 2010). Maximal body size in this group may be driven by the availability of suitably-sized shelters. While related species are known to compete for shelters (Hastings and Galland 2010), competition for suitable shelters may be especially intense in large males of N. blanchardi. This may have played a role in the evolution of the novel features that serve to amplify the gaping display and presumably increase their ability to compete for shelters.

Although this work answers some questions about the evolution of the gaping display in N. blanchardi and some related morphological traits, there are still unanswered questions regarding the evolution of this display. More ecological and behavioral data are needed to quantify the cost of this unusual morphology, and to test the reliability of the signal, such as the size of the AMC as a measure of fighting ability.

Among other fish species with similarly large maxillae is the Long-jawed

Mudsucker, Gillichthys mirabilis (Gobiidae; Barlow, 1963). Like the Sarcastic

Fringehead, males of these gobies have extremely long maxillae that extend to near the posterior margin of the head and they perform a gaping display in which the maxillae are flared outward (Crabtree, 1985; Weisel, 1947). This species also inhabits burrows in the substrate that are likely the foci of aggressive interactions and, like N. blanchardi, it is relatively large compared to closely related species. Convergence in these species may be

21 related to the prevalence of the gaping display in shelter defense in these fishes and peramorphosis as a consequence of their increased body size.

Figures

an outgroup. an

M. sandae M.

the maxilla. the

based on morphological characters with characters morphological on based

Neoclinus Neoclinus

Phylogenetic tree of of tree Phylogenetic

Figure of extent posterior yellow The the show lines 22

Figure 3 Homologous landmarks of three species of Neoclinus fishes shown in upper case letters. (A) anterior tip of nasal bone, (B) base of first dorsal-fin spine, (C) base of first dorsal-fin ray, (D) upper hypural bone, (E) lower hypural bone, (F) base of first anal-fin spine, and (G) base of pelvic-fin spine. Bold lines show truss distance measured for the morphometric analysis.

st 1 mark

nd 2 mark

rd 3 mark

Figure 6 N. blanchardi performing the gaping display to an intruder by opening the mouth and lateral flaring of the maxillae. By opening the mouth, the buccopalatal membrane, which attaches to both upper and lower jaw, unfolds. The edge of buccopalatal membrane has yellow color hypothesized to have evolved to amplify the display.

Figure 7 Lateral view of N. blanchardi performing the gaping display. The red arrow points to the head-bending gesture which is unusual among fishes.

Figure 8 Kinematic diagram showing a flowchart of transitions between behaviors observed in N. blanchardi (N=32).

Figure 9 Reflectance property of the buccopalatal membrane under (A) normal light and (B) UV light.

Figure 11 Frontal view of N. blanchardi while performing a gaping display. This display not only allows the species to show the adductor mandibulae complex but also the well- armed oral dentition.

Figure 13 Cross section of buccopalatal membrane of N. blanchardi under 400X magnification.

Figure 14 Cleared-and-stained maxilla of three species of Neoclinus fishes (N. blanchardi = SIO 60-67-61A; N. uninotatus = SIO uncataloged; N. stephensae = SIO 86 -60). Pink color is Alizarin Z staining of calcified bone. N. blanchardi and N. uninotatus have a unique maxilla in which the posterior part consists of uncalcified (unstained) or weakly calcified bone.

Figure 15 Dissected specimen of 210 mm SL male N. blanchardi (SIO 64-95-61A) showing muscles and bones involved in the gaping display.

Figure 16 Drawing illustrating bones and adductor mandibulae muscles in N. blanchardi. (A) Innermost layer of skull; (B) inner layer of muscles; (C) outermost layer of muscles.

Species Regression slope y-intercept correlation N. blanchardi 0.2984±0.0082 1.2504±0.0105 r2=0.9930 N. uninotatus 0.2668±0.0100 1.1916±0.0094 r2=0.9918 N. stephensae 0.1967±0.0096 1.0713±0.0096 r2=0.9823

Species Regression slope y-intercept correlation N. blanchardi 0.2688±0.0099 1.1563±0.0121 r2=0.9873 N. uninotatus 0.2386±0.0149 1.1106±0.0130 r2=0.9790 N. stephensae 0.1962±0.0096 1.0927±0.0106 r2=0.9823

Species Regression slope y-intercept correlation N. blanchardi (juvenile) 0.0456±0.0962 0.5599±0.1827 r2=0.3176 N. blanchardi (male) 0.3479±0.0729 0.9556±0.0693 r2=0.8895 N. blanchardi (female) 0.2358±0.0308 0.7980±0.0373 r2=0.9302

Figure 18 Scatter plot of log-transformed uncalcified maxilla length against PC1 score for N. blanchardi juveniles, males, and females. Regression line of each stage is shown in the graph with the value of slope, y-intercept and correlation in the table. Blue and red dashed lines show the PC score (size) at which female and male respectively, reach sexual maturity.

Tables

Table 1 List of specimens used in this study from the Marine Vertebrate Collection at Scripps Institution of Oceanography, UCSD. PCA = principal component analysis, M=

Muscle study, C&S= Clear and Stain study, SL= standard length, MX L= Maxilla length

SIO Number Species Sex SL (mm) MX L (mm) Used for 1 SIO 51-24 N. blanchardi Male 27.19 3.14 PCA 2 SIO 55-81 N. blanchardi Male 56.19 9.59 PCA

3 SIO 66-589 N. blanchardi Male 57.83 10.88 PCA 4 SIO 59-376 N. blanchardi Male 82.32 18.79 PCA 5 SIO 14-177 N. blanchardi Male 107.99 27.29 PCA 6 SIO 14-185 N. blanchardi Male 123.71 39.05 PCA 7 SIO 54-79 N. blanchardi Male 140.39 43.66 PCA 8 SIO 51-47 N. blanchardi Male 147.31 36.05 PCA 9 SIO 71-163 N. blanchardi Male 185.83 57.62 PCA 10 SIO 64-95 N. blanchardi Male 210 66.63 PCA 11 SIO 14-199 N. blanchardi Male 210.5 72.63 PCA 12 SIO 50-100 N. blanchardi Male 245 74.66 PCA 13 SIO 60-61 N. blanchardi Female 67.2 11.62 PCA

14 SIO 61-195 N. blanchardi Female 77.19 15.24 PCA 15 SIO 14-178 N. blanchardi Female 91.03 18.51 PCA 16 SIO 60-67 N. blanchardi Female 99.94 22.15 PCA 17 SIO 62-492 N. blanchardi Female 143.66 35.01 PCA 18 W 57-62 N. blanchardi Female 169.78 40.88 PCA 19 SIO 14-198 N. blanchardi Female 173.84 41.65 PCA 20 SIO 14-194 N. blanchardi Female 175 42.87 PCA 21 SIO 14-195 N. blanchardi Female 180 38.14 PCA 22 SIO 93-189 N. uninotatus Male 97.82 17.42 PCA 23 SIO 63-106 N. uninotatus Male 133.1 24.64 PCA 24 SIO 68-585 N. uninotatus Male 134.89 27.07 PCA 25 SIO 14-183 N. uninotatus Male 158.72 31.88 PCA 26 SIO 68-585 N. uninotatus Male 165.14 34.08 PCA

27 SIO 68-585 N. uninotatus Male 165.6 34.86 PCA 28 SIO 14-181 N. uninotatus Female 52.51 7.58 PCA 29 SIO 73-101 N. uninotatus Female 90.01 16.15 PCA 30 SIO 72-368 N. uninotatus Female 104.6 18 PCA 31 SIO 14-175 N. uninotatus Female 126.86 23.08 PCA 32 SIO 82-33 N. uninotatus Female 133.96 25.03 PCA 33 SIO 73-101 N. uninotatus Female 169.12 34.56 PCA 34 SIO 68-585 N. uninotatus Female 182.24 32.32 PCA 35 SIO 59-303 N. stephensae Male 47.42 6.14 PCA

36 SIO 59-303 N. stephensae Male 50.21 6.29 PCA 37 SIO 59-354 N. stephensae Male 53.99 7.59 PCA

38 SIO 14-197 N. stephensae Male 55.62 6.87 PCA 39 SIO 70-130 N. stephensae Male 57.94 7.41 PCA

40 SIO 14-197 N. stephensae Male 58.16 7.04 PCA

Table1: Continued

SIO Number Species Sex SL (mm) MX L (mm) Used for 41 SIO 86-60 N. stephensae Male 62 7.7 PCA+C&S 42 SIO 56-16 N. stephensae Male 62.68 8.08 PCA 43 SIO 64-67 N. stephensae Male 78.15 9.37 PCA 44 SIO 14-193 N. stephensae Female 52.82 6.59 PCA 45 SIO56-16 N. stephensae Female 54.36 6.92 PCA 46 SIO68-32 N. stephensae Female 58.69 7.01 PCA 47 SIO14-196 N. stephensae Female 63.83 7.96 PCA 48 SIO56-16 N. stephensae Female 65.57 8.28 PCA 49 SIO56-17 N. stephensae Female 73 8.82 PCA 50 SIO77-91 N. stephensae Female 81.14 10.2 PCA 51 SIO65-429 N. stephensae Female 82.99 9.74 PCA 52 SIO00-167 N. toshimaesis Male 54.25 6.61 PCA 53 SIO00-167 N. lacunicola Male 43.49 5.07 PCA 54 SIO00-168 N. bryope Male 45.92 6.95 PCA 55 SIO75-404 M. sandae Male 65.6 3.77 PCA+M 56 uncataloged N. uninotatus Unknown ? ? C&S 57 SIO14-194 N. blanchardi Male 220 69.16 M 58 SIO60-67 N. blanchardi male 117.51 31.79 C&S 59 SIO 64-95 N. blanchardi male 210 66.63 M

Table 2 Descriptive ethogram of territorial behaviors in N. blanchardi.

Behavior Definition Visual contact Animals look at each other.

Gaping display Animal shows full gape of mouth in which the head lifts up and the maxillae flare laterally.

Push Animal pushes intruder while showing full gape.

Attack Animal attacks by biting and striking the opponent with the head.

Ignore Animal shows no display or movement.

Escape Animal leaves area immediately after interacting with a conspecific.

Table 3 Percentage of maxilla length (ML) per standard length (SL) from the biggest available specimen of three species of Neoclinus.

Species Sex SL (mm) ML (mm) % (ML/SL) N. blanchardi male 245 74.66 30.47 female 175 49.48 28.27 N. uninotatus male 165.6 34.86 21.05 female 182.27 32.32 17.73 N. stephensae male 78.15 9.37 11.99 female 82.99 9.74 11.73

Table 4 PC1 scores of each studied characters from 55 specimens of Neoclinus.

Characters PC 1

TR1(1-2) 0.22133 TR2(1-7) 0.24038 TR3(2-3) 0.19377 TR4 (2-6) 0.21774 TR5(2-7) 0.23427

TR6(3-4) 0.179 TR7(3-5) 0.18081 TR8(3-6) 0.17963 TR9(3-7) 0.19333 TR10(4-5) 0.19502 TR11(4-6) 0.17206 TR12(5-6) 0.1711 TR13(6-7) 0.21483

Body depth at anal area 0.27181 Standard length 0.1949 Head length 0.252 Eye diameter 0.16956 Predorsal distance 0.23181

Maxilla Length 0.30878 Pelvic base to anal base 0.21376 First anal soft ray 0.15785 5th dorsal fin ray 0.22834

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