STATE UNIVERSITY, NORTHRIDGE

Reproductive Behavior of the , gigas

A thesis submitted in partial fulfillment of the requirements For the degree of Master of Science in Biology By Brian L. Clark

August 2016

The thesis of Brian L. Clark is approved:

______Dr. Brad E. Erisman Date

______Dr. Mark A. Steele Date

______Dr. Robert C. Carpenter Date

______Dr. Larry G. Allen, Chair Date

California State University, Northridge

ii ACKNOWLEDGEMENTS

This project would have not been possible without the effort and support of the following people. A big thanks to the staff at the USC Wrigley Institute for Environmental Science, especially Juan Aguilar, Trevor Odin and Gordon Boivin for their help in the field and knowledge of Catalina Island. I would like to thank Walter Marti and Bill Bushing for sharing information about where giant sea bass occur at Catalina Island. I would like to thank my field assistant, Matt Jelloian, for the many hours he spent with me underwater.

A special thanks to Parker House for coming out with me during my first season and giving me guidance that I couldn’t have gotten from anyone else. I extend my undying gratitude to the Yellowfin Crew and Tim Rowell for their assistance in the field. Tim’s contribution was unmeasurable and I appreciate him taking time away from his own research to help me with mine. Thank you to Dr. Chris Lowe and Connor White for their involvement with the tracking portion of my study. Thank you to the graduate students at

CSUN and the members of the lab who have helped throughout my project: Maureen

Ho, Sam Ginther, Griffin Srednick, Russel Dauksis, Zoe Scott, Sigfrido Zimmerman and

Malek Al-Marayati. I would like to thank my committee members: Dr. Mark Steele for his guidance throughout my research and work in the field, Dr. Brad Erisman and Dr.

Robert Carpenter for their insightful remarks and critics of my rough draft. I would also like to thank my friends and family for their support. Finally, I would like to think my mentor and friend, Dr. Larry Allen for his continual support and guidance over the last few years; I wouldn’t have gotten to this point without it. Thank you for all of the opportunities, knowledge, history and tools necessary to succeed as a scientist. This research was supported by the graduate fellowship at USC Wrigley Institute for

iii Environmental Science, I.W.F.A, 101 Experiment Backers, Nearshore Marine Fish

Research Program, and the Office of Graduate Studies at CSUN.

iv TABLE OF CONTENTS I. Signature page ii

II. Acknowledgements iii

III. Abstract vi

IV. Introduction 1

V. Materials and Methods 5

VI. Results 11

VII. Discussion 17

VIII. Literature cited 27

IX. Appendix A: Figures 32

X. Appendix B: Tables 45

XI. Appendix C: Fish egg COI haplotypes 48

ABSTRACT

Reproductive behavior of the giant sea bass, Stereolepis gigas

by

Brian L.F. Clark Master of Science in Biology

Aspects of the reproductive behavior of giant sea bass, Stereolepis gigas, were observed and monitored at Goat Harbor, Santa Catalina Island, CA from June 2014 to August 2015.

The site was visited daily during the summer months, which is the assumed spawning season and aggregations were not present during the spring and fall months (October –

March). Numbers of giant sea bass ranged from 2 to15 individuals at Goat Harbor. Six individuals were tagged with Vemco V13 tags to determine site fidelity of giant sea bass at Goat Harbor. All 6 were detected at the aggregation site on a daily basis throughout the summer months (July – September) and also detected at the nearby sites of Long Point and Empire Landing. Giant sea bass produced booming sounds, which were often associated with aggressive behavior, but may also to be associated with spawning.

Drumroll chorusing also was recorded at the giant sea bass aggregation with two different frequencies 250 Hz and 350 Hz and may be used to attract mates similar to other fish . These sounds, although not confirmed as giant sea bass vocalizations, coincide with peak giant sea bass activity (1900-2200 hrs) when they are moving about the water column rather than being stationary near the substratum. These vocalizations are not

vi known to be associated with any other in the immediate area, supporting the hypothesis that they are produced by giant sea bass. Courtship was observed during the late afternoons and was most prominent around dusk (1900-2100). Courtship involved sexual dimorphic color change, and displays such as circling in pairs and the nudging of the abdominal area of the presumed female by the snout of the presumed male. The courtship behaviors observed were similar to those observed of giant sea bass in captivity.

Although spawning was not observed the available evidence suggests that spawning occurs just after dusk. Confirmation of spawning at or near the aggregation site was obtained through DNA barcoding with COI primers of eggs sampled from Goat Harbor.

This study has given insight into courtship behavior and how to identify giant sea bass spawning aggregations in the wild, which is crucial for the management of the species.

vii INTRODUCTION

Courtship and spawning behavior is critical for reproductive success, because it increases the likelihood of gamete release and production of viable offspring. These behaviors allow for individuals within the same species to identify one another, portray readiness to reproduce, determine mate quality, and synchronize gamete release in aggregating fishes (DeMartini and Sikkel 2006). Sound production is common behavior observed in soniferous (sound producing) fishes during courtship and reproduction

(Demartini and Sikkel 2006, Aalbers and Drawbridge, 2008, Mann et al. 2009, Walters et al. 2009). It has been hypothesized that fish sounds and vocalizations are used for development of spawning aggregations (Gilmore 2003), a signal reproductive readiness

(Connaughton & Taylor 1996), and gamete release (Lobel 2002).

Fishes are known to form aggregations, which are temporary gatherings for specific purposes (Colin et al. 2003). Fish aggregate for many different reasons such as: for feeding (Heyman et al. 2001), protection and shelter to structure (Castro et al. 2002) and spawning (Sadovy and Domeier 2005). Fish spawning aggregations are the only places many species are able to reproduce (Sadovy and Domeier 2005). Spawning aggregations tend to form at predictable sites and times of the year, and this predictability allows large amounts of fishes to be harvested with minimal effort (Sadovy de Mitcheson et al. 2008). This, in turn, may lead fishers to believe they are harvesting from a healthy population while the actual abundance is declining. This is known as an “illusion of plenty” where fishing continues unrestricted until population collapse and possibly ending the fishery (Sadovy and Domeier 2005, Sadovy De Mitcheson et al. 2008,

Erisman et al. 2011).

1 Studying reproductive strategies is important for creating effective management strategies for fisheries (Sadovy and Eklund 1999) and has lead to regulations for harvesting on spawning aggregations of many fishes, such as the , americanus (Wakefield et al. 2013), hapuku, Polyprion oxygeneios (Wakefield et al.

2010) and the goliath grouper, Epinephelus itajara (Sadovy and Eklund 1999). Spawning often occurs when abundances are highest at aggregation sites, therefore observing high densities of fishes at a specific site only during certain times of the year, indicates where and when a spawning aggregation is going to form (Mann et al. 2010). Another indicator for spawning aggregation sites is the presence of fish eggs and larva (Moser and Watson

2006). Identification with eggs and larvae has generally been done using morphological differences, although many species cannot be identified based on morphology alone

(Watson et al. 1999). Therefore, the use of DNA barcoding (mitochondrial COI sequencing) has been used to identify fish species that cannot be identified by morphological traits (Harada et al. 2015).

The giant sea bass, Stereolepis gigas, is a part of the northeast Pacific nearshore fish fauna, ranging from the Gulf of California to Humboldt Bay (Miller and Lea 1972) but most common south of Point Conception (Crooke 1992). They are thought to form spawning aggregations during the summer months (July – September) (Love 1991, Shane et al. 1996). Giant sea bass are often found in kelp forests on rocky reefs as adults, while juveniles are found at sandy bottom areas (Domeier 2001). These large demersal teleosts reach lengths greater than 2 m, weights greater than 200 kg, and ages of up to 76 years

(Horn and Ferry-Graham 2006, Allen and Andrews 2012, Hawk and Allen 2014, House et al. 2016).

2 Giant sea bass is a member of the Wreckfish family, Polyprionidae, which is represented by five species within two genera; Polyprion (three species) and Stereolepis

(two species) (Nelson 2006) grow to large sizes (>150 cm and >70 kg) and are very long lived (>60 years) (Peres and Klippel 2003, Hawk and Allen 2014).

Although no formal studies have been conducted on the reproductive behavior of members of the wreckfish family, research has been conducted on their reproductive development: age at maturity, gonadal development, and fecundity. Wreckfishes mature between 7 and 14 years of age depending on the species (Sedberry et al. 1999, Peres and

Klippel 2003, Wakefield et al. 2010) and giant sea bass are thought to fall within this range (Fitch and Lavenberg 1971, Domeier 2001, Allen and Andrews 2012). These fishes mature very late compared to most other demersal teleosts that maturing (Wakefield et al.

2013). There are no observations of spawning behavior of Polyprion species in the wild, but studies on the wreckfish and hapuku reproductive physiology suggested spawning in wreckfishes occurs during warm water months (Peres and Klippel 2003). This varies with latitude and is around the Austrial winter both the wreckfish (July to October) (Peres and

Klippel 2003) and hapuku (June-August) (Wakefield et al. 2010). These studies have also shown that males have relatively large testes, suggesting they engage in group spawning (Sadovy et al. 1996, Peres & Klippel 2003, Wakefield et al. 2010).

The giant sea bass is currently listed as Critically Endangered by the International

Union for Conservation of Nature (IUCN; Cornish et al. 2004). It was once commonly found in the Southern California Bight (Crooke 1992). Both commercial and recreational fishers targeted giant sea bass starting in the late 1800’s (Fitch & Lavenberg 1971,

Eschmeyer et al. 1983). The California commercial catch for giant sea bass peaked in

3 1931 at 114 metric tons and crashed within the next few years dropping to less than 7.7 metric tons in 1935 (Figure 1). Giant sea bass were harvested through the 1970s, and in

1982, California Legislature and California Fish and Wildlife put a moratorium on the commercial and recreation catch of giant sea bass in California waters (Allen and

Andrews 2012). Currently, commercial fishers are allowed to land one giant sea bass per trip as bycatch and recreational fishers are allowed to bring two individuals caught in

Mexico back to California. Additional protection of the giant sea bass has been provided by the restrictions on gill and trammel netting in 1994, which stopped all gill and trammel net fishing within 4.8 km of on the mainland and 1.6 km off of the islands (Allen and

Andrews 2012). Although giant sea bass have an effective population size of less than

500 individuals (Chabot et. al 2015), in the last 15 years giant sea bass have been returning to kelp forests off the coast of Southern California (Pondella and Allen 2008,

House et al. 2016) and the number of juveniles reported as caught and released in the recreational fishery has been increasing (Baldwin and Keiser 2008). This return of the giant sea bass in recent years has presented an opportunity to conduct behavioral research on this endangered species.

It has been hypothesized that wreckfishes are group-spawners based on the large testes of hapuku and the wreckfish, although courting pairs of giant sea bass have been observed in the wild during summer months (June – September) (Bushing and Marti pers. observations), which suggests that they are pair-spawning fish. Observations of pair spawning in captivity have been made, although there were only two individuals in the aquarium (Hovey 2001). Spawning was observed on three separate occasions and the

4 aquaria mimicked that of the summer water conditions in California. This only confirms that they are able to pair , but not that it would be preferred in nature.

The primary goal of this study was to investigate the reproductive behaviors of

Stereolepis gigas in the field, which allowed for comparison to the behaviors exhibited in the aquarium by Hovey (2001). The goals of this project were to: 1) determine when giant sea bass abundances are highest throughout the day and explore movement at the putative spawning site 2) describe the general reproductive behavior and if there are associated sounds and vocalizations, and 3) confirm if giant sea bass eggs occurred at the spawning site with DNA barcoding.

5 MATERIALS AND METHODS

Study Site

I conducted this study at Santa Catalina Island, California (Figure 2) during June-

August in 2014 and 2015. The Long Point Marine Protected Area (MPA) at Santa

Catalina Island, California hosts the best-known putative spawning aggregation of giant sea bass in southern California (CA MLPA South Coast Project 2009). Prior to the start of this study, eight different sites around Catalina were examined as potential aggregation sites for giant sea bass. During a study by House et al. (2016), observations of large aggregations were made at Goat Harbor, on the front side of the island, and The V’s, on the backside of the island. At both Goat Harbor and The V’s densities of giant sea bass did not significantly vary throughout the during the study period (June – August 2014 and

2015, no observations were made at The V’s in 2015). Goat Harbor, an east facing cove, was chosen as the primary study site because it was 1) it was within the Long Point MPA,

2) easily accessible by vessel than the other sites, 3) protected from the elements (wind, wave action), and 4) giant sea bass could be observed there on a daily basis during the summer. While The V’s did have a large aggregation, the site was difficult to access and water conditions varied drastically day to day making daily observations infeasible.

Surveys during October, March (2014) and December (2015) showed no presence of giant sea bass at Goat Harbor (Clark pers. observation), providing supporting evidence that Goat Harbor is an aggregation site. Therefore, Goat Harbor is going to be considered an aggregation site for this study. Goat Harbor has a steep slope, going from 10 to 30 m in depth in just under 150 m.

6 Residency at spawning site

To evaluate residency at the putative spawning site, SCUBA divers conducted four, 200-meter transects were at 4 depths (25, 18, 12, 8 m) using dive propulsion vehicles (Figure 3) at Goat Harbor during each survey. These depths were chosen because they allowed for divers to make observations along the entire slope at Goat

Harbor. Surveys were done one to two times per month (5 surveys in 2014, 4 surveys in

2015). Transects were conducted during three different time periods: morning (0800-

1200), afternoon (1300-1600) and evening (1700-2100). The number of individuals were recorded on each transect (n = 108). Distinct individuals were identified based on spot patterns, scaring and standard lengths. The number of surveys conducted at the different time periods differed between the two years (2014 and 2015). In 2014, the same numbers of dives were conducted at each time period (5 dives per period) but no dives could be conducted after 6pm. In 2015 diving was focused around the afternoon and evening time periods (Dives: Morning = 2, Afternoon = 4, Evening = 4) and evening dives were conducted after 6pm. Abundances of zero were common during transects therefore a

Kruskal-Wallis non-parametric test of independence to test for differences in giant sea bass abundances at the study site by time period (data met the assumptions for this test).

Movement to and from the aggregation site at Goat Harbor was something of interest because we assumed the giant sea bass observed here daily were not moving leaving the site. In July 2015, six giant sea bass were tagged by pole spear (n = 3) or spear gun (n = 3) with Vemco V13 tags that ping randomly every 30-90 seconds (to reduce collisions between tags). Three presumed males and three presumed females were tagged. Presumed sexes were based on morphological (Hovey 2001) and behavioral

7 characteristics (Clark pers. observation). Individuals with rotund abdomens, found in a resting position were assumed to be females and the sleeker bodied, swimming individuals were assumed to be males; for this study all individuals are presumed to be either male or female based on those characteristics (further discussed in results). After tagging, given a designation standard lengths measured for each individual using a laser measuring system. Parallel lasers were mounted 10 cm apart and can be used to measure fishes up to 3 m away. All fish measured were be perpendicular to the lasers to get an accurate size estimate. Photographs of every measured fish were taken and then processed to with ImageJ to estimate body lengths (total length, TL).

An array of acoustic receivers was set up with one receiver at each of three sites:

Goat Harbor, Empire Landing and Long Point (Figure 2). The detection range of the receivers was not tested but estimated the range to be at least 200 m based on previous research on Catalina using the same receivers. The three receivers were used to detect the movement of individuals to and from the aggregation site at Goat Harbor. Individuals were either detected continuously with no gaps between or sporadically where there were large gaps in time between detections (1-2 hours) at each site. The instruments were deployed between on July 7th and 8th, 2015 and continued until September 4th, 2015, totaling in 58 days of tagged monitoring

To calculate individual movement, site fidelity was used. Site fidelity was calculated by taking the number of days a fish was detected a at a specific site divided by the number days of the study (58 days) similar to Mason and Lowe (2010). Fish were then placed into three categories for site fidelity low (<33%), moderate (33%-66%) and high (>66%) (Tinhan et al. 2014). Individuals with high site fidelity to a specific site can

8 be assumed to move less than individuals with low site fidelity. To see if individual movements differed, a G2 test of independence was conducted comparing the site fidelity of the individuals. An individual was considered present for a day if it was detected two or more times within a 24-hour period (Mason and Lowe 2010). This method allowed for individuals to be observed at more than one site in a day, but using the site fidelity equation serves as a proxy for movement.

Description of Reproductive Behavior

Visual observations were carried out on SCUBA and documented on dive slates as well as digital video recordings. A total of 63 dives were conducted at Goat Harbor, totaling over 35 hours of underwater observation of observing giant sea bass behavior over a two-year period. Dives averaged 33.7 minutes per dive and were split among three time periods: morning (17 dives, 9.1 hrs), afternoon (35 dives, 17.3 hrs) and evening (11 dives, 9 hrs).

Courtship and aggressive behaviors between individuals were recorded similar to

Erisman and Allen (2006). Behaviors between two individuals (a gravid female and a male) similar to those described by Hovey (2001) were considered courtship behaviors.

Interactions between males where one of them was removed from the act of courting was considered aggression. Outside of courtship, no interactions aggressive between males and females were observed during this study and female to female aggression was not observed. Individuals within 0.5 m of each other were considered either paired (two individuals) or grouped (three or more individuals). Behaviors were then cataloged into

9 an ethogram (catalogue of behaviors by an organism) describing various reproductive behaviors.

Audio and video were paired using GOPRO® (GoPro, Inc. USA) Hero 3 cameras to ground truth vocalizations produced by giant sea bass. A digital spectrogram long-term acoustic recorder (Soundtrap 202: Ocean InstrumentsNZ) also was used to capture vocalizations. This device passively recorded vocalizations for 1 minute every 5 minutes,

24 hours each day (Rowell et al. 2012) during both the 2014 and 2015 field seasons. If a vocalization was heard during the 1-minute clip it was counted as a vocal detection.

Vocalizations during the month of July in 2014 and 2015 were averaged for each hour and then multiplied by five to account for every minute of each hour. Sounds captured by the Soundtrap and Hero 3 then were analyzed using AUDACITY® (AUDACITY Team

2015: free audio editor and recorder) to identify the frequencies produced (Hz) and catalogued into the ethogram table.

Confirmation of Giant Sea Bass Eggs at Aggregation Site

Spawning was not observed during the duration of the study, therefore alternative methods to detect spawning at this site were sued. Fish egg samples were collected from

Goat Harbor, within the Long Point MPA where courtship activity was observed to see if spawning had occurred at or near the site. A 1-m-long zooplankton net (0.505-mm nitex mesh) was used, 3 m from the surface on the evening (2100 hrs – 2215 hrs) of July 7,

2015 and eggs were stored immediately in 95% ethanol. Upon removing the eggs from storage they were rinsed with molecular grade deionized water. Fish eggs were sorted from other zooplankton under a dissecting microscope at 50X. Giant sea bass eggs

10 diameter ranges from 1.5-1.6 mm (Shane et al. 1996) so eggs were separated into two groups, those <1mm diameter and ≥1mm diameter (accounting for eggs that may have decreased in size from the ethanol). Data suggests that giant sea bass hatch around 4.1 mm in length, which is roughly 6 days post spawning (Shane et al. 1996). A subsample of eggs (n = 20) were staged and found to be in the early developmental stages (between the

64 and 256 cells). Individual eggs were then placed into tubes with a buffer (2/3 AE buffer and 1/3 D. I. water) and crushed using a sterile pipette tip similar to Harada et al.

(2015) to release DNA.

To amplify DNA we used universal fish COI primers: COI VF1 forward primer

(5 −TTCTCAACCAACCACAAAGACATTGG− 3) and COI VRI reverse (5

−TAGACTTCTGGTGGCCAAAGAATCA− 3). PCR reaction used a 10μl solution: 5μL

Quigen MasterMix, 0.9μl BSA, 2.1μL water and 1μL primer mix (2μL forward, 2μL reverse primers and 96μL water). PCRs were performed in a GeneAmp 9700 thermal cycler (Applied Biosystems). The thermocycler profile used was 95°C for 15 min, 35 cycles of 95°C for 30 s, 50°C for 45 s and 72°C 1 min and then 72°C for 10 min.

Following PRC samples were run on 10% agarose gel and amplified DNA was detected using ethidium bromide. A subsample was then run on a Quibit 3.0 fluorometer with high sensitivity to see the concentration of DNA for small (< 1mm) and large (≥ 1mm) eggs.

Samples were sent to Laragen Inc. (Culver City, CA) for purification and sequencing and sequencing products were validated by eye and aligned in

SEQUENCHER (Gene Codes Corporation, Ann Arbor, Mich.; Chabot et. al 2015).

Ninety- one unknown egg samples were sequenced with five known samples of giant sea bass DNA. After sequencing results were compared to the NCBI BLAST database.

11 RESULTS

Description of Aggregation Site (Goat Harbor) Giant sea bass aggregated adjacent to the rocky reef over large patches of cobble or sandy bottom with little kelp cover. Aggregations at this site varied between visits to the site from 2 to 19 individuals, although no patterns in variations were observed (Figure

4 shows abundances at Goat Harbor for the 2014 and 2015 field seasons). Giant sea bass were found at depths ranging from 6 - 30 m, although 80% of fish observed were between 18 - 25 m in depth. At Goat Harbor there was a significant difference in abundance was detected among these depths (H = 9.140, df = 3, p = 0.027) (figure 5).

Residency at Aggregation Site

During the 2014 and 2015 field season’s 20 different individuals were observed at

Goat Harbor. In 2014, there was a significant difference in abundance by time period was found (H = 5.99, df = 2, p = 0.05). However, in 2015, no difference was found in abundances by time period (H = 4.37, df = 2, p = 0.11; Figure 6). Abundances throughout the day in 2014 varied significantly from 2015 (H = 10.74, df = 1, p < 0.01) therefore I am unable to combine the data between these years.

The receivers at each site, Empire Landing, Goat Harbor, and Long Point, detected the tagged giant sea bass. In general, the six individuals were detected at Goat

Harbor from 0500 hrs – 2200 hrs (continuously), at Long Point from 2200 hrs - 0300 hrs

(sporadically), at Empire Landing between 0600 hrs – 1000 hrs (sporadically; Figure 7).

Individuals that were detected at Long Point and Empire Landing were detected with lag time between detections (e.g. at 2230 hrs then again at 0230 hrs) before being detected at

12 Goat Harbor again. Half of the individuals (n=3) were detected at all sites while the other half (n=3) were only detected at Goat Harbor and Long Point.

A significant difference in site fidelity was found among the six tracked individuals (G2 = 61.75, df = 10, p < 0.001). Of the individuals tagged only one exhibited high site fidelity to Goat Harbor and one showed low site fidelity for all three sites. Five of the six individuals showed moderate (between 33% - 66% of their detections) site fidelity to both Goat Harbor and Long Point (Figure 8).

Reproductive Behavior

Presumed sexual dimorphism was observed within this species while at aggregation sites. Individuals showed distinct color and morphological differences prior to courting and spawning. While courting, males presented slim lateral profiles and became much lighter in color (Figure 9a) while females possessed much more rotund body shapes, became much darker in color, had a noticeable white patch on the sides above the vent, and had a very dark mask under the eyes (Figure 9b). Individuals were considered males and females based on these assumptions for this study.

Courtship was observed most often in pairs and these behaviors would repeat throughout the afternoon and evening. During surveys, giant sea bass were encountered in pairs 50% of the time, alone or single 31% of the time, and in groups (3 or more) 19% of the time. Females were observed stationary and in a resting position near the , unless being courted by a male. On occasion, pairing would be visible where both a male and female would be resting in close proximity (observed on 2 dives). Unless in close proximity males were not observed in a resting position, they were observed swimming

13 and approaching resting females. If the female reacted to the approach, the female began to swim with the male following closely behind in attempt to initiate courtship (Figure

10a). If the female did not return to a resting position after being followed, she began to swim in a circular pattern with the male following in a similar fashion and this behavior would continue for up to several minutes (30 sec. – 10 minutes; Figure 10b). During circling, the lead male (if more than one male following the female) would rub and bump the abdominal area of the female near the anal fin with its snout often pushing the female upward into the water column (Figure 8c). The individuals would occasionally move up into the water column (>3 m from the substrate) circling much more rapidly (spiral swimming). This behavior would continue until the female swam back to the bottom. The male would either attempt to court the same female again or move to another female in close proximity. Observations of three individuals courting were not uncommon with one female and two males both following the female. If circling behavior commenced, then one of the males would become aggressive toward the other male, essentially displacing the other from the act of courting. The aggressive male then would resume the courting and nudge the female (Table 1). Although spawning was not observed during this study, I have produced a diagrammatic depiction of presumed giant sea bass spawning behavior.

The courting behaviors observed in the wild matched those described by Hovey (2001) so the actual spawning act was adapted from his description (Figure 11).

Courtship behaviors were not observed during the morning period (0600-1200) and appeared to be restricted to the afternoon and evening hours (1200-2100). However, giant sea bass aggregated at the site throughout the day. First observations of the presumed courtship behavior were at 1200 hrs and the last observations were after dusk

14 (2000 hrs). All courtship was observed over depths greater than 12 m. Presumed courtship observed during the afternoons within a meter of the substratum and more than

50% of the females were observed in a resting position. On 18 observations only 3 extended past the pre-courting behavior of following. During the evenings (after 1700 hrs) activity increased with 10% - 20% of females observed in a resting position and individuals not only courting near substratum but also up in the water column 5-15 m above substrate. On 10 of 12 observations of courtship continued passed the following behavior into circling in the evening.

Sound Production and Vocalizations

Giant sea bass produced booms, which were single-pulses with a low frequency

(about 80 Hz) that often were heard when diving among giant sea bass (Figure 12). On two occasions I was able to visually and audibly capture these “booms”. On both occasions the boom was produced because of an interaction between a diver and the individual. The individual would boom at the initiation of swimming rapidly away from the diver causing other fishes in the immediate area to dart away as well. When not induced by a diver, giant sea bass males (presumed) would boom and rush toward another male.

Drum-roll chorusing vocalizations were heard during dives and with the use of passive acoustic hydrophones. This multiple pulse vocalization resembled a drum roll with three to five pulses in sequence. Two distinct drum-rolls where recorded, one with a peak frequency of 250 Hz and the other peaked at 350 Hz (Figure 13). These vocalizations began at 1300 hours continuing through 2400 hours. Vocalizations peaked between 1900-2200 hours (Figure 14).

15 Confirmation of Giant Sea Bass Eggs at Aggregation Site

Of the 91 eggs sequenced from the zooplankton collection, 30% of the eggs could not be verified or lined up with SEQUENCHER (Gene Codes Corporation, Ann Arbor,

Mich.) and contained multiple peaks, leaving 67 samples successfully sequenced (51 large eggs (>1 mm) and 16 small eggs (<1 mm)) and identified eight different species.

For the large eggs, eight haplotypes were identified comprised of four local species including Stereolepis gigas (3%), Seriola lalandi (49%), Sphyraena argentea (19%) and

Scomber japonicus (1%). For small eggs, six haplotypes and four species were identified as (19%), pulcher (3%), Medialuna californiensis

(3%) and Trachus symmetricus (1%) (Table 2). Identifications were based on > 600 bp of sequence data with greater than 90% identity from NCBI BLAST database (Appendix B)

16 DISCUSSION

In 2014, I was able to identify Goat Harbor, within the Long Point MPA, as an aggregation site for giant sea bass because individuals were found there almost daily in numbers of two or greater during transects. 80% of the giant sea bass observed at this site were between 18 m and 25 m in depth, which is consistent with observations made at other locations around Santa Catalina Island (House et al. 2016). A study by House et al.

2016 showed only two consistent aggregations around Santa Catalina Island, with Goat

Harbor being the only one on the front side of the island. Surveys at Goat Harbor during the fall (October 2014, December 2015) and spring (March 2014) showed no observations of giant sea bass at Goat Harbor (Clark pers. observation). These observations provide evidence that Goat Harbor may be a giant sea bass spawning aggregation site.

I hypothesized that densities would be significantly higher around dusk than other times of the day (morning, afternoon and evening) similar to the peak spawning time in many fishes (Adreani et al. 2004, Erisman & Allen, 2006, Mann et. al 2010, Rowell et al.

2012). Abundances of giant sea bass in 2014 and 2015 differed significantly therefore the data could not be combined. Two factors may have caused this discrepancy: 1) In 2015 dives were not restricted to certain hours of the day, unlike 2014 (dives were allowed until 1830 hrs). No activity was observed during morning periods in 2014, therefore we focused our efforts on diving later during the day when more activity was observed.

Although, in 2015 a few dives were conducted during the morning period (before 1200) to confirm if the low activity pattern continued that was observed in 2014 and similar to observations made in 2014, all fish observed were in the resting position. Therefore,

17 dives during this period were not continued. 2) Conditions between the two years varied most likely do to the El Niño observed in 2015. In 2014 the Goat Harbor had large patches of kelp cover which was not there in 2015. Temperature also varied from year to year, 2014 average temperature was 20.4 °C and in 2015 average temperature was

20.8 °C (The Coastal Data Information Program). Although this temperature difference may not differ significantly, temperature can play a central role in the seasonal movement and distribution of fishes (Lowe and Bray 2006). The depth of the thermocline (layer in which the temperature of the water changes rapidly) increased (2014 about 18 m, 2015 about 30 m) (Clark pers. observation). Giant sea bass were observed at about 18 to 24 m in both years therefore the change in the thermocline could possibly affect their behavior.

In 2014, the number of giant sea bass was greatest in the late afternoon (1300-

1600 hr period). In the late afternoon, giant sea bass tended to be most active compared to mornings. Unfortunately, no fish were observed during the evening period in 2014, but this could be due to the constraints mentioned above. In 2015, this pattern was not repeated, as no difference was actually detected in abundance throughout the day.

Although the abundances of giant sea bass differed from year to year, their behavior was similar in both years. Marked differences in behavior did occur throughout the day. In the afternoons, interactions between individuals began (50% of the individuals), but with activity still close to the bottom. In the evenings (after 1600 hours) through dusk (2000 hours) almost all (80% - 90%) of the individuals were not in a resting position, and multiple courtship interactions between males and females were observed throughout the water column. The similarities in behaviors seen in 2014 and 2015 associated with time of day suggest that activity and interactions increased throughout the day: courtship is

18 initiated in the afternoons, it intensifies in the evening and spawning begins around dusk.

This pattern is common in many different species of temperate and tropical fishes (Colin

1992, Erisman and Allen 2006, Aalbers and Drawbridge 2008).

Acoustic telemetry was used to detect if giant sea bass moved away from their aggregation site at Goat Harbor. The majority of the giant sea bass tagged for this study were detected on over 50% of the total days of the study. Based on daily observations of the same individuals at Goat Harbor, I believed that giant sea bass aggregate at sites, such as Goat Harbor, and stay for the entirety of the spawning season. This was not the case for the 6-tagged individuals. Receiver recordings indicated that these giant sea bass are making daily movements from 2.5 – 5 km away from Goat Harbor. The level of site fidelity was different among the six-tagged individuals, implying that the fish were moving independently of one another. This data showed that only one fish (Larry) of the six, exhibited high site fidelity (> 66%) for Goat Harbor, where the majority of the others showed moderate (33% - 66%) to low site fidelity (< 33%). This result shows that giant sea bass may have a large home range and move make predictable movements.

Having moderate to low site fidelity at Goat Harbor (within the Long Point MPA) raises the question as to whether the establishment of MPA’s for this large fish is effective. Detections outside of Goat Harbor began after 2200 hrs, with the first at Long

Point. There was always a lapse in time (two to four hours) before the next detection and the next would always be back at Long Point. This means that either the individuals were swimming passed through the site and continuing east then returning a few hours later or that these individuals were leaving the detection range of the receiver then returning to it before heading west toward Goat Harbor again at after 0300 hrs. Therefore, if individuals

19 were leaving the MPA between 2200 hrs – 1000 hrs then giant sea bass could be protected by the constraints of the MPA because much of the that occurs at Catalina is during daylight (0600-2000 hours) (pers. observation), meaning that the MPA is serving its purpose after all reducing accidental catch of giant sea bass by recreational fishers. Further tracking of more tagged individuals with a larger acoustic array is necessary before strong conclusions may be drawn regarding the how prevalent these dynamic movements actually are for a larger sample of individuals.

During this study, distinct presumed sexual dimorphism was observed in the aggregating giant sea bass. Presumed Females were very rotund (most likely due large filled with hydrated eggs) and would become much darker in color, with a dark mask under the eyes and a noticeable white patch on their sides. The presumed males observed continued to have a slender body shape but became much lighter in color Hovey

(2001) observed of similar color patterns and changes in the aquarium where he was able to verify sex of the individuals via biopsy of gamete tissue thus confirming the sexes of the different morphs. These changes in color and morphology have been seen in many other fishes and are an indication of potential readiness to reproduce, such as kelp bass, and others (Colin 1992, Erisman and Allen 2006).Although spawning by giant sea bass was not observed during this study, many of the courtship behaviors observed in the wild were previously described in observations of two separate spawning events of giant sea bass in a large public aquarium (Hovey 2001). Thus, the behaviors described herein are assumed to be courting behaviors based on their similar nature.

Observations of courtship differed between afternoons (1200 - 1600 hrs) and evenings (after 1700 hrs). During the evening period 83% of observed following lead to

20 further courting behaviors and only 16% of observed following continued on in the afternoon period (1200-1600 hrs). This showed that although courtship does occur during the afternoons, it does not occur as often as in the evenings. Afternoon courtship was observed near the substratum (< 3 m) with many females continuing to be observed in a resting position. One observation of afternoon courtship showed a male following a female and attempting to circle with the female five times. During this the female was observed making a single circle then returning to the resting position after each attempt.

After the fifth attempt the male swam to a nearby female and attempted to court with her.

Just before dusk, giant sea bass were observed throughout the water column in pairs, and sometimes in small groups. The only individuals observed near the benthos were females that were stationary less than one meter above the bottom. Unless startled (by divers or booms) these individuals seemed to move only when approached by a courting male.

When following was observed, females would circle with the males at least 2 times before stopping and 3 occasions 5 circles were observed before the male began following the female out of the eyesight of the divers. The increase in courtship activity may suggest that giant sea bass are spawning just after nightfall. This is similar to what has been seen in a variety of fish species, all of which became active in the evening and reproduce at dusk or just after dark (Colin 1992). Based on the 35 hours of observation of their activity, I hypothesize that spawning in giant sea bass occurs in the evening just after dusk (approximately 2030 hours).

The combination of following, circling and nudging are behaviors commonly seen in marine fishes (Aalbers and Drawbridge 2008, Colin, 1992, Erisman and Allen 2006,

Erisman et al. 2007, Froeschke et al. 2007) and were also seen in the aquarium

21 observations of giant sea bass spawning (Hovey 2001). Following behavior was commonly observed to involve a single female and one or more males. During these interaction males slowly close the gap between them and their potential mate, but no actual contact was ever made. Circling behavior would sometimes begin with multiple males attempting to court a single female, but shortly after circling began, aggression by the lead male directed at the second and third males would invariably lead to a cessation of circling by the additional males, thus leaving the pair to continue courting. The subordinate males would then continue onto other females in the immediate area. Circles would become tighter and increase in speed as the male nudged at the area abdominal area near the anal fin. This nudging behavior is known as a sign of readiness to reproduce by the males, attempting to start a spawning rush similar to what has been observed in (Aalbers and Drawbridge 2008). On two occasions observations were made of females spiraling upward with a single male, but it ended without the release of any gametes. These “false rushes” involved only two individuals, further supporting contention that giant sea bass are primarily pair spawning fishes. Observations of pair spawning have been observed in captivity (Domeier 2001, Hovey 2001), which also supports these observations seen in the wild.

Although the field observations indicate that giant sea bass are primarily pair spawning fishes, the possibility remains of them being group spawners. Many fishes of similar size and egg storage capacity are group spawners (Roberts 1989, Peres & Klippel

2003, Wakefield et al 2010). This could occur with the inclusion of sneaker males or males joining in at the time of the rush after spawning, similar to what has been observed in the temperate sea basses of the genus Paralabrax (Erisman and Allen, 2006) and the

22 tropical pebbled butterfly fish, Chaetodon multicinctus (Lobel 1989). However, observations of such group or aggregate spawning activity in giant sea bass were completely absent.

The booms that the giant sea bass produced were heard frequently but sporadically. In the summer of 2014, I heard constant booming on several dives at site on the backside of the Santa Catalina Island in an aggregation of 15-20 individuals. Booms were not as frequent at Goat Harbor probably due to the fact that the aggregation at that site was smaller. These booms were often heard in association with an aggressive behavior: 1) an individual would produce a boom causing other fishes in the immediate area (including other giant sea bass individuals) to vacate or 2) a single giant sea bass would become startled and boom as it swam away at a rapid pace.

Other large fishes, particularly groupers produce similar sounds during acts of aggression as well as when they are startled, but they are also used as signals for the beginning of a spawning rush (Mann et al. 2009, 2010, Schärer et al. 2012). Giant sea bass could well be using sound for the same purpose during intraspecific interactions.

Although it has yet to be directly observed, booming has been heard around the time of suspected giant sea bass spawning. After dusk and in low light conditions, it is extremely difficult to make direct visual observations.

In the spring of 2015, a necropsy of a large adult giant sea bass that had washed up in La Jolla, CA (L.G. Allen personal observation) revealed a probable mechanism for sound production. Careful dissection revealed striated muscle bands between pleural ribs

#1- #6 immediately dorsal to the anterior portion of the large swimbladder. This tissue

23 was subsequently identified histologically as striated muscle tissue. The first six pleural ribs hinged flexibly on their respective vertebrae and appeared to be functionally capable of independent movement thus allowing them to strike the swimbladder directly below.

This morphological mechanism seems quite capable of producing both the booming and drum-roll chorusing sounds heard and recorded in the field that I am attributing to the courting giant sea bass.

Drum-roll chorusing was heard and recorded almost nightly in both 2014 and

2015 starting around 1300 and peaking from 1900-2200 hours. This coincides with what the peak in giant sea bass activity that was observed during the same time period, further supporting the idea that these vocalizations are being produced by giant sea bass. These vocalizations produced two different frequencies that were always recorded one right after the other with the 250 hz sound first then quickly followed with a 350 hz sound. On occasion the 250 hz vocalization was heard without the 350 hz, but never the other way around. I hypothesize this to be a call and response scenario, where the 350 hz vocalization was a call and the 250 hz vocalization was the response. It is therefore possible that this is a fish calling to attract mates, in a similar manner to frogs where males call attracting females toward them (Price 2007), and then the female responds indicating her readiness for copulation (Wells and Schwartz 2007). Interestingly, each drum-roll calls consisted of 3-5 pulses while the response consisted of 2 pulses. Although underwater audio and visual confirmation of these drum-rolls has yet to be completed, after discounting all known sounds produced by other species common to this area, it is possible this chorusing was produced by giant sea bass in the immediate vicinity.

24 Therefore, I hypothesize that this pattern could be the result of individual ribs striking against the swim bladder independently thus creating this drum-roll type sound.

Because I was unable to directly observe spawning due to low light levels, I tried a new approach to verify whether giant sea bass were actually spawning in the study area.

This approach, in turn, supported to my contention that the behaviors described here, were indeed courtship behaviors and not just normal giant sea bass interactions. Using the universal fish 600 bp COI primers allowed us observe multiple species that were actually spawning at this specific site at Santa Catalina Island. Eight species were identified with variations in percent of eggs sampled. Although transects for all species were not conducted in the study area, on any given day during the summer months high numbers of these eight species could be seen at this site. For example, schools of 100+ California yellowtail swimming through the site was not uncommon and kelp bass were the dominant reef fish in the area. These two species were also the highest percentage of the eggs for their size classes; large (>1 mm) and small (< 1 mm).

Most importantly, giant sea bass eggs were detected making up 3% of the sample.

This relatively low percentage could be because eggs may have already begun to disperse thus reducing my chances of collecting their eggs. With multiple species of broadcast spawners around the same time in this open system, chances of actually finding any giant sea bass eggs was very slim. Finding fertilized eggs at this site does provides support for giant sea bass using this site as a spawning aggregation site and provides more evidence that the observed behaviors are for courtship. The staged eggs were found to be in the 64- cell to 256-cell stage therefore they were recently fertilized (within 2-3 hours). This means that these giant sea bass eggs most likely came from the immediate or near-by

25 adjacent areas. It is very likely that these eggs were produced and fertilized by the giant sea bass observed at Goat Harbor because no other consistent aggregations have been recorded on the front side of Santa Catalina Island (House et al. 2016). This egg-method of monitoring spawning behavior established by Harada et al. (2015) offers an effective way to monitor spawning activity around the islands and could be a useful way to find potential areas where giant sea bass spawning aggregations form as well as other aggregating fishes on the islands and the coast.

Previous research on wreckfishes, family Polyprionidae, has focused on the reproductive physiology (Peres & Klippel 2003, Roberts 1989, Wakefield et al. 2010) and the low population size of giant sea bass has discouraged researchers from studying them (Pondella and Allen 2008). House et al. (2016) showed significant increases in giant sea bass numbers around Santa Catalina Island, thus allowing for this behavioral research to take place. Recent studies have focused on life history traits as well as population status and vulnerability (Allen and Andrews 2012, Hawk and Allen 2014, Chabot et al.

2015). This study attempted to increase our knowledge of wreckfishes by examining the different behaviors that they exhibit in the wild that are associated with reproduction, and it offers the insight on the reproductive behavior of any polyprionid species. The information presented here matches courtship behaviors in the wild to those seen in an aquarium, thus providing new methods for detection of giant sea bass spawning aggregations. Further observations of multiple giant sea bass spawning in captivity could either confirm or the wild are need to confirm the idea that they are pair spawning species.

The annual formation of spawning aggregations is very important to the reproductive success of many fishes and overharvesting these aggregations can cause

26 detrimental effects to their populations. For critically endangered species such as the giant sea bass, identifying spawning aggregations is crucial to their recovery.

Observations of courting, site fidelity, and verification of fertilized giant sea bass eggs reported herein should help positively identify spawning aggregation sites in the future.

Further research identifying other aggregations are needed for better management of giant sea bass populations in California and Mexico.

27 LITERATURE CITED Aalbers, S. A., and M.A. Drawbridge 2008. White seabass spawning behavior and sound Production. Transactions of the American Fisheries Society, 137(2), 542–550. Adreani, M. S., B.E. Erisman, and R.R. Warner 2004. Courtship and spawning behavior in the , Semicossyphus pulcher (Pisces: Labridae). Environmental Biology of Fishes, 71(1), 13–19. Allen, L. G., and A. H. Andrews 2012. Bomb radiocarbon dating and estimated longevity of Giant Sea Bass (Stereolepis gigas). Bulletin of the Southern California Academy of Sciences, 111(1), 1–14. Castro, J. J., J. A. Santiago, and A. T. Santana-Ortega. A general theory on fish aggregation to floating objects: an alternative to the meeting point hypothesis. Reviews in fish biology and fisheries 11, no. 3 (2002), 255-277. Chabot, C. L., H. A. Hawk, and L.G. Allen 2015. Low contemporary effective population size detected in the Critically Endangered giant sea bass, Stereolepis gigas, due to fisheries overexploitation. Fisheries Research, 172, 71–78. Colin, P. L. 1992. Reproduction of the Nassau grouper, Epinephelus striatus (Pisces: ) and its relationship to environmental conditions. Environmental Biology of Fishes 34.4: 357-377. De Mitcheson, Y. S., A. Cornish, , M. Domeier,, P.L. Colin,.,M. Russell, and K.C. Lindeman, 2008. A global baseline for spawning aggregations of reef fishes. Conservation Biology, 22(5), 1233–1244. Colin, P. L., Y. J.Sadovy, M. L. Domeier 2003. Manual for the study and conservation of reef fish aggregations. Soc. Conserv. Reef Fish Agg. Spec. Publ. 1. Cornish, A. 2004. Grouper & Specialist Group Stereolepis gigas. The IUCN Red List of Threatened Species 2004, e.T20795A9230697. DeMartini E.E and P. C. Sikkel 2006. Reproduction. The Ecology of Marine Fishes: California and Adjacent Waters Allen LG, Pondella DJ, Horn MH, eds. University of California Press, Berkeley, CA, 483-523. Erisman, B. E. and L.G. Allen 2006. Reproductive behaviour of a temperate serranid fish, Paralabrax clathratus (Girard), from Santa Catalina Island, California, U.S.A. Journal of Fish Biology, 68(1), 157–184. Erisman, B. E., L. G. Allen, J. T. Claisse, D.J. Pondella, E. F. Miller, J. H. Murray, and C. Walters 2011. The illusion of plenty: hyperstability masks collapses in two recreational fisheries that target fish spawning aggregations. Canadian Journal of Fisheries and Aquatic Sciences, 68(10), 1705–1716. Erisman, B. E., M. L. Buckhorn, and P. A. Hastings 2007. Spawning patterns in the leopard grouper, Mycteroperca rosacea, in comparison with other aggregating groupers. Marine Biology, 151(5), 1849–1861.

28 Froeschke, B., L. G. Allen, and D. J. Pondella 2007. Life History and Courtship Behavior of Black Perch, Embiotoca jacksoni (Teleostomi: Embiotocidae), from Southern California. Pacific Science, 61(4), 521–531. Gaffney, P. M., J. Rupnow, and M. L. Domeier 2007. Genetic similarity of disjunct populations of the giant sea bass, Stereolepis gigas.Journal of Fish Biology, 70(SUPPL. A), 111–124. Gilmore, R. G., Jr. 2003. Sound production and communica- tion in the spotted seatrout. Pages 177–195 in S. A. Bortone, editor. Biology of the spotted seatrout. CRC Press, Boca Raton, Florida. Harada, A. E., E. A. Lindgren, M. C. Hermsmeier, P. A. Rogowski, E. Terrill, and R. S. Burton 2015. Monitoring spawning activity in a Southern California marine protected area using molecular identification of fish eggs. PloS one 10, no. 8, e0134647. Hawk, H. A. 2013. Age, Growth and Genetic Diversity of Giant Sea Bass, Stereolepis gigas. Diss. California State University, Northridge. Hawk, H and L. G. Allen 2014. Age and Growth of the Giant Sea Bass, Stereolepis gigas. CalCOFI Rep., Vol. 55, 2014 Heyman, W. D., R. T. Graham, B. Kjerfve, and R. E. Johannes. Whale sharks Rhincodon typus aggregate to feed on fish spawn in Belize. Marine Ecology Progress Series 215 (2001), 275-282. Hovey, T. 2001. First Observations on Spawning Behavior in the Giant Sea Bass. California Fish and Game 87(0), 61-66 Lobel, P. S. 1989. Spawning behavior of Chaetodon multicinctus (Chaetodontidae); pairs and intruders. Environmental Biology of Fishes, 25(1-3), 125–130. Lobel, P. S. 2002. Diversity of fish spawning sounds and the application of passive acoustic monitoring. Bioacoustics 12, 287–288 Love, M. S. 2011. Certainly More Than You Wanted to Know About The Fishes of The Pacific Coast.. Really Big Press, Santa Barbara, CA, 354-357. Lowe, C. G. and R. N. Bray 2006. Movement ad Activity Patterns. The Ecology of Marine Fishes: California and Adjacent Waters Allen LG, Pondella DJ, Horn MH, eds. University of California Press, Berkeley, CA, 524-553. Mann, D. A., J. V. Locascio, F. C. Coleman, and C. C. Koenig 2009. Goliath grouper, Epinephelus itajara, sound production and movement patterns on aggregation sites. Endangered Species Research, 7(3), 229–236. Mann, D., J. Locascio, M. Schärer, M. Nemeth, and R. Appeldoorn 2010. Sound production by red hind, Epinephelus guttatus, in spatially segregated spawning aggregations. Aquatic Biology, 10(2): 149–154.

29 Mason, T. J., and C. G. Lowe 2010. Home range, habitat use, and site fidelity of barred sand bass within a southern California marine protected area. Fisheries Research, 106(1), 93–101. Moser, H. G. and W. Watson 2006. Ichthyoplankton. The Ecology of Marine Fishes: California and Adjacent Waters Allen LG, Pondella DJ, Horn MH, eds. University of California Press, Berkeley, CA: 269-319. Nelson, J.S. 2006. Fishes of the World, 4th Edition. Wiley Press, NJ, 624 pp. Peres, M. B., and S. Klippel 2003. Reproductive biology of southwestern , Polyprion americanus (Teleostei: Polyprionidae). Environmental Biology of Fishes, 68(2), 163–173. Pondella, D. J., and L. G. Allen, 2008. The decline and recovery of four predatory fishes from the Southern California Bight. Marine Biology, 154(2), 307–313. Price, T. 2007. Species Recognition. Speciation in Birds, 167(1), 273–297. Roberts, C. D. 1989. Reproductive mode in the percomorph fish genus Polyprion Oken. Journal of Fish Biology, 34(1), 1–9. Rowell, T. J., M. T. Schärer, R. S. Appeldoorn, M. I. Nemeth, D. A. Mann, and J. A. Rivera 2012. Sound production as an indicator of red hind density at a spawning aggregation. Marine Ecology Progress Series, 462, 241–250. Sadovy, Y. 1996. Reproductio of reef fishery species. In: N. Poluini % C. Roberts (ed.) Reef Fishereis, Chapmand and Hall, London, 15-59 Sadovy, Y. and A. M. Eklund,1999. Synopsis of biological data on the Nassau grouper, Epinephelus striatus (Bloch, 1792), and the jewfish, E. itajara (Lichenstein, 1822). FAO Fisheries Synopsis, 68. Sadovy, Y. and M. Domeier 2005. Are aggregation-fisheries sustainable? Reef fish fisheries as a case study. Coral Reefs, 24(2), 254–262. Sadovy de Mitcheson, Y., A. Cornish, M. Domeier, P. Colin, M. Russell, and K. C. Lindeman 2008. A global baseline for spawning aggregations of reef fishes. Conserv. Biol. 22(5), 1233– 1244. Schärer, M. T., M. I. Nemeth,D. Mann, J. Locascio, R. S. Appeldoorn, & T. J. Rowell 2012. Sound Production and Reproductive Behavior of Yellowfin Grouper, Mycteroperca venenosa (Serranidae) at a Spawning Aggregation. Copeia, 2012(1), 135–144. Shane, M. A., W. Watson, and H. G. Moser 1996. Polyprionidae: Giant sea basses and wreckfishes. In The early stages of fishes in the California Current Region. H. G. Moser, ed. Coop. Fish. Invest. Atlas No. 33. Allen Press Inc., Lawrence, Kansas. 873–875.

30 TinHan, T., B. Erisman, O. Aburto-Oropeza, A. Weaver, D. Vazquez-Arce, and C. Lowe. 2014. Residency and seasonal movements in Lutjanus argentiventris and Mycteroperca rosacea at Los Islotes Reserve, Gulf of California. Marine Ecology Progress Series 501, 191 Wakefield, C. B., S. J. Newman, and B. W. Molony 2010. Age-based demography and reproduction of hapuku, Polyprion oxygeneios, from the south coast of Western Australia: implications for management. ICES Journal of Marine Science, 67(6) Wakefield, C., S. Newman, and D. Boddington 2013. Exceptional longevity, slow growth and late maturation infer high inherent vulnerability to exploitation for bass groper, Polyprion americanus (Teleostei: Polyprionidae). Aquatic Biology, 18(2) Wells, K. D., and J. J. Schwartz 2007. The behavioral ecology of anuran communication. Hearing and sound communication in amphibians. Springer New York. 44-86.

31 APPENDIX A: Figures

Giant Seabass Commercial Catch (pounds) 1928-2007 300,000

250,000

200,000

150,000

Catch (pounds) Catch 100,000

50,000

0

1932 1992 1928 1936 1940 1944 1948 1952 1956 1960 1964 1968 1972 1976 1980 1984 1988 1996 2000 2004 Year

Figure 1: Commercial catch (pounds landed) over the period from 1928 to 2007 from California waters (data courtesy of California Department of Fish and Game by L.G. Allen).

32

Empire Landing Goat Harbor

Long Point

Larry Allen

Figure 2: Map showing Santa Catalina Island, California. Stars indicate sites where this study took place.

33

Figure 3: Image of a dive propulsion vehicle (DPV) with mounted parallel calibrated lasers (10 cm apart) for measuring organisms and GoPro Hero 3 Black Edition video camera used for giant sea bass surveys.

34

20

18

16

14

12

10

8

6

Abundance Abundance of giant sea bass 4

2

0

Figure 4: The count of giant sea bass observed at Goat Harbor for 2014 (blue) and 2015 (green)

35

6

(H = 9.140, df = 3, p = 0.027) 5

4

3

2 Mean abundanceMean

1

0 6 12 18 24 Depth (m)

Figure 5: The average number of giant sea bass per transect (N= 9) at four depths (m) at Goat Harbor (± SE) in 2015.

36 10 2014 (H = 5.990, df=2, p=0.05) 9 2015 (H= 4.371, df=2, p=0.11) 8

7

6

5

4

3 Mean abundance of giant sea bass sea giant of abundance Mean 2

1

0 0 0600-1200 1300-1600 1700-2000

Figure 6: The average number of individuals per time of day from diver surveys within Goat Harbor for both 2014 (blue) and 2015 (green) (± SE).

37 Empire

Goat 38 Harbor

Long Point

Figure 7. Sample of daily detections for all 6 individuals tagged. Each color indicates a different individual, black bars indicate multiple detections at a site. Gray bars indicate night and white indicate daytime occurrences.

100

Empire Landing 90 G2= 61.747, df= 10, p< 0.001 Goat Harbor 80 Long Point 70

60

50

Percent time(days) Percent 40

30

20

10

0 0 0 0 Ali (f) Calvin (m) Lorna (f) Larry (m) Steph (f) Brian (m)

Figure 8: Percent of time each individual was detected by acoustic receivers at each of the three sites

39

A

B

Figure 9: Sexual dimorphism between male (A) and female (B) giant sea bass in the wild during spawning season, with both in situ photographs and drawings of these individuals to show greater contrast. Arrows depict key features in males (A) a slim body profile lighter in color with concave abdominal profile and in females (B) a very dark mask under the eyes, a more rotund body shape, and a noticeable white patch on the sides above the vent (left to right).

40

A. Following

B. Circling

C. Nudging

Figure 10: Diagrammatic depictions the three common courting behaviors: Following (A), Circling (B) and Nudging (C.

41

42

Figure 11: Hypothetical diagrammatic depiction of spawning behavior exhibited by giant sea bass. The final stages of courtship and actual spawning is believed to occur near and just after dusk as represented by the light to dark gradient (left to right) in the illustration.

Figure 12: A spectrograph displaying the range of sound of the giant sea bass “boom” right around 80 hz. Hertz on the y-axis. Images produced with Audacity®

B A

Figure 13: Spectrograph displaying the range of sound for both the call (A, 250 hz) and the response (B, 350 hz) of the drumroll chorusing. Hertz on the y-axis. Images produced with Audacity®

43

60

50

40

30

20

10 44

Average Average vocaldetections (min) 0

Time of day

Figure 14. Average minutes of vocal detections of the drumroll chorusing per hour over a 24-hour day produced by giant sea bass. Data from July 2015 and 2016 (± SE)

APPENDIX B: Tables

Table 1. Catalogue of observed behaviors during the spawning season in giant sea bass, Stereolepis gigas

Behavior Frequency Description

Individuals loosely grouped over sandy bottom Aggregating Common adjacent to and rocky reefs in numbers of 5-20 individuals.

Throughout the entire day, presumed females were commonly observed hovering < 1m Resting Common above the substratum unless approached by a courting male.

A male rests in close proximity to a gravid Pairing Common female, both hovering < 1m above the substrate

Male advances towards a presumed gravid Approach Common female to begin courtship behaviors

One to three males swim behind a gravid Following Common female, no contact with females; common during early courtship

Gravid female begins to swim in a circular pattern, while the one to two males follow in Circling Common the same motion; common during courtship up to several minutes

The lead presumed male following/circling approaches the female from the side or underneath and begins rubbing and bumping Nudging Common his snout against the presumed gravid female’s abdominal area near the anal fin pushing the female upward

45

Behavior Frequency Description (cont’d)

Male and female begin to circle rapidly Spiral Rare upward into the water column; only observed swimming after dusk

Male and female continue circling near surface, beating with caudal fins, pair tilts Spawning Not observed slightly along dorsal-ventral axis with the release of eggs, followed closely by release of sperm (Hovey 2001)

Drumroll Only produced during the after dusk till late Common chorusing evening (7pm-2am); possible courting sounds Produced in aggregations, when a male would move toward a group of individuals causing Boom Rare others to scatter; also observed when individual is aggravated, causing it to swim away rapidly

46

Table 2: Shows the eight different species that were genetically (CO1) identified in the sample from the plankton tow at Goat Harbor done on July 7th, 2015 at 9pm.

Species (Common name) Total eggs % of eggs

Stereolepis gigas (giant sea bass) 2 3%

Seriola dorsalis (=lalandi) (California 33 49% yellowtail)

Sphyraena argentea (Pacific barracuda) 13 19%

Scomber japonicus (Pacific mackerel) 1 1%

Semicossyphus pulcher (California 2 3% sheephead)

Paralabrax clathratus (kelp bass) 13 19%

Medialuna californiensis (halfmoon) 2 3%

Trachurus symmetricus (jack mackerel) 1 1%

47

APPENDIX C: Fish egg COI 600BP haplotypes (Samples sharing the same haplotype are listed in parentheses, s < 1mm, b >1mm) Medialuna californiensis:

GU440401.1 (9s) CGCCCTCCTTGGAGACGACCAGATTTATAATGTAATCGTTACAGCACATGCCTTTGTAATAATTT TCTTTATAGTAATACCAATTATGATTGGAGGGTTTGGAAACTGACTGATTCCCCTTATGATCGGT GCACCAGACATGGCATTTCCCCGAATAAACAACATGAGCTTTTGACTGCTCCCCCCTTCTTTCCT GTTACTCCTTGCTTCTTCTGGTGTTGAAGCTGGGGCCGGCACCGGCTGAACCGTCTATCCCCCTC TCGCTGGTAACTTAGCCCATGCAGGAGCCTCAGTAGACCTAACAATCTTCTCCCTTCACTTGGCA GGTATTTCTTCCATCCTTGGGGCTATTAATTTCATTACAACTATCATTAACATGAAACCCCCTGCC ATCTCCCAATATCAAACCCCTCTCTTTGTATGGGCAGTTCTAATCACAGCGGTTCTCCTCCTCCTC TCCCTTCCCGTCCTCGCTGCCGGTATTACAATACTTCTCACAGACCGAAATCTTAACACAACCTTC TTCGACCCAGCAGGA

Paralabrax clathratus:

JN600315.1 (12,11,15,40,22,6,13,20s) CGCTCTCTTAGGGGACGACCAGATCTACAATGTAATTGTTACGGCACACGCTTTCGTAATAATTT TCTTTATAGTAATACCAATTATGATTGGGGGTTTCGGAAACTGACTTATCCCTCTAATGATTGGG GCTCCCGATATAGCATTCCCTCGAATAAATAACATAAGCTTTTGACTCCTCCCTCCCTCTTTCCTTC TTCTCTTAGCTTCCTCAGGGGTTGAAGCAGGAGCAGGTACAGGCTGAACAGTTTACCCTCCATT ATCCGGTAACTTAGCCCACGCCGGAGCTTCTGTTGATCTAACAATCTTCTCTCTCCACTTAGCAG GTATCTCTTCAATTCTAGGGGCAATTAATTTTATTACTACAATTATTAACATGAAGCCCCCTGCTA TTTCTCAATACCAAACACCTTTATTTGTTTGAGCTGTTCTAATTACTGCAGTACTGCTTCTTCTTTC CCTGCCAGTCCTTGCCGCAGGTATTACAATACTTTTAACCGATCGAAATTTAAATACAACATTCTT TGATCCTGCAGGA

JN600315.1 (29s) CGCTCTCTTAGGGGACGACCAGATCTACAATGTAATTGTTACGGCACACGCTTTCGTAATAATTT TCTTTATAGTAATACCAATTATGATTGGGGGTTTCGGAAACTGACTTATCCCTCTAATGATTGGG GCTCCCGATATAGCATTCCCTCGAATAAATAACATAAGCTTTTGACTCCTCCCTCCCTCTTTCCTTC TTCTCTTAGCTTCCTCAGGGGTTGAAGCAGGAGCAGGTACAGGCTGAACAGTTTACCCTCCATT ATCCGGTAACTTAGCCCACGCCGGAGCTTCTGTTGATCTAACAATCTTCTCTCTCCACTTAGCAG GTATCTCTTCAATTCTAGGGGCAATTAATTTTATTACTACAATTATTAACATGAAGCCCCCTGCTA TTTCTCAATACCAAACACCTTTATTTGTTTGAGCTGTTCTAATTACTGCAGTACTGCTTCTTCTTTC CCTGCCAGTCCTTGCCGCAGGTATTACAATACTTTTAACCGATCGAAATTTAAATACAACATTCTT TGATCCTGCAGGA

48 GU440445.1 (4b) GCCTTCTTATTCGAGCCGAGCTCAGCCAACCGGGCGCTCTCTTAGGGGACGACCAGATCTACAA TGTAATTGTTACGGCACACGCTTTCGTAATAATTTTCTTTATAGTAATACCAATTATGATTGGGG GTTTCGGAAACTGACTTATCCCTCTAATGATTGGGGCTCCCGATATAGCATTCCCTCGAATAAAT AACATAAGCTTTTGACTCCTCCCTCCCTCTTTCCTTCTTCTCTTAGCTTCCTCAGGGGTTGAAGCA GGAGCAGGTACAGGCTGAACAGTTTACCCTCCATTATCCGGTAACTTANCCCACGCCGGAGCTT CTGTTGATCTAACAATCTTCTCTCTCCACTTANCAGGTATCTCTTCAATTCTAGGGGCAATTAATT TTATTACTACAATTATTAACATGAAG

Scomber japonicus:

JQ354331.1 (58) CTGGACTTGACCTCATCTGTTGCGGGGGTTTGCTTTTCCATGNGCTTCTTTGTNNACNCGCAAGT TAAAATTGGAGGGTTCGGAAACTGACTGATCCCCCTAATGATCGGAGCCCCCGACATGGCATTT CCCCGAATAAATAACATAAGCTTCTGACTTCTGCCCCCCTCTCTCCTGCTGCTCCTGTCTTCTTCG GCAGTTGAAGCCGGTGCCGGAACTGGCTGAACAGTTTATCCTCCCCTCGCTGGGAACCTAGCAC ACGCCGGGGCATCAGTTGATTTGACCATCTTCTCACTCCACCTAGCAGGTGTTTCCTCAATCCTT GGGGCCATTAACTTCATCACAACAATCATTAACATAAAACCTGCAGGTGTATCCCAATACCAAAC CCCTCTGTTCGTCTGAGCAGTCCTAATTACAGCTGTCCTTCTCCTTCTATCCCTACCAGTTCTTGCT GCCGGCATTACAATGCTCCTAACAGACCGAAATCTAAATACTACCTTCTTCGACCCTG

Semicossyphus pulcher:

JQ934979.1 (4,5s) CGCTCTCCTGGGAGACGACCAAATTTACAATGTAATCGTTACGGCGCATGCGTTCGTAATAATCT TCTTTATAGTAATACCAATCATGATCGGTGGCTTCGGAAACTGACTCATCCCCTTGATAATTGGG GCCCCCGACATAGCCTTTCCCCGAATAAATAATATGAGCTTCTGGCTTCTCCCCCCATCCTTCCTC CTCCTGTTAGCCTCTTCTGGGGTAGAAGCTGGAGCCGGGACCGGATGAACCGTCTACCCACCAT TAGCGGGCAATCTAGCCCACGCAGGGGCCTCAGTCGATCTAACCATTTTCTCGCTCCATTTGGCA GGGATCTCCTCAATCTTAGGAGCAATCAATTTTATTACAACTATTATTAACATAAAACCACCAGC CATTTCTCAGTACCAAACTCCTCTGTTCGTCTGATCCGTTTTAATTACAGCCGTACTTCTCCTACTT TCACTCCCAGTCCTTGCCGCTGGTATTACAATGCTTCTAACCGACCGAAACTTAAATACCACCTTC TTTGACCCGGCAGGG

Seriola dorsalis (lalandi):

KM877629.1 (67,19,21,56,62,72b) TGCTCTTCTGGGGGACGATCAAATTTATAACGTAATCGTTACAGCGCACGCGTTTGTAATAATTT TCTTTATAGTAATACCAATTATGATTGGAGGGTTTGGGAACTGACTCATCCCCTTGATGATTGGG GCTCCCGATATAGCATTCCCCCGAATAAACAATATGAGCTTCTGACTCCTTCCCCCCTCGTTCCTC CTACTTTTAGCCTCTTCAGGCGTTGAAGCCGGGGCCGGAACGGGTTGAACAGTCTACCCGCCTC TAGCCGGCAACCTCGCTCACGCAGGAGCATCCGTAGACTTAACAATTTTTTCCCTTCATTTAGCT GGAATCTCCTCAATTCTAGGGGCTATTAACTTTATTACGACCATCATCAACATGAAACCCCATGC CGTTTCCATGTACCAAATTCCCCTATTCGTCTGAGCTGTTCTAATCACGGCCGTGCTCCTGCTCCT

49 ATCGCTTCCAGTTCTAGCCGCTGGCATTACGATGCTTCTGACAGACCGAAACTTAAATACTGCCT TCTTTGACCCAGCTGGA

KM877634 (25b) TGCTCTTCTGGGGGACGATCAAATTTATAACGTAATCGTTACAGCGCACGCGTTTGTAATAATTT TCTTTATAGTAATACCAATTATGATTGGAGGGTTTGGGAACTGACTCATCCCCTTGATGATTGGG GCTCCCGATATAGCATTCCCCCGAATAAACAATATGAGCTTCTGACTCCTTCCCCCTTCGTTCCTC CTACTTTTAGCCTCTTCAGGCGTTGAGGCCGGGGCCGGAACGGGTTGAACAGTCTACCCGCCTC TAGCCGGCAACCTCGCTCACGCAGGAGCATCCGTAGACTTAACAATTTTTTCCCTTCATTTAGCT GGAATCTCCTCAATCCTAGGGGCTATTAACTTTATTACGACCATCATCAACATGAAACCCCATGC CGTTTCCATGTACCAAATTCCCCTATTCGTCTGAGCTGTTCTAATCACGGCCGTGCTCCTGCTCCT GTCGCTTCCAGTTCTAGCCGCTGGCATTACGATGCTTCTGACAGACCGAAACTTAAATACTGCCT TCTTTGACCCAGCTGGA

KM877634.1 (13,22,65,47,64,45,49,2,26,27,61,42,18,11b, 1s) TGCTCTTCTGGGGGACGATCAAATTTATAACGTAATCGTTACAGCGCACGCGTTTGTAATAATTT TCTTTATAGTAATACCAATTATGATTGGAGGGTTTGGGAACTGACTCATCCCCTTGATGATTGGG GCTCCCGATATAGCATTCCCCCGAATAAACAATATGAGCTTCTGACTCCTTCCCCCTTCGTTCCTC CTACTTTTAGCCTCTTCAGGCGTTGAGGCCGGGGCCGGAACGGGTTGAACAGTCTACCCGCCTC TAGCCGGCAACCTCGCTCACGCAGGAGCATCCGTAGACTTAACAATTTTTTCCCTTCATTTAGCT GGAATCTCCTCAATTCTAGGGGCTATTAACTTTATTACGACCATCATCAACATGAAACCCCATGC CGTTTCCATGTACCAAATTCCCCTATTCGTCTGAGCTGTTCTAATCACGGCCGTGCTCCTGCTCCT GTCGCTTCCAGTTCTAGCCGCTGGCATTACGATGCTTCTGACAGACCGAAACTTAAATACTGCCT TCTTTGACCCAGCTGGA

KM877656.1 (23,63,41,37b) TGCTCTTCTGGGAGACGATCAAATTTATAACGTAATCGTTACGGCGCACGCGTTTGTAATAATTT TCTTTATAGTAATACCAATTATGATTGGAGGGTTTGGGAACTGACTCATCCCCTTGATGATTGGG GCTCCCGATATAGCATTCCCCCGAATAAACAATATGAGCTTCTGACTCCTTCCCCCTTCGTTCCTC CTACTTTTAGCCTCTTCAGGCGTTGAAGCCGGAGCCGGAACGGGTTGAACAGTCTACCCGCCTC TAGCCGGCAACCTCGCTCACGCAGGAGCATCCGTAGACTTAACAATTTTCTCCCTTCATTTAGCT GGAATCTCCTCAATTCTAGGGGCTATCAACTTTATTACGACCATCATCAACATGAAACCCCATGC CGTTTCCATGTACCAAATTCCCCTATTCGTCTGAGCTGTTCTAATCACGGCCGTGCTCCTGCTCCT GTCACTCCCAGTTCTAGCCGCTGGCATTACAATGCTTCTGACAGACCGAAACTTAAATACTGCCT TCTTTGACCCAGCTGGA

50 KM877625.1 (20b) TGCTCTTCTGGGGGACGATCAAATTTATAACGTAATCGTTACAGCGCACGCGTTTGTAATAATTT TCTTTATAGTAATACCAATTATGATTGGAGGGTTCGGGAACTGACTCATCCCCTTGATGATTGGG GCTCCCGATATAGCATTCCCCCGAATAAACAATATGAGCTTCTGACTCCTTCCCCCTTCGTTCCTC CTACTTTTAGCCTCTTCAGGCGTTGAGGCCGGGGCCGGAACGGGTTGAACAGTCTACCCGCCTC TAGCCGGCAACCTCGCTCACGCAGGAGCATCCGTAGACTTAACAATTTTTTCCCTTCATTTAGCT GGAATCTCCTCAATTCTAGGGGCTATTAACTTTATTACGACCATCATCAACATGAAACCCCATGC CGTTTCCATGTACCAAATTCCCCTATTCGTCTGAGCTGTTCTAATCACGGCCGTGCTCCTGCTCCT GTCGCTTCCAGTTCTAGCCGCTGGCATTACGATGCTTCTGACAGACCGAAACTTAAATACTGCCT TCTTTGACCCAGCTGGA

KM877629.1 (43b) TGCTCTTCTGGGGGACGATCAAATTTATAACGTAATCGTTACAGCGCACGCGTTTGTAATAATTT TCTTTATAGTAATACCAATTATGATTGGAGGGTTTGGGAACTGACTCATCCCCTTGATGATTGGG GCTCCCGATATAGCATTCCCCCGAATAAACAATATGAGCTTCTGACTCCTTCCCCCCTCGTTCCTC CTACTTTTAGCCTCTTCAGGCGTTGAAGCCGGGGCCGGAACGGGTTGAACAGTCTACCCGCCTC TAGCCGGCAACCTCGCTCACGCAGGAGCATCCGTAGACTTAACAATTTTTTCCCTTCATTTAGCT GGAATCTCCTCAATTCTAGGGGCTATTAACTTTATTACGACCATCATCAACATGAAACCCCATGC CGTTTCCATGTACCAAATTCCCCTATTCGTCTGAGCTGTTCTAATCACGGCCGTGCTCCTGCTCCT ATCGCTTCCAGTTCTAGCCGCTGGCATTACGATGCTTCTGACAGACCGAAACTTAAATACTGCCT TCTTTGACCCAGCTGGA

Sphyraena argentea:

EU752211.1 (16,54,32,57,38b, 30s) ATCACTCCTGGGGGATGACCAGATCTATAACGTAATTGTAACGGCGCATGCTTTTGTAATAATCT TTTTCATAGTAATACCTATCATAATTGGAGGCTTTGGGAACTGACTTATTCCACTAATAATTGGC GCCCCCGACATGGCCTTTCCACGAATAAATAACATAAGCTTTTGACTTCTCCCTCCCTCTTTCCTG CTTCTGCTCTCCTCCTCAGCAGTAGAATCAGGTGCAGGTACGGGGTGAACAGTTTACCCCCCTTT ATCAGCCAATCTAGCTCATGCAGGTGCATCTGTTGACCTAACAATTTTCTCCCTCCACCTAGCCG GAATTTCGTCAATTCTAGGGGCTATTAACTTCATCACTACTATTATCAACATAAAACCAACAATTA CCTCAATATACCAAATCCCACTCTTTGTATGATCCGTGCTAATTACTGCTGTTCTCCTCCTCCTCTC TCTCCCTGTTCTAGCTGCCGGAATCACAATACTATTAACTGACCGAAATCTAAACACTGCATTCTT CGACCCTGCAGGA Stereolepis gigas:

GU440534.1 (1,14b) CGCCCTCTTAGGGGACGACCAAATTTATAACGTAATTGTTACAGCACACGCATTTGTAATAATTT TCTTTATAGTAATGCCAATTATAATCGGAGGATTCGGAAACTGACTTGTCCCCCTAATGATCGGG GCCCCAGACATAGCATTCCCTCGAATGAATAATATAAGCTTTTGACTTCTTCCCCCATCCTTCCTC CTCCTTCTTGCTTCCTCAGGAGTAGAGGCTGGCGCTGGCACCGGATGAACAGTCTACCCTCCCCT AGCTGGTAATTTAGCCCACGCAGGGGCCTCCGTTGACTTGACAATTTTTTCTCTACACTTAGCAG GGATTTCCTCAATTCTCGGAGCCATTAACTTCATTACAACCATCATTAACATAAAACCCCCTGCCA

51 TCTCCCAATATCAGACTCCTCTCTTTGTATGAGCCGTACTAATTACCGCCGTCCTTCTCCTCCTCTC CCTCCCAGTTCTCGCTGCTGGCATTACAATACTTCTTACAGATCGAAACCTCAACACCACCTTCTT CGACCCCGCAGGA

Trachurus symmetricus:

JQ354520.1 (45s) CGCCCTTCTAGGGGATGACCAAATTTACAACGTAATTGTTACGGCCCACGCTTTCGTAATAATTT TCTTTATAGTAATGCCAATTATGATTGGAGGCTTTGGAAACTGACTGATTCCACTAATGATCGGA GCCCCTGATATAGCCTTCCCTCGAATGAATAACATGAGCTTCTGACTACTCCCTCCCTCCTTCCTT TTGCTTTTAGCCTCTTCAGGTGTTGAAGCCGGGGCCGGAACTGGTTGAACAGTCTATCCCCCACT GGCTGGGAACCTTGCCCACGCCGGAGCATCCGTAGATTTAACCATCTTCTCCCTTCACCTAGCAG GGGTCTCATCAATTCTAGGGGCTATTAACTTTATTACCACTATTATCAACATGAAACCTCCTGCA GTCTCAATATATCAAATCCCACTATTTGTTTGAGCTGTCTTAATTACAGCTGTCCTTCTTCTTCTCT CTCTTCCTGTCCTAGCTGCTGGCATTACAATACTTTTAACAGACCGAAATCTAAATACTGCTTTCT TTGACCCAGCAGGC

52