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Reproduction of the Sandbar Shark, carcharhinus Plumbeus, in the Western North Atlantic Ocean and Gulf of Mexico

Permanent Link: http://ufdc.ufl.edu/UFE0024745/00001

Material Information

Title: Reproduction of the Sandbar Shark, carcharhinus Plumbeus, in the Western North Atlantic Ocean and Gulf of Mexico
Physical Description: 1 online resource (119 p.)
Language: english
Creator: Piercy, Andrew
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: demography, elasmobranch, fish, reproduction, sandbar, shark
Forest Resources and Conservation -- Dissertations, Academic -- UF
Genre: Fisheries and Aquatic Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: The reproduction of the sandbar shark, Carcharhinus plumbeus, in the western North Atlantic Ocean and Gulf of Mexico was examined. Specimens were collected through fishery-dependent and ?independent sampling programs. Morphological measurements of the sharks and reproductive organs were taken. Indices of maturity were constructed using measurements of gonads, genital ducts, and claspers. Sharks were shown to mature between 140 and 160 cm fork length. Gonadosomatic indices and variation in genital duct condition were used to determine seasonal trends in reproduction of mature sharks. Sandbar sharks have discrete seasonal reproductive cycles: males produce sperm from January to May with a peak in May and females develop eggs from January to May with ovulation occurring in June. Females were shown to exhibit a greater than two year reproductive cycle. Embryonic development was assessed through measurements of weights and lengths of uterine contents. Gestation was 12 months, from July to the following June, with parturition in late June. Trends in embryonic and maternal relative conditions were noted. Embryos from larger female sharks exhibited less variation in relative condition. Demographic analysis was used to determine the magnitude of effect that variations in reproductive parameters can have on population growth. Variation in age of maturity and seasonality of reproduction were shown to have small effects on intrinsic population growth. Survival of juvenile age classes was shown to have the greatest impact on population growth.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Andrew Piercy.
Thesis: Thesis (Ph.D.)--University of Florida, 2009.
Local: Adviser: Murie, Debra J.
Local: Co-adviser: Snelson, Franklin F.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2011-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0024745:00001

Permanent Link: http://ufdc.ufl.edu/UFE0024745/00001

Material Information

Title: Reproduction of the Sandbar Shark, carcharhinus Plumbeus, in the Western North Atlantic Ocean and Gulf of Mexico
Physical Description: 1 online resource (119 p.)
Language: english
Creator: Piercy, Andrew
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: demography, elasmobranch, fish, reproduction, sandbar, shark
Forest Resources and Conservation -- Dissertations, Academic -- UF
Genre: Fisheries and Aquatic Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: The reproduction of the sandbar shark, Carcharhinus plumbeus, in the western North Atlantic Ocean and Gulf of Mexico was examined. Specimens were collected through fishery-dependent and ?independent sampling programs. Morphological measurements of the sharks and reproductive organs were taken. Indices of maturity were constructed using measurements of gonads, genital ducts, and claspers. Sharks were shown to mature between 140 and 160 cm fork length. Gonadosomatic indices and variation in genital duct condition were used to determine seasonal trends in reproduction of mature sharks. Sandbar sharks have discrete seasonal reproductive cycles: males produce sperm from January to May with a peak in May and females develop eggs from January to May with ovulation occurring in June. Females were shown to exhibit a greater than two year reproductive cycle. Embryonic development was assessed through measurements of weights and lengths of uterine contents. Gestation was 12 months, from July to the following June, with parturition in late June. Trends in embryonic and maternal relative conditions were noted. Embryos from larger female sharks exhibited less variation in relative condition. Demographic analysis was used to determine the magnitude of effect that variations in reproductive parameters can have on population growth. Variation in age of maturity and seasonality of reproduction were shown to have small effects on intrinsic population growth. Survival of juvenile age classes was shown to have the greatest impact on population growth.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Andrew Piercy.
Thesis: Thesis (Ph.D.)--University of Florida, 2009.
Local: Adviser: Murie, Debra J.
Local: Co-adviser: Snelson, Franklin F.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2011-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0024745:00001


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REPRODUCTION OF THE SANDBAR SHARK, CARCHARHINUS PLUMBEUS, IN THE WESTERN NORTH ATLANTIC OCEAN AND GULF OF MEXICO By ANDREW PIERCY A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2009 1

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2009 Andrew Piercy 2

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To my parents, Donald and Cynthia Piercy 3

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ACKNOWLEDGMENTS I wish to thank my advisor Dr. Debra Murie for countless hours of guidance and encouragement. I wish to thank my committee co-chair Dr. Franklin Snelson Jr. for continuing to be a mentor and friend and taking the time to help me even though he is retired. I also wish to thank my committee members Dr. Mike Allen and Dr. Colette St. Mary for much encouragement and guidance. Many undergraduate student volunteers helped with this research particularly Stephanie Boyer, Emily Kunihiro, and Ashley Jennings. I wish to thank Alexia Morgan, Taylor Chapple, and Peter Cooper along with other fishery observers for assistance in collecting specimens. I thank George Burgess for hiring me back in 2002 and contributing funding for specimen collection through the National Shark Research Consortium. Dr. Jim Gelsleichter was always available for advice and continues to be a great mentor and friend. Lastly, I thank my parents for their tireless support over the many years of graduate school. 4

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TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 ABSTRACT...................................................................................................................................11 CHAPTER 1 THE SANDBAR SHARK......................................................................................................13 Introduction................................................................................................................... ..........13 Objectives...............................................................................................................................16 2 REPRODUCTIVE CYCLE OF SANDBAR SHARKS IN THE NORTHWESTERN ATLANTIC OCEAN AND GULF OF MEXICO..................................................................17 Introduction................................................................................................................... ..........17 Methods..................................................................................................................................20 Sandbar Shark Collections..............................................................................................20 Sampling for Male Reproductive Biology......................................................................21 Sampling for Female Reproduction.................................................................................22 Regional Comparison......................................................................................................23 Results.....................................................................................................................................24 Male Reproduction..........................................................................................................24 Female Reproduction.......................................................................................................25 Regional Differences.......................................................................................................26 Discussion...............................................................................................................................27 Male Reproduction..........................................................................................................27 Female Reproduction.......................................................................................................29 Regional Comparison......................................................................................................32 Summary..........................................................................................................................33 3 MATERNAL INFLUENCE ON SANDBAR SHARK EMBRYONIC DEVELOPMENT...................................................................................................................59 Introduction................................................................................................................... ..........59 Methods..................................................................................................................................62 Results.....................................................................................................................................64 Embryonic Condition......................................................................................................64 Lipid Analysis..................................................................................................................65 Discussion...............................................................................................................................65 5

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4 THE EFFECTS OF REPRODUCTIVE VARIATION ON SANDBAR SHARK POPULATION GROWTH.....................................................................................................83 Introduction................................................................................................................... ..........83 Methods..................................................................................................................................85 Mortality..........................................................................................................................86 Reproductive Parameters.................................................................................................88 Results.....................................................................................................................................89 Natural M ortality.............................................................................................................8 9 Population Growth..........................................................................................................89 Elasticities................................................................................................................... .....90 Discussion...............................................................................................................................91 5 EPILOGUE..................................................................................................................... ......105 APPENDIX ADDITIONAL FATTY ACID DATA................................................................107 LIST OF REFERENCES.............................................................................................................109 BIOGRAPHICAL SKETCH.......................................................................................................119 6

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LIST OF TABLES Table page 3-1 Relationships between maternal reproductive parameters and relative condition (Kn) of embryos in their litters for sandbar sharks in the northwestern Atlantic Ocean and Gulf of Mexico during the placental stage of gestation. ....................................................70 3-2 Regression analysis of maternal (N=5) and embryonic (N=15) parameters and concentrations of three essential fatty acids. ......................................................................71 4-1 Sandbar shark reproductive parameters used in population models. .................................96 7

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LIST OF FIGURES Figure page 2-1 Measurement of outer clasper length in sandbar shark. Measurement is taken from the margin of the pelvic fin (P) to the tip of the clasper ( c). Line represents the plane of measurement. .................................................................................................................34 2-2 Histological cross-section of sandbar shark testis, showing germinal zone (G) and epigonal tissue (E). Dotted line demarcates the area of testis tissue and dashed line the area occupied by late stage spermatocysts. Bar represents 1mm. ................................35 2-3 Sandbar shark proportional outer clasper length (mm) as a function of FL (cm) for sharks caught in the Commercial Shark Fishery Observer Program from 2003-2005 (N=1973). ........................................................................................................................36 2-4 Proportion of male sharks mature based on proxy clasper calcification data. Solid line represents logistic model, dotted lines represent 95% confidence intervals; closed circles represent binary data. ..................................................................................37 2-5 Epididymal width (mm) for sandbar sharks caught in all months. ....................................38 2-6 Mean gonadosomatic index for male sandbar sharks by month. Error bars representstandard error, numbers represent sample size. ...................................................39 2-7 Mean testis width (mm) for sandbar sharks by month; error bars represent standard error, numbers represent sample size. ................................................................................40 2-8 Proportion of mature spermatocysts (stage VI) in sandbar shark testis by month (N=3 samples per month) error bars represent +SE. ..................................................................41 2-9 Sandbar shark mean (+ SE) epididymal width (mm) by month for mature sharks. Numbers above bars indicate sample size. ........................................................................42 2-10 Tissue architecture of the epididymis of sandbar sharks ...................................................43 2-11 Sandbar shark seminal vesicle width (m) by month (N=3 samples per month). Error bars represent standard error. .............................................................................................44 2-12 Seminal vesicles of sandbar sharks ....................................................................................45 2-13 Nidamental gland width of sandbar sharks collected in the northwestern Atlantic Ocean and Gulf of Mexico ................................................................................................47 2-14 Proportion of female sandbar sharks classified as mature based on presence of uterine contents or large nidamental glands. Solid line represents logistic model, dotted lines represent 95% confidence intervals; closed circles represent binary data. ...48 8

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2-15 Sandbar shark litter size by fork length (cm) for specimens collected in the northwestern Atlantic Ocean and Gulf of Mexico. ............................................................49 2-16 Non-pregnant mature sandbar shark mean oocyte diameter (mm) by month. Error bars represent standard deviation, numbers are sample size. .............................................50 2-17 Oocyte diameters (mm) of individual non-pregnant mature sandbar sharks by month showing bimodality in diamters starting in March. Data points represent one measured oocyte for one mature shark. .............................................................................51 2-18 Non-pregnant mature sandbar shark nidamental gland weight (g/FL) by month. Error bars represent standard error, numbers are sample size. ....................................................52 2-19 Monthly mean maximum embryo stretch total length of sandbar shark litters. Error bars represent standard error; numbers are sample sizes (numbers of litters examined). ..........................................................................................................................53 2-20 Histological preparation of sandbar shark nidamental gland. Area where sperm would be present if stored shown by the arrow, as well as connective tissue ( c) and outer tunic of the gland (o). Bar represents 10 micrometers. .............................................54 2-21 Mean testis gonadosomatic index by month for sandbar sharks sampled in the northwestern Atlantic Ocean and Gulf of Mexico. Error bars represent standard error; numbers are sample size. .........................................................................................55 2-22 Mean epididymal widths by month comparing northwestern Atlantic Ocean and Gulf of Mexico sandbar sharks. Error bars represent standard error; numbers are sample size. ....................................................................................................................................56 2-23 Mean nidamental gland weight (g) by month, comparing northwestern Atlantic Ocean and Gulf of Mexico sandbar sharks. Error bars represent standard error; numbers are sample size. ...................................................................................................57 2-24 Non-pregnant mature sandbar shark oocyte diameters (mm) by month, comparing northwestern Atlantic Ocean and Gulf of Mexico sandbar sharks. ...................................58 3-1 Litter specific embryonic weights from sandbar sharks collected in the northwestern Atlantic Ocean and Gulf of Mexico ...................................................................................72 3-2 Relationship between sandbar shark embryo weight (g) as a function of embryo stretch total length (cm) for sharks in the northwestern Atlantic Ocean and Gulf of Mexico. ..............................................................................................................................75 3-3 Mean embryonic relative condition by litter as a function of embryonic age for sandbar sharks in the northwestern Atlantic Ocean and Gulf of Mexico. ......................76 9

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3-4 Range of embryonic relative conditions and maternal fork lengths (cm) for sandbar sharks caught in the third trimester of gestation in the northwestern Atlantic Ocean and Gulf of Mexico. ...........................................................................................................77 3-5 Percent concentration (by weight) of lipids in sandbar shark embryonic liver samples during the gestation period. ................................................................................................78 3-6 Percent concentration (by weight) of lipids in sandbar shark embryonic liver samples by embryonic mass (g). ......................................................................................................79 3-7 Sandbar shark embryonic concentration of Arachidonic Acid (AA) compared to embryonic mass (g). One outlying datum is circled. ........................................................80 3-8 Sandbar shark embryonic concentration of Eicosapentaoic Acid (EPA) compared to embryonic mass (g). One outlying datum is circled. ........................................................81 3-9 Sandbar shark embryonic concentration of Docosahexanoic Acid (DHA) compared to embryonic mass (g). One outlying datum is circled. ....................................................82 4-1 Fishing mortality schedule for sandbar sharks in the northwestern Atlantic Ocean and Gulf of Mexico based on catch data. .................................................................................97 4-2 Indirect estimates of sandbar shark natural mortality. .......................................................98 4-3 Intrinsic population growth rates r of sandbar sharks under various fishing mortality and changes to age at 50% maturity assuming a biennial and a triennial reproductive cycle and overlaying both cycles. .................................................................99 4-4 Survival elasticity values of sandbar sharks assuming a biennial and triennial reproductive cycle. ...........................................................................................................101 4-5 Sandbar shark survival elasticities assuming a 10year age of 50% maturity and a biennial and triennial reproductive cycle. ........................................................................102 4-6 Fertility elasticities of sandbar shark assuming a 10 year age of 50% maturity and a biennial and triennial reproductive cycle. ........................................................................103 4-7 Fertility elasticities of sandbar shark assuming an 18 year age of 50% maturity and a biennial and triennial reproductive cycle. ........................................................................104 10

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Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy REPRODUCTION OF THE SANDBAR SHARK, CARCHARHINUS PLUMBEUS, IN THE WESTERN NORTH ATLANTIC AND GULF OF MEXICO By Andrew Piercy August 2009 Chair: Debra Murie Cochair: Franklin F. Snelson Jr. Major: Fisheries and Aquatic Sciences The reproduction of the sandbar shark, Carcharhinus plumbeus, in the western North Atlantic Ocean and Gulf of Mexico was examined. Specimens were collected through fishery-dependent and independent sampling programs. Morphological measurements of the sharks and reproductive organs were taken. Indices of maturity were constructed using measurements of gonads, genital ducts, and claspers. Sharks were shown to mature between 140 and 160 cm fork length. Gonadosomatic indices and variation in genital duct condition were used to determine seasonal trends in reproduction of mature sharks. Sandbar sharks have discrete seasonal reproductive cycles: males produce sperm from January to May with a peak in May and females develop eggs from January to May with ovulation occurring in June. Females were shown to exhibit a greater than two year reproductive cycle. Embryonic development was assessed through measurements of weights and lengths of uterine contents. Gestation was 12 months, from July to the following June, with parturition in late June. Trends in embryonic and maternal relative conditions were noted. Embryos from larger female sharks exhibited less variation in relative condition. Demographic analysis was used to determine the magnitude of effect that variations in reproductive parameters can have on population growth. Variation in 11

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age of maturity and seasonality of reproduction were shown to have small effects on intrinsic population growth. Survival of juvenile age classes was shown to have the greatest impact on population growth. 12

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CHAPTER 1 THE SANDBAR SHARK Introduction The sandbar shark, Carcharhinus plumbeus, is a member of one of the largest and most commercially important shark families (Carcharhinidae) (Castro, 1983). This species is widely distributed in tropical and subtropical regions of the Pacific, Indian, and Atlantic Oceans, as well as the Mediterranean Sea (Joung et al., 2004). In North American waters, C. plumbeus ranges from Cape Cod to Florida (Bigelow and Schroeder, 1948; Sminkey and Musick, 1995), throughout the Gulf of Mexico (Springer, 1960; Castro, 1983), and around Hawaii (Daly-Engel et al., 2007). Tagging studies (Casey and Kohler, 1991) and genetic studies using mitochondrial DNA (Heist et al., 1995) and microsatellite markers (Heist and Gold, 1999) have shown sandbar sharks in the eastern Gulf of Mexico and northwest Atlantic Ocean to represent one population. The sandbar shark is a large species, reaching a maximum size of approximately 2 m fork length (FL) (Castro, 1983). Sandbar sharks are commercially harvested throughout much of their range, particularly in the waters of the U.S. east coast. As shark fin soup is the primary product developed from harvested sharks in the commercial shark fishing industry, this species is highly sought after for its large fins. In recent years, sandbar sharks have been heavily fished in the northwestern Atlantic, representing 35.9% of the targeted catch in the coastal bottom longline shark fishery (Burgess and Morgan, 2005). Additionally, this species comprises approximately 20% of the large shark fauna and is second only to the blue shark, Prionace glauca, in recreational catches along the U.S. east coast (Hoff, 1990; Sminkey and Musick, 1995). In recent years there has been a worldwide decline in fish stocks due to over-exploitation. This decrease has been seen in elasmobranchs (sharks, skates, and rays), which are highly vulnerable to overfishing due to their general slow growth and low reproductive output. In the 13

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U.S., shark stocks in the northwest Atlantic Ocean have decreased dramatically over the last 20 years (NMFS, 1993, 2003; Camhi, 1998, 1999). In 1993, the National Marine Fisheries Service (NMFS) adopted a management plan to protect U.S. shark populations (NMFS, 1993). This plan grouped sharks into three categories based on their size and habitat preference: Large Coastal, Small Coastal, and Pelagic. Sandbar shark stocks have previously been managed as part of the Large Coastal species complex. Recent stock assessments of the Large Coastal Shark Complex have shown that some species in this group (e.g., C. limbatus) may be improving while others continue to be over-fished (NMFS, 2003). Musick et al. (1993) documented a two-thirds decline in abundance of sandbar sharks in Virginia waters from 1974 to 1991. However, the majority of U.S. commercial shark fishing occurs in Florida, which has the largest and most active recreational and commercial shark fishery of any Atlantic or Gulf coastal state (Camhi, 1998). Consequently, Florida shark populations have also declined dramatically since the late 1970s (Camhi, 1998). A recent species-specific stock assessment for the sandbar shark shows that the northwestern Atlantic and Gulf of Mexico population is currently over-fished and that over-fishing is still occurring (NMFS, 2006). New commercial shark fishing regulations proposed by NMFS, if adopted, will greatly reduce fishing for sandbar sharks during the 2008 season (NMFS, 2008). These measures may essentially shut down the bottom-longline shark fishery, as sandbar sharks represent the majority of the targeted catch in that fishery. The possibility of a shark fishing closure reinforces the need for updated and improved reproductive data on this commercially important species. More accurate assessment models, developed with updated life history data (age, growth, and 14

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reproduction), will allow fisheries managers to establish more reliable catch quotas to ensure sustainability of the fishery. Age and growth of C. plumbeus in the northwestern Atlantic has been well studied, albeit with conflicting results. Casey et al. (1985) reported a maximum age of 30 years, while a later study by Casey and Natanson (1992) reported maximum age of 40 years. A recent age and growth study has reported a maximum age of 27 for sandbar sharks in the northwestern Atlantic and Gulf of Mexico (Romine, 2008). However, none of the aging studies for sandbar sharks have been validated. Recent attempts to validate the age and growth of the sandbar shark through bomb-radio carbon isotope analysis have been unsuccessful (F.F. Snelson, unpublished data). Variation in reported age and growth has lead to variation in reported ages of maturity. Casey et al. (1985) reported the age of maturity for males to be 13 years, and 12 years for females. Age of maturity relevant for both sexes has been variously reported as being as young as 15 years (Sminkey and Musick, 1995) and as old as 30 years (Casey and Natansan, 1992). However, size at maturity for both male and female C. plumbeus in the northwest Atlantic has been consistently reported to be 150 cm fork length (FL) (Casey and Natanson, 1992; Sminkey and Musick, 1995). To date, Springer (1960) is the most complete study on sandbar shark reproduction in northwestern Atlantic Ocean and Gulf of Mexico. Springers study, now 48 years old, presented some data on the timing of mating, litter size and a few observations on gestation. However, there remains a need for improved and more contemporary data on key components of the reproductive cycle, including the timing of mating and seasonality of reproduction. Additionally, many general reproductive parameters have not been defined including, vitellogenesis (yolk development), timing of sperm production and storage, and timing of 15

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ovulation and parturition. Determining if sandbar shark reproduction is seasonal and how often reproduction occurs (annual, biennial, triennial...etc) is important for fisheries management. The schedule of reproduction impacts the yearly reproductive output of the shark population. If reproduction occurs only every third year then reproductive output would be very low and fisheries managers may need to implement more stringent fishing quotas or other management techniques. Knowledge of the timing of gestation may allow fishery managers to establish time-area closures to protect pregnant sharks from fishing mortality. Additionally, contemporary estimates of the sizes of maturity are needed for sandbar sharks in northwestern Atlantic and Gulf of Mexico as density-dependent variation in the onset of maturity may have occurred as the population has declined. Variation in sizes of maturity can lead to variation in population growth, as lifetime reproductive output is influenced by the number of years a shark is able to reproduce. The ability of fisheries managers to develop more accurate assessments using better estimates of sexual maturity will lead to better management efforts to ensure sustainability of sandbar shark fishing. Objectives The overall goal for this dissertation research was to examine facets of the reproductive biology of sandbar sharks in the Gulf of Mexico and northwest Atlantic Ocean that are critical in developing sustainable management of the species. The specific objectives were to: 1) determine the reproductive cycle of female and male sandbar sharks, including the timing of mating, vitellogenesis, ovulation, gestation, parturition, spermatogenesis, and sperm storage; 2) examine the relationship between maternal nourishment of embryos during gestation and the effect of maternal size on embryonic condition; 3) determine size at maturity; and 4) examine the potential effects of variation in female fitness and reproduction on population growth using suitable life-history models. 16

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CHAPTER 2 REPRODUCTIVE CYCLE OF SANDBAR SHARKS IN THE NORTHWESTERN ATLANTIC OCEAN AND GULF OF MEXICO Introduction Knowledge of reproductive timing and behavior is important for management and assessment of fish stocks. The ability of fisheries managers to develop management policies to ensure sustainability of fisheries is dependent on the accuracy and availability of biological data on the species in the fisheries. Knowledge of the size/age of maturity, in addition to other life history data such as the periodicity of reproduction, can be incorporated into assessment models by fisheries managers to determine the potential population growth of a species and sustainable levels of fishing. Knowledge of the timing of gestation can be used by fishery managers to develop conservation measures, such as time area closures to protect pregnant female sandbar sharks. The presence of a seasonal reproductive cycle in elasmobranchs has only been examined in a few well-studied species, such as the Atlantic stingray, Dasyatis sabina (e.g. Piercy et al., 2003), Blacknose shark, Carcharhinus acronotus (e.g. Sulikowski et al., 2007), Spiny dogfish, Squalus acanthias (Tsang and Callard, 1987), and Bonnethead shark, Sphyrna tiburo (Manire et al., 1995). Gonadosomatic indices (GSI), changes in gonad mass relative to body mass, have been used to determine seasonal changes in the gonads of elasmobranchs (Parsons and Grier, 1992; Johnson and Snelson, 1996). Ovarian GSI and changes in ovarian egg size have been used to determine the timing of vitellogenic activity (yolk development) and ovulation (e.g., Snelson et al., 1988). Ovary weight increases during vitellogenesis as the oocytes accumulate yolk. Ovulation occurs just after a peak in oocyte diameter and peak GSI (Driggers et al., 2004). Observations of ovarian follicle size can also clarify whether female sandbar sharks reproduce 17

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every year or have a period of reproductive inactivity, as has been shown in Atlantic populations of C. acronotus (Driggers et al., 2004). The timing of mating can be determined from the presence of recent mating scars and sperm in the female reproductive tract. However, the timing of fertilization may not correlate with mating. Fertilization in S. tiburo has been shown to occur 6 months after mating (Parsons and Grier, 1992). This possible de-coupling of sperm production, fertilization, and mating may be due to protracted sperm storage in females. Long-term storage of sperm in the female nidamental gland has been shown for S. lewini, C. obscurus (Pratt, 1993) and M. canis (Hamlett et al., 2002). Sperm storage in female sandbar sharks can be analyzed through histological preparations of sandbar shark nidamental glands. Seasonality in the reproduction of male elasmobranchs has also only been explored in a limited number of studies. Seasonal sperm production has only been identified thus far in a handful of shark species, such as Squalus acanthias (Simpson and Wardle, 1967), Scyliorhinus canicula (Dobson, 1974), Mustelus manazo and M. griseus (Teshima, 1981), Sphyrna tiburo (Parsons and Grier, 1992), and C. acronotus (Sulikowski et al., 2007). Increases in GSI of testes typically occur during the period of sperm production. Changes in testes GSI have been reported for several shark species occurring in Florida waters, including Rhizoprionodon terraenovae (Parsons, 1982), S. tiburo (Parsons, 1987), and C. limbatus (Killam, 1987). However, other species such as Prionace glauca (Pratt, 1979) have been shown to exhibit little changes in sperm production and GSI, despite having a seasonally defined mating period. A few studies have shown that peak testes activity is not directly correlated to an increased GSI (Maruska et al., 1996; Tricas et al., 2000). In addition, peak mating has been shown to occur well after peak GSI in M. manzo and M. grieseus (Teshima, 1981) and in the Atlantic stingray (Snelson et al., 1988). 18

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In the case of D. sabina, males store sperm in the seminal vesicles over a protracted period (Piercy et al., 2003). Characterizing the spermatogenic cycle in relation to changes in GSI of testes, and examining the seminal vesicles for sperm storage, will clarify the male reproductive cycle for sandbar sharks. There is the potential for regional variation in their reproductive cycle, as has been shown for several shark species. Latitudinal variation in reproductive parameters has been demonstrated in the bonnethead shark (S. tiburo). Bonnethead sharks in the northwestern Gulf of Mexico exhibited larger asymptotic sizes, larger sizes at maturity, and larger near-term embryos than bonnethead sharks in Tampa Bay or South Florida (Lombardi et al., 2003). Also, while Driggers et al. (2004) reported that C. acronotus reproduce biennially in the Northwestern Atlantic, a recent study suggests that populations of this species in the eastern Gulf of Mexico reproduce annually (Sulikowski et al., 2007). While previous genetic studies suggest that sandbar sharks of the northwestern Atlantic and Gulf of Mexico represent one genetic subpopulation (Heist and Gold, 1999), regional variation in reproductive parameters may still occur. Incorporating possible regional differences in reproduction of sandbar sharks into demographic models will allow insight into how this variation can affect population growth and if these regions should be managed separately. The goal of this study was to gather contemporary data on the reproduction of the sandbar shark in the northwestern Atlantic Ocean and Gulf of Mexico. Specific objectives were to determine the size of maturity for male and female sandbar sharks, determine the timing of reproductive events (e.g. sperm production, vitellogenesis, ovulation, mating, and gestation), and determine if regional variations exist in reproductive parameters. 19

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Methods Sandbar Shark Collections Sandbar sharks were captured throughout the year from January 2003 to November 2007 by personnel in the Commercial Shark Fishery Observer Program (CSFOP) sampling the catch in the bottom-longline shark fishery. This sampling covered commercial shark fishing boats from Louisiana east to Florida and north to North Carolina (Burgess and Morgan, 2005) (N=598 sharks sampled). In addition, sharks were also sampled in the Florida Program for Shark Researchs (FPSR) fishery-independent sampling program in both the northwestern Atlantic and eastern Gulf of Mexico (NSRC, 2007) (N=143 sharks sampled). Bottom-longline shark fishing vessels typically use between 8 to 16 km (5 to 10 mi) of mainline (monofilament or cable) with gangions consisting of a large circle hook (typically 14/0 to 18/0 in size), short steal leader (61cm) and a longer section of monofilament (1.8 m) attached to the main line with a clip. Hooks are baited with a plethora of bait types, including various teleosts (mullet, Mugil cephalus, is common) and smaller sized elasmobranchs. Baited longlines are soaked overnight, usually 8 to 12 hours, and the catch is then hauled in. The CSFOP placed trained scientists on bottom-longline commercial shark fishing vessels to monitor the catch and sample biological materials from a subset of shark specimens, including shark vertebrae, jaws, livers, and reproductive tracts. During times of the year that the bottom-longline commercial shark fishing season was closed, samples were obtained from the FPSR fishery-independent sampling program, thus resulting in near year-round sampling. Fishery-independent sampling was conducted through leasing of several commercial bottom-longline shark fishing vessels utilizing gear and fishing techniques that mirror those used during the commercial season, with the exception of shorter soak times (4-6 hours) to minimize mortality to sharks and any bycatch. 20

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Sampling for Male Reproductive Biology Upon capture, sandbar shark gender was determined and measurements of FL (straight line from the tip of the snout to fork of caudal fin) and total length (TL) (straight line from tip of snout to a perpendicular line drawn from the tip of the upper caudal lobe when in a natural position) were taken. Outer clasper (paired intromittent organs) length was measured for males (Figure 2-1). Reproductive organs and tissue samples were immediately removed from a subset of male sharks (N=303) and placed in seawater buffered 10% formalin solution. Reproductive samples included the right testis (whole), the right epididymis, and a section of the seminal vesicles. Size at maturity of male sandbar sharks was assessed through examination of clasper length as a proportion of shark FL, and epididymal width as a function of FL. Both parameters have been correlated with the onset of maturity (Jensen et al., 2002; Driggers et al., 2004), which is visualized by a marked increase in the sizes of the claspers and epididymides as FL increases. The calcification of claspers also corresponds with maturity in male sharks. While calcification data were not available for specimens in this study, comparison of clasper size in proportion to body size and related calcification data from a previous study on sandbar sharks in Australia (McAuley et al., 2007) were used as a proxy to assess maturity of specimens in the present study (claspers were fully calcified at clasper lengths > 8% FL). A logistic model was used to develop a maturity ogive based on the above parameters. Epididymal width data were analyzed to assess sizes of maturity and seasonal variation in the activity of epididymides. A testis gonadosomatic index (GSI) was developed by dividing the testis weight (g) by FL (mm) 100. Fork length was used in the GSI calculation instead of body mass because sharks are not weighed on the vessels. A Kruskal-Wallis test was used to assess statistical significances of any monthly variations in GSI. 21

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Tissue samples from a subset (N= 32) of mature male reproductive tracts (maturity was determined based on the parameters listed above) were analyzed using standard histology techniques at the University of New Hampshire Histology Lab. Briefly, transverse sections of the testis, epididymis, and seminal vesicle (~2-3 mm) were dehydrated in a graded series of alcohol (80-100% ethanol), cleared in a limonene-based solvent, infiltrated in molten paraffin, and processed for routine paraffin histology. Tissue sections (5 m thick) for use in histological analyses were prepared using a rotary microtome, adhered to poly-L-lysine-coated slides, and stained with Harris hematoxylin and counterstained with eosin. Observations of the prepared slides were conducted with a Leica DM LB2 compound scope. An index of mature spermatocysts present in the testis was calculated by dividing the area of the testis occupied by late stage spermatocysts with the total area of the testis in cross-section (Figure 2-2). Stages of spermatogenesis followed those proposed by Maruska et al. (1996). Area measurements of testes were calculated using Image Pro Express imaging software (Media Cybernetics Inc., Bethesda, MD). Sampling for Female Reproduction Upon capture, female sharks were measured for FL and TL and reproductive organs and tissues were removed from a subset (N= 438) and stored on ice or fixed in seawater buffered 10% formalin. Female reproductive samples included the right ovary (only the right ovary is functional), right nidamental gland (shell gland), and a mid-section of the right uterus. If the female shark was pregnant, then the contents of both uteri were placed on ice and stored at C. Additionally, liver samples were taken from female sandbar sharks and were also placed on ice and later stored at C until processed (see Chapter 3). Uterine contents were examined later in the laboratory and embryos were sexed, measured (FL and stretch TL) and liver samples 22

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dissected and stored at C. After 2 weeks in the formalin solution, reproductive samples were rinsed in water for 24 hours, dehydrated in 50% ethanol for 1 hour, and stored in 70% ethanol. For females, the diameter of a single haphazardly selected ovarian egg and nidamental gland (shell gland) width were measured using digital calipers (Fowler Inc., Newton, MA). A marked increase in nidamental gland width has been shown to indicate maturity of carcharhinid sharks (e.g. Driggers et al., 2004). Maturity assessments were made based on a marked increase in the size of the nidamental gland in relation to female shark size and the presence of uterine contents. The timing of reproductive events and the seasonality of the reproductive cycle were determined through analysis of temporal changes in the morphology of the genital ducts of mature (based on the parameters listed above) sandbar sharks, i.e., changes in oocyte diameter, nidamental gland width, and uterine contents. Tissue samples of nidamental glands (N= 60) from mature sandbar sharks were also prepared using the same standard histological techniques described previously but were processed at Mote Marine Laboratory, FL. Cross-sections of nidamental glands were examined under a compound scope for evidence of sperm storage in the lumen and connective ducts. Regional Comparison Potential differences in reproduction between sandbar sharks in the northwestern Atlantic and Gulf of Mexico were assessed through qualitative analysis. Possible variation in the reproductive cycle was determined through analysis of the timing of reproductive events in the two regions. Regional variations in fecundity were examined by comparing litter sizes with t-tests statistical analysis. 23

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Results Male Reproduction Maximum proportional outer clasper length occurs at fork lengths greater than 130 cm (Figure 2-3). Using proportional clasper sizes of calcified claspers in McAuley et al. (2007) as a reference, sandbar claspers in the present study were predicted to begin calcifying at 4% FL and be fully calcified at 8% FL, corresponding to shark lengths between 90 and 160 cm FL, respectively. Assuming that a shark with a proportional clasper length of 8% or greater is mature, sizes of projected maturity can be determined. Logistic regression analysis of proxy calcification data indicated approximately 50% of sharks at 140 cm FL are mature (p < 0.001; Figure 2-4). At a length of 150 cm FL, 89% of sharks would be considered mature with the remaining 11% of the sharks classified as maturing. At a length interval of 170 cm FL, 100% of sharks would be considered mature. Sandbar shark epididymides begin to increase in size around 140 cm FL (Figure 2-5). Maximum epididymal width was in a 150 cm FL shark. The gonadosomatic index (GSI) for male sandbar sharks showed a significant increase beginning in February with a peak in April (Kruskal-Wallis; F =37.82 p < 0.01; Figure 2-6). Testicular width also showed a marked increase in the spring with a peak in April (Figure 2-7). Histological analysis of testicular tissue showed an increase in the presence of late stage spermatocysts in the spring months with a maximum in May (Figure 2-8). These data indicated that sperm production occurred in the months of March through May, with maximum gonadal activity for male sandbar sharks occurring in May. Epididymal activity followed a similar trend with increases in epididymal width seen in the months of March, April, and May, with maximum width in May (Figure 2-9). Spermatozoa were present in the epididymides during the spring, with a declining presence after the peak in 24

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May (Figure 2-10). Histological architecture of the epididymides showed variation during periods of sperm maturation and inactivity. Epididymal ducts were enlarged with little connective tissue during the spring and early summer months (Figure 2-10b). As spermatozoa presence decreased in the epididymis, epididymal ducts decreased in size and the amount of inter-duct connective tissue increased (Figure 2-10c). Sandbar shark seminal vesicle activity also exhibited a trend of increasing activity during the spring months. Seminal vesicle widths increased in March, April, and May, with a dramatic decrease in width in June (Figure 2-11). Histological analysis showed that spermatozoa were present in the seminal vesicle in April and May, with declining amounts of spermatozoa present in June, and no spermatozoa present in the following months (Figure 2-12). During the period of sperm storage, seminal vesicles exhibited less connective tissue and thinner invaginations projecting into the lumen (Figure 2-12). As sperm storage declined, seminal vesicle invaginations thickened and connective tissue increased. These data indicate that mature male sandbar sharks produce sperm in the months of March, April, and May, with a peak in sperm production occurring in May. Sperm storage in the seminal vesicle occurs during April and May. The decline in the presence of sperm in the genital ducts of males in June suggests that mating actually occurs in late May or June. Female Reproduction Female sandbar sharks exhibit marked increases in nidamental gland widths at fork lengths of 140 cm to 160 cm (Figure 2-13). Also, pregnant sandbar sharks ranged in size from 140 cm to 182 cm FL. To develop a maturity ogive, therefore, females were considered mature if they were pregnant or had large nidamental glands consistent in size as those of pregnant sharks (width > 26 mm). Sizes at which 50% and 100% of the population were mature was 148 cm and 165 cm FL, respectively (p < 0.001; Figure 2-14). Of 20 near-term litters examined, 25

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litter sizes ranged from 6 to 14 pups with a mean of 9.65. Embryonic sex ratio did not vary significantly from 1:1 (t-test; t = 0.55; p = 0.58). Litter size was not correlated with maternal FL (r 2 = 0.017) (Figure 2-14). Mature female sandbar sharks exhibited a seasonal reproductive cycle. The oocytes began vitellogenesis (the accumulation of yolk) in February and showed increasing size and variation in oocyte diameters through the month of June, after which mean oocyte diameter decreased (Figures 2-15). To understand the large variation in oocyte diameter observed over the months of February to June, individual oocyte diameters were plotted as a function of month, with the data indicating a bimodal distribution in each month (Figure 2-16). This bimodality in oocyte diameter indicated that two conditions of mature female sharks occur during the months of vitellogenesis: mature but non-egg developing (smaller oocytes) and mature with egg developing (larger oocytes). Pregnant female sharks do not exhibit concurrent vitellogenic activity during gestation. Marked increases in weight of the nidamental gland occurred in the months of June and July (Figures 2-17). This increase corresponded with the presence of fertilized eggs in the uteri and indicated that sandbar shark ovulation occurs in late June. The gestation period for sandbar shark embryos is approximately 12 months, with the placental stage beginning in late September after approximately 3 months of development, and parturition occurring in late June (Figure 2-19). No evidence of sperm storage was seen in any of the sampled nidamental glands (Figure 2-20). Regional Differences No difference in the timing of sperm production, as indicated by testis GSI, was observed between male sandbar sharks in the Gulf of Mexico and northwestern Atlantic Ocean (Figure 2-21). Males from both regions exhibited increases in epididymal widths during the months April 26

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and May (Figure 2-22). However, a more pronounced increase in epididymal width was observed for Atlantic sharks during this time period (Figure 2-22). The nidamental gland showed similar patterns of increasing weight in the late winter and spring months for both Gulf of Mexico and northwestern Atlantic Ocean groups (Figure 2-23). Females from both regions showed a peak in nidamental gland weight in June and July. Oocyte diameter data indicated that female sandbar sharks in both regions exhibited highly variable values in the spring months (Figure 2-24). Elevated oocyte diameter measurements for the Atlantic region in the month of May are a product of small samples size (N=3). The presence of mature females with small oocytes was noted in both regions (Figure 2-24). Of the 20 late-term litters examined, no significant difference in litter size was observed between sandbar sharks in the Gulf of Mexico and Atlantic Ocean (t-test: t = -0.52, p = 0.62). Discussion Male Reproduction Marked increases in proportional clasper lengths of sandbar sharks were observed to begin at 120 cm FL. Merson (1998) reported increases in clasper lengths at slightly larger sizes, beginning at 130 cm FL and reaching an asymptote at 145 cm FL. Clasper length data in the present study, using calcification data from McAuley et al.s (2007) study as a proxy, suggests that 50% of sharks are mature at a length of 140 cm FL, and 100% of sharks were mature at 165 cm FL. Based on clasper length and calcification, Merson (1998) reported a 50% likelihood of maturity at 147 cm FL. Other studies have consistently reported 50% maturity lengths of 150 cm FL (Casey and Natanson, 1992; Sminkey and Musick, 1995) The onset of increased epididymal size occurs at slightly longer fork lengths than the onset of increasing clasper growth. However, this 10 cm difference in fork lengths may not be significant. Further, inadequate sampling of epididymides from smaller size classes may hinder 27

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analysis of trends in epididymal growth. Also seasonal variation in epididymal width due to sperm production may confound analysis as mature males exhibit small epididymides during reproductively inactive months. These issues suggest that for this study, epididymal width data are not appropriate for assessing maturity. As with other elasmobranchs, mature male sandbar sharks have a defined reproductive cycle and show seasonal variation in the size and activity of their gonads and accessory sex organs (Garnier et al., 1999; Conrath and Musick, 2002; Piercy et al., 2003; Sulikowski et al., 2007). Sandbar sharks, like other carcharhinid sharks, show elevated testicular weight and activity just prior to mating (Sulikowski et al., 2007). However, peak sperm production was not correlated with peak testis width or weight, the latter peaking a month before peak sperm production. This temporal disconnect between peak sperm production and testis size, while short in time span, has been previously reported in other elasmobranch fishes (Maruska et al., 1996; Piercy et al., 2003). The seasonal variation in gonad size is likely an effort to conserve energy through reducing gonad size during times of inactivity. Energy conservation through reduced gonad size is a common strategy not limited to fish species and has been reported in other taxa including birds (e.g. Wikelski et al., 2003). The timing of peak epididymal and seminal vesicle activity also occurs after peak gonad size. As these organs are involved in sperm maturation and transport, their activity should increase as sperm production peaks. This lagging of peak genital duct activity after peak testis size has also been reported in a recent study on sandbar sharks (Merson, 1998) and other elasmobranch fishes (e.g. Piercy et al. 2003). No evidence of long-term sperm storage was observed in sandbar shark seminal vesicles. The absence of sperm in the lower genital ducts of sandbar sharks caught after the month of July, coupled with a decrease in seminal vesicle width 28

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in June, would indicate that mating likely occurs in June. Springer (1960) also reported that mating occurred in June for this species. Seasonal changes in the histological architecture of genital ducts of male sandbar sharks were similar to those reported for the Atlantic stingray (Piercy et al., 2003). Both species show decreases in the amount of connective tissue present in epididymal and seminal vesicle cross sections as the epithelium grows during periods of sperm production. Further, as seen in the Atlantic stingray (Piercy et al. 2003), seminal vesicle invaginations in mature sandbar sharks decrease in thickness as sperm presence increases. Female Reproduction In the present study, the marked increase in nidamental gland width at lengths of 140 to 160 cm FL in female sandbar sharks was similar to that noted by Merson (1998) (140 cm to 150 cm). The smallest pregnant shark in the present study was 140 cm FL. Merson (1998) reported the smallest pregnant sandbar shark to be 156 cm FL. Collectively, these data indicate that female sandbar sharks are maturing at sizes of 140 cm to 160 cm FL. A maturity ogive indicates that at a size of 148 cm 50% of females are mature. This size of maturity for females was consistent with previously reported maturity lengths of 150 cm from other sandbar shark studies (Casey and Natanson, 1992; Sminkey and Musick, 1995; Merson, 1998). As oocyte development has been observed in immature sharks with undeveloped uteri (e.g. White et al., 2002), oocyte diameter was not utilized as a sole marker for maturity in this study. Oocytes may develop without being ovulated, resulting in the resorbtion of the yolk protein by the shark. While other indicators of maturity can be assessed (e.g. presence of hymen, mating scars, presence of sperm in genital ducts) the accuracy of these indicators can be problematic depending on the reproductive behavior of the species. Many indicators may only reveal the occurrence of mating, not maturity. If sandbar shark mating is coercive and initiated by males, 29

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as suggested by Portnoy et al. (2006), then females that have mated may not necessarily be mature. Alternatively, if sandbar shark mating is not coerced by males, but initiated by females, then these markers may hold true. This presents a circular argument in that the validity of many proposed markers of maturity is dependent on the belief that only mature females mate. Further, the ability to accurately categorize a mating wound as fresh, or detecting the presence of sperm or hymen, may be hindered by the use of more than one person sampling the sharks and the available training. Female sandbar sharks have a defined seasonal reproductive cycle. Oocyte diameter data for mature non-pregnant females indicate that vitellogenesis, the accumulation of yolk, occurs between February and June. Merson (1998) reported similar timing of yolk development for sandbar sharks. No concurrent vitellogenesis with gestation was observed in any specimen in the present study. In combination with a protracted gestation period, this suggests that sandbar sharks have a reproductive cycle that is longer than one year. Springer (1960) and Merson (1998) have also previously reported that the reproductive cycle for female sandbar sharks was longer than one year. The bimodality in oocyte size of mature female sandbar sharks indicated that a portion of the mature population may exhibit a resting phase, whereby they are neither increasing their oocyte size through vitellogenesis nor are they pregnant. This results in three reproductive conditions present during the spring: pregnant, mature vitellogenic sharks, and mature resting sharks. This indicates the potential for an entire reproductive cycle for female sandbar sharks in this region to be greater than 2 years. While Springer (1960) proposed a 2 year reproductive cycle for sandbar sharks, Merson (1998) also noted bimodal oocyte data indicating a 3 year reproductive cycle. 30

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Reproductive cycles greater than 2 years have been reported in other fishes. Dusky sharks, Carcharhinus obscurus, are believed to have a 3 year reproductive cycle, based on the presence of a protracted gestation period (Musick et al., 1993; Dudley et al., 2005). Further, green sturgeon Acipenser medirostris, which have growth characteristics similar to sharks, have been shown to skip-spawn with intervals of 2, 3, and 4 years in between reproductive events (Erickson and Webb, 2007). The potential reduction in sandbar shark reproductive output from a more protracted reproductive schedule may impact population growth. Assessment models utilizing biennial reproductive data may be over estimating sandbar shark reproductive output. This reinforces the need for assessing how variation in reproductive parameters can affect potential population growth (See Chapter 4). An alternative explanation for the presence of resting females is that these sharks were not really mature. However, the size range of females in this resting mode was 146 to 172 cm FL, well within the range of maturity. Further, the widths of the nidamental glands from these females fell well within the range of nidamental widths of pregnant females, which further suggests that these females were indeed mature. While uterine width measurements can be used as another indicator for maturity (Whitney and Crow, 2007), reliable measurements of uterine widths were not available for the specimens in this study. Another alternative explanation for these bimodal data was that the resting females were pregnant, but gave birth early or aborted during capture. No evidence of abortion (e.g., presence of partial litters in utero, partial birth) due to stress of capture was noted during the sampling in this study. Further, the time period of parturition was short based on embryonic data in this study, and the presence of free swimming neonates (Mote Marine Lab survey, Tyminski pers. comm.) Observations of neonates in other studies of sandbar sharks are largely restricted to 31

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the months of June to August (Nichols and Murphy, 1916; Breeder, 1925; Bigelow and Schroeder, 1948; Osarno, 1992; Merson, 1998). Fishery-independent surveys in the northwestern Atlantic and Gulf of Mexico have reported small numbers of neonate sandbar sharks captured earlier than June: NMFS Panama City reports two in May (Baremore, pers. comm.); and Mote Marine Laboratorys survey reports one undersized in April (Tyminski, pers. comm.). Additionally, while the Virginia Institute of Marine Science fishery-independent surveys report 42 neonates captured in the month of May, this number is far less than the 143 captured in the month of June (Romine, pers. comm.). These studies and data support the observation in the present study that parturition for the majority of sandbar harks occurs in June, but there is some variability in this timing. A mean litter size of 9.6 pups was observed in the present study, a value similar to litter sizes reported in previous studies. Springer (1960) reported a mean litter size of ten ( + 2) for sandbar sharks caught in the Atlantic and Gulf of Mexico. Further, Springer (1960) reported no relationship between sandbar shark litter size and maternal size. In contrast, Joung and Chen (1995) reported increasing sandbar shark litter sizes with maternal size for sandbar sharks in Taiwanese waters, albeit it was a very weak relationship (r = 0.41). No evidence of sperm storage by females was observed in this study. While Pratt (1993) found sperm in some female sandbar shark nidamental glands, his samples were only from months just after mating. Furthermore, Pratt (1993) reported that this presence of sperm was likely not long-term storage and that the long reproductive cycle of the sandbar shark would require sperm to be stored for a protracted period of time that may be unfeasible. Regional Comparison While regional and latitudinal variation in age, growth, and reproduction has been noted for other elasmobranch species (e.g. Tricas et al., 2000; Lombardi et al., 2003; Sulikowski et al., 32

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2007), no regional variation in the timing of reproductive events was observed for sandbar sharks in the Gulf of Mexico and northwest Atlantic Ocean. As little genetic variation is seen between sandbar sharks in these two regions (Heist et al., 1995; Heist and Gold, 1999), the highly migratory nature of sandbar sharks may inhibit regional variation in population genetics, which in turn may conserve reproductive traits. Indeed, a tagging study has shown that sandbar sharks migrate between the Gulf of Mexico and the Atlantic Ocean (Casey and Kohler, 1991). Summary While this study report slightly smaller sizes of maturity for males, the sizes of maturing female sandbar sharks is similar to lengths of maturity reported in previous studies. The potential impact that small variations in maturity sizes can have on recruitment levels may be minor depending on the age structure. Contrary to previous studies, the reproductive cycle of female sandbar sharks may be more protracted than conventionally believed. The data presented in this study indicate a 3 year reproductive cycle as opposed to the previously reported 2 year reproductive cycle. This difference in reproductive timing may have major impacts on sandbar shark stock assessments. Modeling the potential changes in population growth due to updated reproductive knowledge will allow for the evaluation of previous management practices and better development of future stock assessments (see Chapter 4). 33

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Figure 2-1. Measurement of outer clasper length in sandbar shark. Measurement is taken from the margin of the pelvic fin (P) to the tip of the clasper ( c). Line represents the plane of measurement. 34

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35 Figure 2-2. Histological cross-section of sandbar shark testis, showing germinal zone (G) and epigonal tissue (E). Dotted line demarcates the area of testis tissue and dashed line the area occupied by late stage spermatocysts. Bar represents 1mm.

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02468101214165060708090100110120130140150160170180190200210Fork Length (cm)Outer clasper length (%FL) Calcifying Calcified 36 Figure 2-3. Sandbar shark proportional outer clasper length (mm) as a function of FL (cm) for sharks caught in the Commercial Shark Fishery Observer Program from 2003-2005 (N=1973). Boxes represent specimens that would have calcifying and calcified claspers based on the proxy clasper data from McAuley et al. (2007).

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00.10.20.30.40.50.60.70.80.910102030405060708090100110120130140150160170180190200210220Fork Length (cm)Proportion mature )1/(1)1666.03019.23(FLey 37 Figure 2-4. Proportion of male sharks mature based on proxy clasper calcification data. Solid line represents logistic model, dotted lines represent 95% confidence intervals; closed circles represent binary data.

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0510152025303540507090110130150170190Fork Length (cm)Epididymis width (mm) 38 Figure 2-5. Epididymal width (mm) for sandbar sharks caught in all months.

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23131661331015020406080100120140160180200JanFebMarAprMayJunJulAugSeptOctNovDecMonthGonadosomatic Index 39 Figure 2-6. Mean gonadosomatic index for male sandbar sharks by month. Error bars representstandard error, numbers represent sample size.

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231922291061362205101520253035JanFebMarAprMayJunJulAugSeptOctNovDecMonthMean testis width (mm) 40 Figure 2-7. Mean testis width (mm) for sandbar sharks by month; error bars represent standard error, numbers represent sample size.

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00.050.10.150.20.250.3JanFebMarAprMayJunJulAugSeptOctNovMonthProportion of mature spermatocyst (stage VI) by area 41 Figure 2-8. Proportion of mature spermatocysts (stage VI) in sandbar shark testis by month (N=3 samples per month) error bars represent + SE.

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2318226147926190510152025JanFebMarAprMayJunJulAugSepOctNovDecMonthEpididymal width (mm) 42 Figure 2-9. Sandbar shark mean ( + SE) epididymal width (mm) by month for mature sharks. Numbers above bars indicate sample size.

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A B s c c C 43 c Figure 2-10. Tissue architecture of the epididymis of sandbar sharks: A) during reproductively inactive months (e.g. November); B) during peak sperm production (May); and C) after sperm production (June); e and arrow: epididymal duct; s: spermatozoa; c: connective tissue. Bar represents 1mm.

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0200400600800100012001400JanFebMarAprMayJunJulAugSepOctNovMonthMean seminal vesicle width (um) Figure 2-11. Sandbar shark seminal vesicle width (m) by month (N=3 samples per month). Error bars represent standard error. 44

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Figure 2-12. Seminal vesicles of sandbar sharks: A) tissue architecture during sperm storage (May; magnification 8x); B) tissue architecture during mating period (June; magnification 12.5x); C) tissue architecture during the months following mating (e.g. August; magnification 8x); lower case "c": connective tissue; s: seminal fluid with spermatozoa; arrow: seminal vesicle invaginations. Bar represents 1mm. 45

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46 C B A s s c c c

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01020304050606080100120140160180200Nidamental gland width (mm)Fork Length (cm) Pregnant Vitellogenic Resting Other 47 Figure 2-13. Nidamental gland width of sandbar sharks collected in the northwestern Atlantic Ocean and Gulf of Mexico.

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00.10.20.30.40.50.60.70.80.910102030405060708090100110120130140150160170180190200Fork length (cm)Proportion mature )1/(1)1878.09656.27(FLey 48 Figure 2-14: Proportion of female sandbar sharks classified as mature based on presence of uterine contents or large nidamental glands. Solid line represents logistic model, dotted lines represent 95% confidence intervals; closed circles represent binary data.

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y = 0.0324x + 4.2477R2 = 0.01760246810121416150155160165170175180185Maternal Fork Length (cm)Litter size 49 Figure 2-15. Sandbar shark litter size by fork length (cm) for specimens collected in the northwestern Atlantic Ocean and Gulf of Mexico.

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41610122543171722323044051015202530JanFebMarAprMayJunJulAugSepOctNovDecMonthMean oocyte diameter (mm) 50 Figure 2-16. Non-pregnant mature sandbar shark mean oocyte diameter (mm) by month. Error bars represent standard deviation, numbers are sample size.

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05101520253035404501234567Month (January June)Oocyte diameter (mm) 51 Figure 2-17. Oocyte diameters (mm) of individual non-pregnant mature sandbar sharks by month showing bimodality in diamters starting in March. Data points represent one measured oocyte for one mature shark.

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816816204317131624244002468101214161820JanFebMarAprMayJunJulAugSeptOctNovDecMonthMean weight (g)/FL 52 Figure 2-18. Non-pregnant mature sandbar shark nidamental gland weight (g/FL) by month. Error bars represent standard error, numbers are sample size.

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Figure 2-19. Monthly mean maximum embryo stretch total length of sandbar shark litters. Error bars represent standard error; numbers are sample sizes (numbers of litters examined). 1574315411835010203040506070JanFebMarAprMayJunJulAugSepOctNovDecMonthMean max embryo STL (cm)* 53

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54 Figure 2-20. Histological preparation of sandbar shark nidamental gland. Area where sperm would be present if stored shown by the arrow, as well as connective tissue ( c) and outer tunic of the gland (o). Bar represents 10 micrometers. o cc

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264456923131039146050100150200250JanFebMarAprMayJunJulAugSepOctNovDecMonthMean gonadosomatic index GSI Atlantic GSI Gulf of Mexico 55 Figure 2-21. Mean testis gonadosomatic index by month for sandbar sharks sampled in the northwestern Atlantic Ocean and Gulf of Mexico. Error bars represent standard error; numbers are sample size.

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710467519132313123944760510152025JanFebMarAprMayJunJulAugSepOctNovDecMonthEpididymal width (mm)/fork length (cm) Atlantic Gulf of Mexico 56 Figure 2-22. Mean epididymal widths by month comparing northwestern Atlantic Ocean and Gulf of Mexico sandbar sharks. Error bars represent standard error; numbers are sample size.

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792131622639121134763519109812136051015202530JanFebMarAprMayJunJulAugSepOctNovDecMonthNidamental gland weight(g)/FL(cm) Atlantic Gulf of Mexico 57 Figure 2-23. Mean nidamental gland weight (g) by month, comparing northwestern Atlantic Ocean and Gulf of Mexico sandbar sharks. Error bars represent standard error; numbers are sample size.

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05101520253035404501234567MonthOocyte diameter (mm) Atlantic Gulf of Mexico Figure 2-24. Non-pregnant mature sandbar shark oocyte diameters (mm) by month, comparing northwestern Atlantic Ocean and Gulf of Mexico sandbar sharks. 58

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CHAPTER 3 MATERNAL INFLUENCE ON SANDBAR SHARK EMBRYONIC DEVELOPMENT Introduction Elasmobranch fishes (sharks, skates, and rays) utilize internal fertilization, and exhibit several modes of reproduction: oviparity (egg laying); ovoviviparity (live birth without a placental connection); and placental viviparity (live birth with placental connection). All of these modes result in potentially fewer offspring in a given reproductive event compared to fishes that have external fertilization. However, most sharks produce large young that exhibit a higher survival rate than the progeny of teleost fishes (Pratt and Casey, 1990). The most fecund sharks are the oviparous species whose fecundity is not hindered by offspring size. Embryonic development of oviparous sharks is dependent on the energy stored in the yolk of the egg. Thus, the young of oviparous shark species are generally smaller than viviparous species (Pratt and Castro, 1990). Embryonic development of ovoviviparous species is initially fueled by egg yolk. However, ovoviviparous elasmobranchs have developed several methods for further nourishment of embryos, such as the secretion of a nutrient rich milk in the uterus (histotroph) (e.g. Snelson et al., 1988), the ovulation of unfertilized eggs for embryonic ingestion (oophagy) (e.g. Jensen et al., 2002), and inter-uterine cannibalism (adelphagy) (e.g. Hamlett and Koob, 1999). The most advanced mode of reproduction exhibited by sharks is placental viviparity (Pratt and Castro, 1990). In this mode, embryos are initially nourished by egg yolk. Once the yolk sac is empty, it interdigitates with the maternal uterine wall and forms a yolk sac placenta (Hamlett and Wourms, 1984; Pratt and Castro, 1990). This connection serves several functions: transfer of nutrients; transfer of macromolecules, such as immunoglobulins; respiration; and osmotic and ionic regulation (Hamlett and Koob, 1999). In placental shark species, as initial yolk stores in the yolk sac are depleted, the maternal liver produces yolk 59

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protein precursors that are secreted into the blood, where they are utilized by developing oocytes for the next reproductive cycle (Hamlett and Koob, 1999). These yolk precursors can also be transported across the uterus and fetal placenta to nourish embryos during gestation (Hamlett and Koob, 1999). It is also probable that in addition to yolk protein precursors, the maternal liver mobilizes maternal fat stores for embryo nourishment. Livers of pregnant elasmobranchs, such as Trygon violacea (Lo Bianco, 1919; Ranzi, 1933), Torpedo ocellata (Reach and Widakowitch, 1912; Ranzi, 1933), and Acanthias blainvillei and Mustelus vulagris (Ranzi, 1933) are reduced in size as embryos develop in utero. Ranzi (1933) suggests that mobilization of lipids may be a major factor in embryonic growth and maternal lipid reduction. Analysis of the fatty acid composition of the maternal liver and embryo livers may give insight into which fatty acids are mobilized for embryonic growth and whether the mobilization of these fatty acids is reflected in maternal fatty acid composition. The presence of essential fatty acids, those fatty acids that are not synthesized but derived wholly from diet, would provide positive support for the presence of a mechanism for maternal sharks to transfer fatty acids to embryos. Further, the roles of fatty acids in growth and development of sharks is poorly understood. However, three essential fatty acids, docosahexanoic acid (DHA), eicosapentanoic acid (EPA), and arachidonic acid (AA), have been reported as important in teleost fish growth and development (Copeman et al., 2002). Determining the presence and concentration of fatty acids, particularly DHA, EPA, and AA, in shark embryos may support or refute the idea that fatty acids are important in embryonic development. The sandbar shark exhibits the placental viviparous mode of reproduction. Yolk-filled eggs are ovulated out of the gymnovarian-type ovary, move through the oviduct, are fertilized in the nidamental glands, and then are retained in the paired uteri for the duration of embryonic 60

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development (Pratt and Castro, 1990). The gestation period for sandbar sharks in the northwestern Atlantic and Gulf of Mexico is 12 months (see Chapter 2). The placental stage of embryonic development is reached approximately 3 months after fertilization (see Chapter 2). Some shark species exhibit trends in fecundity with larger females producing larger young or more pups per reproductive event than smaller females (e.g. blue sharks Prionace glauca and thresher sharks Alopias sp.) (Pratt and Casey, 1990). However, these trends are not well studied for most shark species. Joung and Chen (1995) and Sminkey and Musick (1996) noted a weak correlation of increasing litter size with increasing maternal size for sandbar sharks in Taiwanese waters and northwest Atlantic, respectively. Other studies including the present study (see Chapter 2), however, have not reported this relationship in Atlantic and Gulf of Mexico populations of sandbar sharks (Springer, 1960; Merson, 1998). Further, no study has examined the potential for larger sandbar sharks to produce larger young. On the assumption (or basis) that larger young have higher survival (Wourms, 1977), this ultimately would result in an increase in the fitness of the female (Brockelman, 1975). In the present study, runts were observed in many sandbar shark litters after the onset of the placental connection. Although the presence of shark runts in viviparous species has been noted in other studies (e.g. Jensen et al., 2002), none have thoroughly examined the mechanism or cause for such variation in embryonic size. Portnoy et al., (2007) have shown that sandbar sharks in Florida waters are polyandrous in their mating habits. Therefore, variation in pup condition could be related to paternal contribution to embryonic DNA. However analysis of the genetic makeup of runts indicated they were not genetically distinct from non-runt siblings, i.e. male sharks that fathered runts also fathered non-runt sharks (Portnoy and Piercy, unpublished data). This suggests that the occurrence of runts may not be related to paternal genetic 61

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contribution but rather some maternal mechanism, such as nutritional partitioning. Further analysis of the relationship between maternal condition or size with embryonic condition, and the mechanisms of embryonic nourishment, is certainly merited (Wourms et al., 1988). The objectives for this study were therefore to: 1) determine if there is a relationship between maternal length and embryonic condition for sandbar sharks caught in the northwestern Atlantic Ocean and Gulf of Mexico; 2) determine if female sandbar sharks mobilize essential fatty acids to developing embryos during the placental stage of gestation; and 3) determine if embryonic fatty acid concentration and lipid levels vary with embryonic weight. Methods Pregnant sandbar sharks were collected through fishery-dependent and fishery-independent sampling (see Chapter 2). Upon collection, measurements (to nearest cm) were taken of maternal fork length (straight-line distance from snout to fork of the tail) and total length (tip of the snout to the posterior tip of the tail, with the tail in the natural position, taken along the body midline to a point intersected by a perpendicular dropped from the posterior tip of the upper lobe of the tail). Embryos from each litter were frozen and later measured for embryonic fork length and stretch total length (straight-line distance from snout to end of upper lobe of the caudal fin as stretched in a straight line), and weighed to the nearest 0.1 g in the lab. Relative condition (Kn) (Le Cren 1951) values were calculated for each embryo using the equation: Kn = W/aL b ; with W being weight in grams, L being length (cm), and coefficients a and b derived from the allometric relationship of weight as a function of length for all sandbar shark embryos. Variation in relative condition of embryos due to litter size, maternal length, and month of capture (embryonic age) were examined using linear regression analysis. Maternal and embryonic liver samples were also collected from pregnant females and their litters and stored at -29 C. A subset (N = 5 litters) of liver samples were analyzed for fatty acid 62

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composition at the Analytical Toxicology Core Laboratory, University of Florida. Briefly, lipids were extracted from each sample using a solution of 2:1 chloroform/methanol with 0.01% butylated hydroxytoluene (Folch et al., 1957, as modified by Iverson et al., 1997). Fatty acid methyl esters (FAMEs) were produced using acid-catalyzed methylation. Methyl esters of the fatty acids were analyzed using gas chromatography (Hewlett-Packard 6890) with mass spectral detection (Hewlett-Packard 5973). The analytes, separated across a CP-WAX column (Varian, 25 m x 0.25 mm inside diameter, 0.2 m film), were identified by comparing the retention times and m/z (mass to charge ratio) of selected ions from analytes in the samples to those of authentic standards (NuCheckPrep, Supelco). Fatty acids were named using International Union of Pure and Applied Chemistry nomenclature of number of carbons: number of double bonds, where n-x indicates the position of the first double bond in relation to the terminal methyl end of the fatty acid. Concentration of fatty acids was determined against five-point standard curves and reported as ng FA/ ug total FA weight. To determine % total lipid in the tissue, the lipids in a weighed portion of tissue sample (1-2 g) were extracted using a hexane-acetonitrile mixture (Szabo pers. comm.). The solvent was then separated from the lipid by evaporation and the lipid content was determined gravimetrically. Fatty acid levels and lipid concentrations were determined for a subset of maternal (N=5) and embryonic (N=15) liver samples. Three embryos of varying weights (minimum, middle, and maximum) from five litters were chosen along with corresponding maternal samples for analysis. Analysis focused on variation in concentrations of docosahexanoic acid (DHA, 22:6n-3), eicosapentanoic acid (EPA, 20:5n-3), and arachidonic acid (AA, 20:4n-6) among the samples as these fatty acids have been shown to be important in fish growth. 63

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Results Embryonic condition Large intra-litter variations in embryonic weights were observed in sandbar shark throughout the gestation period, particularly during later stages of gestation (Figure 3-1). Runts, embryos that were visibly smaller than litter mates were observed in several litters examined (Figure 3-1). The maximum variation in embryonic weight was observed in one runt of a litter captured in January that weighted 66% less than the mean litter weight. For some pregnant sandbar sharks runts were detected in both uteri. No significant inter-uterine variation in the number of embryos present in right and left uteri was observed (paired t-test: t = -1.374; p = 0.187). Sandbar shark embryos exhibited an allometric relationship between weight (WT) and stretch total length (STL) (Figure 3-1) described by WT=0.0044STL 3.08 (Figure 3-2). Relative condition values were calculated for all embryos of the 26 litters examined, with a mean calculated per litter. Analysis of embryos in the placental stage of gestation showed a significant relationship between the month of gestation and mean condition of embryos in the litters (Figure 3-3; r 2 = 0.33, F = 5.79, p = 0.032). When accounting for the trimester of gestation when the embryos were captured, no significant relationship between maternal FL and mean embryonic condition was detected (Table 3-1). Further, no relationship between the range of mean embryonic condition (maximum minimum mean values) and maternal FL was detected for the first and second trimesters (Table 3-1). However, a significant relationship was detected between maternal FL and the range of embryonic condition observed in sandbar sharks in the third trimester (Table 3-1; Figure 3-4; r 2 = 0.91, F = 42.44, p < 0.01). No significant relationship was detected between litter size and the mean or range in mean embryonic condition in any trimester (Table 3-1). 64

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Lipid analysis A significant positive relationship between the duration of gestation and mean embryonic liver percent lipid concentration was observed (Figure 3-5; r 2 = 0.93, F = 39.07, p < 0.01). While a positive significant relationship between embryonic mass and percent lipid concentration of the liver was observed (Figure 3-6; r 2 = 0.86, F = 81.69, p < 0.001), no significant relationship was detected between maternal fork length and maternal liver lipid concentration (r 2 = 0.24, N=5, F = 0.96, p = 0.39). One maternal liver sample exhibited much lower lipid levels than the other samples tested (2.92% versus an average of 61.9%). Essential fatty acids (fatty acids wholly derived from maternal diet) were detected in embryonic liver samples (Appendix A). During analysis of fatty acid data, one outlier was detected (see Figures 7, 8, and 9) and not used in regression analysis. Maternal concentrations of AA, EPA, and DHA did not vary significantly with maternal FL (Table 3-2). The mean and range of embryonic concentrations of AA, EPA, and DHA also did not vary significantly with maternal FL or duration of gestation (Table 3-2). However, embryonic AA, EPA, and DHA concentration decreased as embryo mass increased (Table 3-2; Figures 7, 8, and 9). Discussion While variation in embryonic growth has been observed in previous studies of viviparous sharks, no study has examined potential mechanisms causing this variation. Studies of sandbar shark reproduction have previously focused on basic reproductive knowledge, including age/size of maturity, reproductive cycle (e.g. Springer 1960; McAuley et al. 2007), and observations of the stages of gestation (Baranes and Wendling, 1981). The presence of runts and large variations in embryonic relative condition throughout the year indicated that this phenomenon was not limited to specific stages of gestation or specific developmental events. Further, the occurrence of runts in June, the month of parturition, suggests that these smaller embryos are indeed born at 65

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a smaller size. Relative condition suggested that while maternal size does not influence litter size, smaller pregnant sandbar sharks may be less able to provide nourishment to embryos during the placental stage of gestation. The decreasing range in relative embryonic condition with increasing maternal length in the third trimester suggested that while larger female sandbar sharks do not have higher mean embryonic relative conditions, larger females are better able to provide equal nutrients to all embryos present. This decrease in range of embryonic condition may also be related to maternal age. Smaller/younger female sharks with large ranges of embryonic condition may be reproducing for the first time. While no study on sandbar sharks has shown reduced embryonic fitness for younger mothers, some female teleost fishes have been shown to produce smaller eggs during their first reproductive cycle (e.g. Abodi et al. 2005). Further, some teleost species exhibit a positive correlation between maternal size and egg size (e.g. Morita and Takashima, 1998). Larger egg sizes in teleosts have been shown to have a positive influence on juvenile survival (Hutchins, 1991). Further, runts of the litter in swine have been shown to have slower post natal growth than non-runt pigs (Powell and Aberle, 1980). The effect of birth size on post-natal growth in sharks has not been studied. Also, the potential for growth compensation may allow for undersized neonates to catch up with their cohort as seen in some teleosts (e.g. Ali and Wootton, 2001). A form of compensatory growth, specifically density-dependent increases in juvenile growth rates, has been reported for sandbar sharks in the northwestern Atlantic Ocean (Sminkey and Musick, 1995). However, the potential negative costs to compensatory growth, as seen in some fish (e.g. Royle et. al., 2006), has not been studied in sharks. The ability of larger sandbar sharks to give birth to pups that are uniformly large may result in higher per litter survival rates, 66

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as smaller neonates may exhibit greater mortality, following the traditional view that bigger is better regarding fish size and natural mortality. Large body sizes in fish have been shown to provide fitness benefits, such as access to more prey types (e.g. Juanes and Conover, 1994) and decrease in numbers of potential predators (e.g. Parker, 1971). Alternatively, Dibattista et al. (2007) reported that selection against larger sizes and relative conditions may be present for juvenile lemon shark (Negaprion brevirostris), as larger sizes may hinder survival as increased foraging to maintain an increase in size may result in increased exposure to predators. The potential for variation in body size and condition to provide either a benefit or hindrance to shark neonate survival rates warrants consideration, particularly in assessment models using indirect estimates of neonate natural mortality, as these models often utilize age and growth data (e.g. Chen and Watanabe, 1989). Further, mortality estimates based on length at age data or weight at age data, using mean values at birth may result in inaccurate estimates of the mortality of neonates if variations in size at birth are present. Intra-litter variation in embryonic lipids levels and condition may be influenced by the position of the pup in utero. While data on the influence of pup position on embryonic development is sparse for sharks, studies on other live bearing animals suggest potential trends. Studies on mice and swine gestation have shown that fetuses occupying anterior uterine positions received greater amounts of nutrient rich blood and were born at greater weights than litter mates (Perry and Rowell, 1969; Ryan and Vandenbergh, 2002; Barr et al., 2005). Further, in mice the position of the sexes in utero was shown to influence embryonic levels of sex hormones through absorption in amniotic fluid and from the maternal blood source (Drickamer, 1996; Ryan and Vandenbergh, 2002). This endocrine variation was shown to influence later behavior (aggression) and reproductive success (Ryan and Vandenbergh, 2002). Low condition sandbar 67

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shark embryos may have inadequate placental connections or connections at poorly vascularized regions of the maternal uterus. Unfortunately in the present study, the position of the embryos in the uteri was not noted. While the energetic cost of reproduction has not been fully assessed, the cost of developing embryos for 12 months is likely high. The amount of resources available to pregnant sharks would likely influence maternal energy stores. If prey density declines, larger females may be out competing smaller sharks for ever increasingly limited resources. Further studies examining these potential relationships in shark development are currently needed. While paternally contributed genetic material appears to not influence the formation of runts in sandbar sharks (Portnoy and Piercy, unpublished data), further genetic analysis is needed to detect if particular genotypes are more likely to develop into runts. The results of the present study suggest maternal control over embryo condition. The mechanism of this control is yet to be determined. Fatty acids essential for embryo growth are transferred from the pregnant females to the embryos via the placental connection. Embryo weight was shown to be a significant factor in embryonic concentrations of EPA, AA, and DHA. While the duration of gestation was not a statistically significant factor on embryonic concentrations of these fatty acids, this parameter covaries with embryo weight, which hinders accurate assessment of its significance. Analysis of the relationship between the duration of gestation and embryonic fatty acids levels exhibited p-values that while not statistically significant (p > 0.05) were nearing statistical significance, further supports the notion of covariation The lack of additional samples for each month (i.e. samples from multiple litters within a month) in the lipid analysis precludes elucidating the covariation or interactions between these two parameters. Covariation aside, levels of important essential fatty acids (EPA, AA, and DHA) appear to decline in the 68

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69 livers of the embryos as they increase in size. This may reflect the embryos every increasing use of these fatty acids for growth. Alternatively, it may reflect an inability of the pregnant sandbar shark to maintain high levels of these fatty acids throughout gestation. Also variation in embryonic fatty acids levels may be related to th e quality or position of the plancetal connection (see above). A larger sample size taken over the entire prot racted gestation period noting placental position may provide further insight. No tably, within litters, lower weight embryos did not appear to have consistently lower levels of lipids or lower concentrat ions of essential fatty acids. This would suggest that li pid and fatty acid levels are not adequate predictors for embryo weight. Mortality estimates are inherent ly difficult to calculate for fish found in open systems (e.g. marine species). If neonate size influences surviv al rates, then stock asse ssment models utilizing a mean size at birth may be underestimating neonate mortality if large vari ations in birth size occur. Further, the potential for embryonic condition to affect survival rates in neonates warrants future research. The possibil ity of these low condition neonate s to have increased mortality, slower growth, or future reproductive issues should be examined. Future, telemetry studies on habitat utilization of various condition neonates in nursery areas may pr ovide insight regarding neonate condition with regards to fora ging effort and mortality rates.

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Table 3-1. Relationships between maternal reproductive parameters and relative condition (Kn) of embryos in their litters for sandbar sharks in the northwestern Atlantic Ocean and Gulf of Mexico during the placental stage of gestation. 1st Trimester (N=5 litters) 2nd Trimester (N=5 litters) 3rd Trimester (N=7 litters) Parameterr 2 Fp-valuer 2 Fp-valuer 2 Fp-valueMaternal FL vs. Mean Kn0.361.700.280.412.160.240.150.700.44Maternal FL vs. Range Kn0.503.000.180.8415.330.030.9142.44<0.01Litter Size vs. Mean Kn0.482.820.190.020.050.840.221.130.34Litter Size vs. Range Kn0.492.960.180.190.680.470.010.010.96 70

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Table 3-2. Regression analysis of maternal (N=5) and embryonic (N=15) parameters and concentrations of three essential fatty acids. Docosahexanoic acid Eicosapentaoic acid Arachidonic acid 22:6n-3 20:5n-3 20:4n-6 Parameter testedr2Fp-valuer2Fp-valuer2Fp-valueMaternal length vs. Maternal concentration0.050.160.720.030.070.790.020.080.79Maternal length vs. Mean embryonic concentration0.040.130.740.060.170.700.100.340.60Maternal length vs. Range of embryonic concentrations0.020.050.830.020.060.820.040.120.75Duration of gestation vs. Mean embryonic concentrations0.706.920.080.696.760.080.738.230.06Duration of gestation vs. Range embryonic concentrations0.665.850.090.665.800.090.696.700.08Duration of gestation vs. Maternal concentration0.030.090.780.030.100.77<0.01<0.010.95Embryo mass vs. embryonic concentration0.4611.31<0.010.4911.56<0.010.5917.15<0.01 71

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0501001502002503003504004500123456Litter numberEmbryonic weight (g) Normal RuntsA 72 Figure 3-1. Litter specific embryonic weights from sandbar sharks collected in the northwestern Atlantic Ocean and Gulf of Mexico, illustrating the presence of runts in the; A) first trimester, B) second trimester, C) third trimester.

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01002003004005006007008009000123456Litter numberEmbryonic weight (g) Normal RuntsB 73 Figure 3-1. Continued.

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0200400600800100012001400012345678Litter numberEmbryonic weight (g) Normal RuntsC 74 Figure 3-1. Continued.

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y = 0.0044x3.083802004006008001000120014001600010203040506070Embryo Stretch Total Length (cm)Mass (g) 75 Figure 3-2. Relationship between sandbar shark embryo weight (g) as a function of embryo stretch total length (cm) for sharks in the northwestern Atlantic Ocean and Gulf of Mexico.

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y = 0.0326x + 0.7181R2 = 0.821900.20.40.60.811.21.402468101214Embryonic age (months)Mean embryonic Kn 76 Figure 3-3. Mean embryonic relative condition by litter as a function of embryonic age for sandbar sharks in the northwestern Atlantic Ocean and Gulf of Mexico. Data symbol square denotes embryos shifting from yolk nourishment to placenta. The conditions of these embryos were considered artificially elevated and were not used in the regression analysis.

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y = -0.0002x + 0.0398R2 = 0.913900.0020.0040.0060.0080.010.012155160165170175180185Maternal Fork Length (cm)Range of Kn values 77 Figure 3-4. Range of embryonic relative conditions and maternal fork lengths (cm) for sandbar sharks caught in the third trimester of gestation in the northwestern Atlantic Ocean and Gulf of Mexico.

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y = 3.0434x + 43.168R2 = 0.9287010203040506070809002468101214Duration of gestation (months)Mean lipid percentage 78 Figure 3-5. Percent concentration (by weight) of lipids in sandbar shark embryonic liver samples during the gestation period.

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y = 0.0248x + 52.658R2 = 0.862901020304050607080901000200400600800100012001400Embryo mass (g)Lipid percentage 79 Figure 3-6. Percent concentration (by weight) of lipids in sandbar shark embryonic liver samples by embryonic mass (g).

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00.511.522.533.50200400600800100012001400Embryo weght (g)Embryonic concentration of AA (ng/ug) Litter 1 Litter 2 Litter 3 Litter 4 Litter 5 80 Figure 3-7. Sandbar shark embryonic concentration of Arachidonic Acid (AA) compared to embryonic mass (g). One outlying datum is circled.

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00.511.522.50200400600800100012001400Embryo weight (g)Embryonic concentration of EPA (ng/ug) Litter 1 Litter 2 Litter 3 Litter 4 Litter 5 81 Figure 3-8. Sandbar shark embryonic concentration of Eicosapentaoic Acid (EPA) compared to embryonic mass (g). One outlying datum is circled.

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Figure 3-9. Sandbar shark embryonic concentration of Docosahexanoic Acid (DHA) compared to embryonic mass (g). One outlying datum is circled. 0123456789100200400600800100012001400Embryo weight (g)Embryonic concentration of DHA (ng/ug) Litter 1 Litter 2 Litter 3 Litter 4 Litter 5 82

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CHAPTER 4 THE EFFECTS OF REPRODUCTIVE VARIATION ON SANDBAR SHARK POPULATION GROWTH Introduction Historically, the sandbar shark has been the key species in coastal commercial shark longline fisheries in the U.S., representing over a third of the total landings (Morgan and Burgess, 2004). Efforts towards a sustainable fishery for this commercially important shark species have been largely unsuccessful, as sandbar stocks along the U.S. east coast were recently deemed overfished (NMFS, 2006). Failure of previous management practices may partially be due to insufficient or incorrect biological data concerning the life history and reproduction of the sandbar shark. An earlier assessment was optimistic regarding sandbar shark population status (NMFS, 2002), but changes in reported life history parameters specifically the age at 50% maturity (13y changed to 19y) showed that these sharks were less productive than originally determined (NMFS, 2006). Variation in reproductive parameters of the sandbar shark presented in Chapters 2 and 3 demonstrate that additional revisions to life history data will be necessary in stock assessment models for this species. In particular, the potential for female sharks to exhibit a 3 year reproductive cycle in contrast to the long held belief of a 2 year reproductive cycle, warrants research into the possible impact parameter changes of this magnitude may have on population growth. Also, plasticity in the sizes/ages of maturity may have strong impacts on population growth. To explore these issues, I used demographic models while applying sandbar shark reproductive data detailed in Chapters 2 and 3. Demographic models have been widely used to evaluate the validity of parameter estimates and management strategies in many animals. For example, models of sea turtle demography have lead to more emphasis on reducing adult mortality, as adult age classes contributed more to population growth than younger age classes 83

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(Crouse et al., 1987). Grusha (2005) reported that parameter values from published life history studies for the cownose ray, Rhinoptera bonasus, in the Chesapeake Bay seemed reasonable but led to a life table prediction that the population would crash under no fishing pressure unless survival of neonates exceeded 100% (Gedamke et al., 2007). This outcome showed that the life history parameters of the cownose ray were not plausible. Life tables have been used by many biologists to estimate population growth in elasmobranch fishes (e.g., Hoenig and Gruber, 1990; Cailliet, 1992; Simpfendorfer, 2005). Life tables use existing life history data (e.g., fecundity, number of female pups per litter, mortality rates, frequency of reproduction) to calculate values for r (intrinsic rate of population increase). Alternatively, Leslie matrix models have been slow to be utilized for demographic studies via the use of matrix algebra and the extra computational requirements (Simpfendorfer, 2005). Leslie matrix models have recently begun to be favored for demographic analyses, however, due to increases in available computer programs for solving the matrix algebra equations (Simpfendorfer, 2005). The development of Leslie matrices allows estimation of r values for various scenarios of fishing mortality, natural mortality, and reproductive parameters. Further, Leslie matrices allow for the calculation of elasticity values of the matrix elements (survival and fertility). Elasticity is a measure of proportional effect, i.e., the effect that a change in a given matrix element has as a proportion to the change in that element. The elasticity values allow for the determination of the magnitude of the effect various life-history stage or age transitions can have on population dynamics or potential population growth (dominant eigen value). Previous studies have utilized elasticity calculations to elucidate important parameters that affect shark population growth. Goldman (2002) reported that variation in age of first reproduction in salmon sharks, Lamna ditropis, accounted for more 84

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influence on population growth than maximum age. A lack of adequate data has previously hindered these kinds of analyses for the sandbar shark. In this study, Leslie matrix models constructed from life tables were used to answer several questions: 1) does frequency of reproduction influence potential population growth? 2) does variation in age of maturity influence population growth? 3) do the impact of these reproductive variations change under different fishing mortalities? and 4) at what age group is survival most important for population growth? Methods Sandbar shark reproductive data from Chapter 2 were incorporated into the demographic models. Ages were assigned to length classes using the von Bertalanffy growth equation from the most recent life history data available for the sandbar shark in the northwestern Atlantic Ocean (Romine, 2008). Life tables and Leslie matrix models were constructed using Poptools, a Microsoft Excel add-on (Hood, 2005). Leslie matrices were constructed from life tables and used the birth-pulse model as reproduction is limited to a short breeding season for this species. A post-breeding census was used, i.e., data are collected after the birth pulse. In this census technique, all offspring are counted but not all females will be alive for the next birth pulse. In a post breeding census, survival and fertility parameters are estimated as: 1iiillP Where P i = probability of survival to age i; l = probability at birth of surviving to age i. iiiPmF Where F i = fertility at age i; m = fecundity of age class i. Life table and Leslie matrix analysis are dependent on several assumptions. These models assume that the population in question is closed, i.e. no immigration or emigration. These 85

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models also assume an unlimited, homogeneous environment, age-specific survival and fecundity rates (density independent), a stable age distribution, and animals of a given age are identical (Caswell, 2001). The discrete Lotka-Euler equation was used to estimate intrinsic rates of population increase r. xxrxmle1 Where r = intrinsic rate of increase, x = age class, l x = age specific survival rate, m x = age specific fertility rates (annual number of female offspring). Elasticities for survival and fertility parameters were calculated as: 1,,jiijijjiijaae Where = the dominant eigen value; e ij = elasticity value for the (i,j) th element of the matrix; a ij = entry for the (i,j) th element of the matrix. Mortality Age specific estimates of natural mortality were derived from two indirect methods proposed by Chen and Watanabe (1989) and Lorenzen (2000). The Chen and Watanabe (1989) method utilizes two equations: one for the early and middle life stages that vary with age, and a second equation of constant mortalities for later ages greater than a s (age of senescent) (Roff, 1992; Cortes and Brooks, 2005). oKtsteKao1ln*1 where K is the growth coefficient from the von Bertalanffy growth equation, and t o = theoretical age at zero length (Chen and Watanabe, 1989). Prior to age a s mortality at age a is estimated as: 86

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)1()(otaKaeKM For ages after a, mortality is estimated as: )))(ln(())(1(1max1maxosoKtKaKtKaseeeeaaM The Lorenzen (2000) method of estimating natural mortality follows: arraLLMM)*( where M a = mortality at length a, M r = mortality at reference length r, L r = reference length, and L a = length at age a. Multiple reference mortalities (max age mortality of 0.01, 0.05, 0.075, 0.1 and birth reference) were used to determine the predicted mortalities of the Lorenzen model. Neonate mortality was assigned a value of 0.4 as reported in previous stock assessments (NMFS, 2006). While other methods for estimating mortality from life history data exist, these techniques are most appropriate as input values can be taken directly from a von Bertalanffy growth curve output and reference mortalities. Further, other techniques utilize weight parameters and temperature data. As these sharks are highly migratory, water temperature values of the surrounding environments would be highly variable. Also, the nature of the sampling program precluded weights of the specimens from being obtained. Additionally, set levels of fishing mortality were added to natural mortality to test the effect zero, low (0.12), medium (0.25), and high (0.50) levels of fishing mortality has on the elasticities of sandbar shark age classes commonly caught in the coastal bottom longline fishery. Low and medium fishing mortality levels were based on catch data from commercial fishing 87

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(Brooks, 2007). The high level of fishing mortality was applied to explore how elasticities responded to extreme fishing pressure. In order to apply fishing mortality to appropriate age classes, Commercial Shark Fishery Observer Program data were analyzed to determine at what size class and corresponding age class sandbar sharks enter the coastal bottom longline shark fishery. Sharks were considered fully recruited into the fishery at a size class of 120 cm FL and an age of 7 years. Proportion of fishing mortality was added according to the proportion of the age class recruited into the fishery (Figure 4-1). Reproductive parameters Variation in fecundity and age of first reproduction of sandbar sharks was modeled using reproductive data from Chapter 2 (Table 4-1). Fecundity does not appear to be highly correlated with maternal size (Chapter 2; Springer, 1960; Joung and Chen, 1995). Therefore, a constant fecundity value was used across all mature age classes (mean litter size = 9.6 pups per event; Chapter 2). Annual realized fecundity, taking into account the reproductive periodicity (2 years versus 3 years) of female sandbar sharks was used in the demographic models. Values based on either a biennial and triennial reproductive cycle were used [e.g., 9.6 pups per event would equal 4.8 pups per year (biennial) or 3.2 pups per year (triennial)]. Assuming a 1:1 sex ratio (see Chapter 2) would further reduce effective reproductive output to half these values (i.e. 2.4 for biennial and 1.6 for triennial) as only female offspring are used in assessment modeling. To assess how variation in age at maturity affected model outputs, Leslie matrices were constructed with ages of 50% maturity set between 10 and 18 years. To provide a more biologically realistic model, fecundity values were assigned following the corresponding ages from the length of maturity ogive (see Chapter 2). For example, the length of 25%, 50%, and 75% maturity was 142 cm, 148 cm, and 155 cm FL which corresponded to ages of 12, 14, and 16 years. 88

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Intrinsic population growth rates were compared among ages of maturity (10-18 yr), between reproductive cycles (2 or 3 yr), and among fishing mortality rates (0-0.5). Fertility elasticity values were also graphically compared for the extremes of age of maturity (10 versus 18 yr), among fishing mortalities (0 to 0.5), and for biennial versus triennial reproductive cycles. Results Natural mortality Life history parameters used to estimate natural mortality of sandbar sharks were: k = 0.106, t o = -3.24, L = 178 cm FL (Romine, 2008). Both indirect mortality estimate techniques resulted in decreased mortality with increasing age (Figure 4-2). However, variation in the reference mortality used in the Lorenzen technique resulted in large variability in mortality estimates. While mortality estimates from the Lorenzen technique using a mortality reference value of 0.1 resulted in reasonable values, the accuracy of the reference value is unknown. The Chen and Watanabe (1989) method produced mortality estimates that also appear to be biologically reasonable and were based on known empirical life history data (Figure 4-2). Therefore, estimates of natural mortality from the Chen and Watanabe (1989) method were utilized in the demographic analysis. Population growth Variation in age of maturity resulted in a high degree of variability in intrinsic rates of population increase r (Figure 4-3, A-C). When a triennial reproductive cycle was assumed, variation in population rates among the ages of maturity declined slightly (Figure 4-3 B,C). An 8-yr change in age of maturity resulted in roughly a 10% change in population growth, with older ages of maturity resulting in decreased population growth (Figure 4-3 A-C). Reducing the age of maturity had a slightly greater impact on population growth than increasing the age of maturity (Figure 4-3 A-C). Increasing fishing mortality (F) resulted in a decrease in variability 89

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of population growth (Figure 4-3 A-C). The frequency of reproduction had a small effect on population growth. Changing from a biennial reproductive cycle to a triennial cycle resulted in a maximum observed difference in population growth rate of 3.2 % and a minimum difference of 1.8% (Figure 4-3 C). At an age of 50% maturity of 14 years, and a triennial reproductive cycle, the population growth was on average 2.28% less than that observed with a biennial reproductive cycle across the four fishing scenarios. Elasticities Survival elasticities derived from the Leslie matrices indicate that survival of early age classes had the greatest proportional effect on the intrinsic population growth (Figures 4-4 A, B). However, increasing the age of maturity resulted in a reduction of elasticity values of early age classes and a slight increase in elasticities of older age classes (Figures 4-4 A, B). Changing from a biennial reproductive cycle to triennial reproductive cycle (Fig. 4-4, A, B; Fig. 4-5, A, B), or an increase in fishing mortality (Fig. 4-5, A, B) also resulted in a slight decrease in elasticities of early age classes and a slight increase in elasticities of older age classes. Fertility elasticity values were elevated around the ages of maturity with a decline in older age classes (e.g. Figure 4-6A). As age of maturity was increased, fertility elasticities shifted with elevated levels occurring in later age classes (e.g. Figures 4-7A). However, elasticities were overall reduced with an increased age of maturity. Fertility elasticities decreased when increased fishing mortality was applied (e.g., Figures 4-6A). However, variation in elasticity values declined in older age classes and when age of maturity was increased (Figures 4-6A, 4-7A). Changing the reproductive cycle from biennial to triennial also resulted in a decrease in elasticity values (e.g., Figures 4-6A versus 4-6B), but the relative decrease was much more apparent at an age of maturity of 10 yr versus 18 yr (Figs. 4-6, 4-7). 90

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Discussion A lack of empirical studies estimating shark natural mortality, with the exception of two studies on lemon (Negaprion brevirostris; Gruber et al. 2001) and blacktip (C. limbatus; Heupel and Simpfendorfer 2002) sharks, has lead to debate regarding ways to accurately estimate mortality rates for sharks. Estimates of natural mortality for sharks have historically relied on the use of indirect methods based on life history parameters (Cortes, 2007). Many have argued as to which of the many methods of indirect estimation of natural mortality is best utilized in shark demographic studies (e.g., Chen and Watanabe, 1989; Walker, 1998; Cortes, 2007). In this study, I chose the Chen and Watanabe (1989) and Lorenzen (2000) methods because they have previously been used in shark demography studies and stock assessments (e.g. Goldman, 2002; NMFS, 2006). However, uncertainty in reference mortality lead to high variability in mortality estimates derived from the Lorenzen method. While both methods resulted in declining mortality as age increase, the Chen and Watanabe method relied on more empirical data (life history traits) and was deemed more appropriate. However, errors in age and growth data may introduce bias in mortality estimates based on von Bertalanffy growth parameters. Determining the accuracy of either method in predicting mortality rates is currently not feasible without comparison with empirically derived estimates, which do not exist for this species (Cortes, 2007). The intrinsic rate of population increase reported in this study may be artificially elevated when compared to full stock assessments (e.g., NMFS, 2006) because the models developed in this study used assumed levels of fishing mortalities and applied these mortalities across ages of sharks caught in one fishery only (commercial longline). However, recreational fishing mortality may impact earlier age classes not at risk of being caught in commercial longlines due to differences in gear selectivity. Alternatively, reported population growth may be below actual 91

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growth as the models developed in this study were density-independent. Studies on density-dependence compensation in shark populations are sparse. Sminkey and Musick (1995) reported compensation in juvenile sandbar shark growth rates in response to over-exploitation. While Carlson and Baremore (2003) proposed density-dependent compensation in Atlantic sharpnose sharks (Rhizoprionodon terraenovae) manifested in greater growth rates and younger ages of maturity, no study has shown density-dependent compensation in reproductive parameters of sandbar sharks. Au and Smith (1997) constructed demography models of the leopard shark (Triakis semifasciata) with assumptions that density-dependent compensation was manifested in increased juvenile survival, however without data to support this assumption, this strategy may be flawed. Wood et al., (1979) suggested that density-dependence in spiny dogfish (Squalus acanthias) is likely mediated through changes in natural mortality. Without documentation of how density-dependence may affect reproductive parameters, we are left with the option of assuming uncertain compensation levels or factoring variation in reproductive parameters and modeling their outputs. Intrinsic population growth rate of sandbar sharks observed in the present study, using reproductive data derived from Chapter 2 (age of maturity 14 y, 9.6 pups per litter, triennial reproductive cycle), was 17.7% at a fishing mortality of zero. This value is similar to those reported for other carcharhinid sharks (e.g., 16% for C. brevipinna) (Chen and Yuan, 2006). The 2002 sandbar shark stock assessment (NMFS, 2002) reported an intrinsic rate of increase of 23%, using an equal weighted Bayesian surplus production model analysis and assuming a biennial reproductive cycle. In this study, I observed similar intrinsic rates of increase between 20% and 17.2% under various fishing mortalities, a biennial reproductive cycle, and an age of 92

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maturity of 14 yr. This similarity is likely due to the small difference in age at 50% maturity, i.e. 13y vs 14y. Only minor changes in intrinsic population growth rates were observed when switching from a biennial reproductive cycle to a triennial cycle. Further, this variation decreased slightly as fishing mortality was increased or when age of maturity was increased. When modeling a biennial reproductive cycle, small changes in the age of maturity resulted in similar variation in intrinsic rates of increase as those observed when shifting to a triennial cycle. Model simulations therefore indicated that small variation in age of maturity will add little error in intrinsic rates of increase. Further, the small variation in rates of increase due to changes in the periodicity of reproduction suggested that the incorporation of a biennial reproductive cycle in previous stock assessments introduced only limited error. The over-fished status of sandbar sharks (NMFS, 2006) would further reduce this potential error as variation in intrinsic rates of increase were shown to decrease as fishing mortality was applied. This low variability in rates of increase also negates the speculation that sandbar shark populations should have crashed if the reproductive cycle is indeed 3 yr rather than 2 yr, as seen in the dusky shark. However, the currently over-fished status of the sandbar shark in the northwestern Atlantic and Gulf of Mexico has lead to a recent closure of the commercial bottom longline shark fishery (NMFS, 2008). Recent studies have suggested that sustainable levels of F are typically no more than 0.8*M which in the present study would be a modest exploitation level of 0.096 (Walters and Martell, 2004; Allen et al. 2009). Elasticity analyses in this study indicated that survival of juveniles was the most important parameter for population growth. Goldman (2002) reported elevated elasticity values for juvenile salmon sharks (Lamna ditropis) in the Pacific Ocean and juvenile sand tiger sharks 93

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(Carcharias taurus) in the western north Atlantic Ocean, and suggested that juveniles would be the most important stage to focus management efforts. Smith et al. (2008) also reported that higher elasticity values of juvenile diamond stingrays (Dasyatis dipterura) strongly influenced population growth. A review of additional elasmobranch literature suggest a common trend of greater importance of juvenile survival to population growth in many species e.g. Sphyrna lewini (Cortes, 2002), Carcharodon carcharias (Cortes, 2002), Carcharhinus isodon (Carlson et al., 2003), C. falciformis (Beerkircher et al., 2003). This trend in elevated juvenile elasticities has also been noted in other studies on long-lived marine animals. For example, Heppel et al. (1998) also reported that juvenile elasticity values were higher than adult values for loggerhead sea turtles (Carretta carretta). Fertility elasticity values in this study indicate that reproduction of maturing age classes is more important than later age classes in sandbar shark population growth. The reduction in fertility elasticity values in the older age classes may be due to the decrease in the numbers of these older ages present in the population. Further, the lack of significant increase in litter size with maternal age or size may also reduce the significance of older age classes toward fertility. The difference in magnitude of elasticities of fertility versus survival may reflect the low reproductive output of these sharks. Indeed, previous studies on elasmobranchs have shown a trend of changes in fertility being far less important than changes in survival of juveniles in population growth (e.g. Cortes, 2002; Goldman, 2002; Beerkircher et al., 2003). Elasticity analysis has been used to aid in developing conservation measures in marine species management (e.g. Heppell and Crowder, 1998). Elasticity analysis of loggerhead sea turtle population trends has lead to a change in conservation focus from reducing nesting mortality toward reduction of free swimming sea turtle mortality through the inclusion of sea 94

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turtle excluder devices in shrimp trawl nets (Crowder et al., 1994). Results from this study suggest that efforts to reduce juvenile sandbar shark mortality would provide the most benefit toward rebuilding populations. Further, efforts to protect young mothers may enhance stock levels. In conclusion, the frequency of reproduction was shown to have limited influence on population increase. Additionally, small variations in the age of maturity also introduced only limited variation in population growth. However, extreme changes in ages of maturity resulted in higher variation in population growth. Increases in fishing mortality further reduced the observed effect that variation in reproductive parameters had on population growth. Elasticity analysis indicated that survival of juvenile sharks had the most influence on sandbar shark population growth. 95

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Table 4-1. Sandbar shark reproductive parameters used in population models. ParameterValueSourceFecundity9.6Chapter 2Reproductive cycle2y/3yChapter 2Age at first reproduction10-18Chapter 2Pup Survival0.6SEDAR11 (NMFS 2006) 96

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0%10%20%30%40%50%60%70%80%90%100%01234567891AgePercent of fishing mortality applied 0 Figure 4-1. Fishing mortality schedule for sandbar sharks in the northwestern Atlantic Ocean and Gulf of Mexico based on catch data. 97

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00.050.10.150.20.250.30.350.40.45051015202530Age (year)Natural Mortality Chen and Watanabe Lorenzen 0.01 Lorenzen 0.05 Lorenzen 0.075 Lorenzen 0.1 Figure 4-2. Indirect estimates of sandbar shark natural mortality. 98

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00.050.10.150.20.250.3101112131415161718Age at 50% MaturityIntrinsic growth rate (r) r at F = 0 r at F=0.12 r at F=0.25 r at F=0.5 A 00.050.10.150.20.250.3101112131415161718Age at 50% MaturityIntrinsic growth rate (r) r at F = 0 r at F=0.12 r at F=0.25 r at F=0.5 B Figure 4-3. Intrinsic population growth rates r of sandbar sharks under various fishing mortality and changes to age at 50% maturity assuming a biennial (A) and a triennial reproductive cycle (B) and overlaying both cycles (C). 99

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00.050.10.150.20.250.3101112131415161718Age at 50% MaturityIntrinsic populaiton growth rate (r) r at Biennial and F=0 r at Biennial and F=0.5 r at Triennial and F=0 r at Triennial and F=0.5 C Figure 4-3. continued. 100

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00.010.020.030.040.050.060.070.0805101520253035Age (year)Survival Elasticity values Age of Maturity =10 Age of Maturity =12 Age of Maturity =14 Age of Maturity =16 Age of Maturity =18 00.010.020.030.040.050.060.070.0805101520253035Age (year)Survival Elasticity Values Age of Maturity =10 Age of Maturity =12 Age of Maturity =14 Age of Maturity =16 Age of Maturity =18 A B Figure 4-4. Survival elasticity values of sandbar sharks assuming a biennial (A) and triennial (B) reproductive cycle. 101

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00.010.020.030.040.050.060.070.0805101520253035Age (year)Survival Elasticity Values F=0 F=0.12 F=0.25 F=0.5 00.010.020.030.040.050.060.070.0805101520253035Age (year) Survival Elasticity Values F=0 F=0.12 F=0.25 F=0.5 A B Figure 4-5. Sandbar shark survival elasticities assuming a 10 year age of 50% maturity and a biennial (A) and triennial (B) reproductive cycle. 102

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00.0010.0020.0030.0040.0050.0060.0070.0080.0090.01051015202530Age (year)Fertility Elasticities F=0 F=0.12 F=0.25 F=0.5 00.0010.0020.0030.0040.0050.0060.0070.0080.0090.01051015202530Age (year)Fertility Elasticities F=0 F=0.12 F=0.25 F=0.5 A B Figure 4-6. Fertility elasticities of sandbar shark assuming a 10 year age of 50% maturity and a biennial (A) and triennial (B) reproductive cycle. 103

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00.0010.0020.0030.0040.0050.0060.0070.0080.0090.0105101520253035Age (year)Fertility Elasticities F=0 F=0.12 F=0.25 F=0.5 00.0010.0020.0030.0040.0050.0060.0070.0080.0090.0105101520253035Age (year)Fertility Elasticity F=0 F=0.12 F=0.25 F=0.5 A B Figure 4-7. Fertility elasticities of sandbar shark assuming an 18 year age of 50% maturity and a biennial (A) and triennial (B) reproductive cycle. 104

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CHAPTER 5 EPILOGUE The sandbar shark is a commercially important species that has seen decline in recent years. Current assessment models indicate that this shark is currently overfished in the northwestern Atlantic Ocean and Gulf of Mexico. Stock assessment scientists have predicted that with a stop in fishing for sandbar sharks that the rebuilding of the stocks will take over 60 years (NMFS, 2006). Previous, management efforts have been ineffective largely due to a lack of accurate data on the life history of sandbar sharks in the fishery. Changes in life history parameters used in sandbar shark stock assessment models, particularly the age of maturity, resulted in more pessimistic results when compared to previous assessment (NMFS, 2002; 2006). Accurate stock assessments require contemporary data concerning the biology of the species. The goal of this study was to provide updated and novel data on the reproduction of the sandbar shark in the northwestern Atlantic Ocean and Gulf of Mexico. This study has provided updated sizes of maturity and fecundity as well as new information on the timing of the reproductive cycle (Chapter 2). While previous studies have speculated on the timing of egg and sperm development (e.g. Springer, 1960), this study provides specific data on this timing and a frame work for future correlations with endocrine studies. Further, use of immunocytochemical techniques may provide information on the timing of activity of hormone receptors in sandbar shark reproductive tissue. The change from a 2 yr to a 3 yr or greater reproductive cycle represents a fundamental shift from the historic view of reproductive output for this species. This change has only recently been elucidated (Chapter 2; Merson, 1998) and may lead to speculation on the accuracy of our current knowledge concerning the reproductive cycles of other sharks. Studies on the energetics of reproduction for this species may provide insight into the mechanisms governing this protracted reproductive cycle. Demographic analysis presented 105

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in this study (Chapter 4) illustrates how variation in reproductive parameters may influence population growth. Small shifts in the age of maturity or a switch from a 2 yr to a 3 yr reproductive cycle had limited impact on population growth. Also, increases in fishing mortality further reduced the impact that reproductive variation had on population growth. The lack of accurate data on natural mortality of large migratory sharks should be the focus of future research efforts, as the use of indirect estimates for mortality may lead to error. This study also provides novel analysis of maternal influences on embryonic development of sandbar sharks (Chapter 3). The presence of high variation in embryonic weight and condition has not been previously examined. The data present in this study indicate that larger females are better able to produce uniformly fit young. The potential for variation in birth size to impact neonate mortality is currently not known and merits further research. The analysis of fatty acids and their roles in embryonic development have not previously been examined in sharks. The presence of essential fatty acids in the livers of gestating embryos indicates that these acids are transferred through the placental connection from maternal stores. Future research should focus on the energetics of embryonic development and with the addition of further litters may allow for elucidation of the roles that embryonic size or gestation stage may have on levels of fatty acids present. This dissertation represents the most up to date analyses and largest study on the reproduction of the sandbar shark in the northwestern Atlantic Ocean and Gulf of Mexico. The future incorporation of these data in stock assessment models may allow for more accurate models and better management policies. 106

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107 APPENDIX ADDITIONAL FATTY ACID DATA

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F A StandardsMAA1A2A3MBB1B2B3MCC1C2C3MDD1D2D3MEE1E2E3C18:2w66.566.917.907.4511.867.638.198.639.857.169.048.348.758.128.556.9810.587.065.877.61C18:3w6NDNDNDND1.31NDNDND1.03NDNDND1.24NDNDND1.320.240.080.14C18:3w3NDNDNDND3.42NDNDND2.23NDNDND3.00NDNDND3.790.260.12NDC20:2w68.520.590.950.556.250.750.830.844.310.741.101.104.181.021.050.545.291.120.560.83C20:3w6NDND0.05ND3.390.02NDND2.130.06ND0.042.39ND0.01ND2.580.180.080.09C20:4w65.900.170.910.3235.470.440.540.8011.280.080.110.1117.110.350.130.0720.912.990.841.15C20:3w3ND0.03NDND1.09NDNDND0.66NDNDND0.87NDNDND1.260.11ND0.08C20:5w3ND0.070.350.1322.130.220.260.394.510.060.060.128.750.210.090.0515.122.060.450.57C22:4w62.620.050.160.067.940.090.100.153.730.030.020.043.610.050.020.024.130.400.100.14C22:5w3ND0.110.530.1726.470.340.400.628.620.070.070.0815.210.310.150.0818.142.110.480.74C22:6w34.590.191.320.33100.070.630.711.0020.310.110.130.1633.460.700.340.0661.479.371.672.31Total FA Wt (ug)4.58499.44645.21905.83708.26884.26787.56903.38365.58544.60647.26413.77394.451163.631210.461159.91297.72552.12396.36355.01Tissue Wt (mg)1.2321.2311.8801.5602.2231.8521.8732.1191.3061.2551.3451.0541.6872.7142.8102.3010.9071.9201.1401.122 Essential fatty acids detected in embryonic and maternal liver tissue: (M) denotes maternal sample; (number) denotes embryo. 108

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BIOGRAPHICAL SKETCH Andrew Piercy was born and raised in eastern Virginia. Living on a waterfront community reinforced his interest in marine science. Andrew graduated from Christopher Newport University in December 1998 with a B.S. in Biology with English and biology honors. In 2002 Andrew graduated from the University of Central Florida with a M.S. in biology. His thesis topic focuses on reproduction of the Atlantic Stingray. In 2002 Andrew was hired as a research biologist at the newly formed Florida Program for Shark Research at the Florida Museum of Natural History. In 2004 Andrew entered the Ph.D. program in the Department of Fisheries and Aquatic Sciences at the University of Florida under the direction of Dr. Debra Murie while continuing full time employment at the museum. 119