AGE AND GROWTH OF THE PACIFIC BLUE , NIGRICANS:

A COMPARISON OF GROWTH ZONES IN OTOLITHS, VERTEBRAE,

AND DORSAL AND ANAL FIN SPINES

A Thesis presented to the Faculty

of

California State University, Stanislaus

and

Moss Landing Marine Laboratories

in Partial Fulfillment

Of the Requirements for the Degree

Master of Science in Marine Science

By

Kevin T. Hill

November, 1986 ABSTRACT

Age estimation of the Pacific blue marlin is still in the developmental stages, with most data resulting from otoliths. The pu;pose of this study was to examine, interpret, and quantitatively compare growth patterns found in the otoliths, vertebrae, and dorsal· and anal fin spines. Each hardpart was evaluated for usefulness in terms of legibility and interpretability of growth patterns, ease of collection and processing, and the comparative precision of the resultant age data. Blue marlin were sampled at various billfishing tournaments held in Kailua-Kana, between Augusts of 1982 and 1984. Skeletal hardparts and morphometric data were collected from 213 male and 104 female marlin. Individual round weight (kg), eye-fork length (EFL em) and sex were recorded. Males ranged from 95.4 to 222.0 em EFL (19.1 to 138.8 kg) and females ranged from 125.7 to 398.8 em EFL (20.9 to 748.0 kg). Otoliths, and dorsal and anal fin spine sections all contained growth zones which were interpreted as annual events, and the number of these zones increased with the size of the . Vertebrae contained minute incremental growth marks which may represent some type of weekly or bi-weekly events, however, no "annual" events were apparent. There was a direct linear relationship between age estimates of corresponding otoliths, dorsal and anal fin spines. Age estimates of spine samples from larger fish included a statistical replacement of inner growth zones which were destroyed by matrix expansion. Paired T-tests revealed no significant difference between age estimates of corresponding hardparts. There was a stronger relationship between corresponding dorsal and anal fm spine age estimates than between otoliths and these two hardparts. Dorsal and anal fin spines were more practical ageing structures in terms of ease of collection, processing, legibility, and interpretation. Although more difficult to dissect out and work with, otoliths were often more useful for providing detailed age information from daily and other incremental patterns. Mean length-at-estimated age values were similar for otoliths, and dorsal and anal fm spines. Males appear to grow to an average size of 17 6 em EFL at 6 years, after which their growth levels off rapidly. The largest male sampled (222.0 em EFL) is the largest male on record, yet was only estimated at 9 years. The oldest male was estimated to be 18 years at 193.8 em EFL. Growth of female marlin does not level off until a

i i much later age than males. The largest female sampled (398.8 em EFL) was also one of the oldest at 26 years.

I I I ACKNOWLEDGEMENTS

I owe a debt of gratitude to my graduate committee members, Drs. Greg Cailliet, Pamela Roe, and Jay Christofferson, as w!!ll as Dr. Richard Radtke, for their unending encouragement, support, and patience throughout the term of this project. My special thanks go to Greg Cailliet for being an inspiring teacher, advisor, and good friend. A number of organizations made it possible to collect samples for this study, including the fishermen and fishing teams of the billfishing tournaments, the Hawaiian International Billfishing Association, the Pacific Gamefish Research Foundation, Hawaiian Fish Distributors, Jerry Kinney of Volcano Isle Fish Wholesalers, and the Hawaiian Fishing Agency. Greg Cailliet, Bruce Welden, Lisa Natanson, Carol Hopper, and Allison Mallory provided much needed assitance in collecting samples at the tournaments. Pele supplied additional support at the ice house. Dr. Eric Prince and Dennis Lee of NMFS Southeast Fisheries Center, Miami, donated additional spine samples from the largest female marlin, and contributed valuable input as well. Additional personell and students from Moss Landing Marine Laboratories contributed much needed assistance and technical advise through all phases of this project, including: Kevin Lohman for computer assistance, Signe Lundstrum for SEM training, and Jim Brennan and Farran Wallace for sample cleaning sessions. Sheila Baldridge and Sandi O'Neill provided invaluable library research assistance. I would like to thank my parents, Randall and Geraldine Hill, for their never-ending tolerance and support from the very start. This project was conceived by and made possible through the cooperative efforts of Greg Cailliet and Richard Radtke, both of whom I owe a debt of gratitude to. The research was funded in part by a grant from NOAA, Office of Sea Grant, Department of Commerce, #R/F-84. The Packard Foundation granted additional support enabling an additional collecting trip.

v TABLE OF CONTENTS

ABSTRACT 11

1HESIS APPROVAL IV

ACKNOWLEDGEMENTS v

LIST OF TABLES V Ill

LIST OF FIGURES ix

LIST OF APPENDICES Xi i

INTRODUCTION 1

MATERIALS AND ME1HODS 4 Morphometric Analyses 4 Otoliths 4 Vertebrae 5 Dorsal and Anal Fin Spines 6 Hardpart Growth 8 Assessment of Ageing Techniques 8 Age-Length Relationship 9

RESULTS 10 Summary of Sampled 10 Morphometries 10 Otoliths as Ageing Material 10 Vertebrae as Ageing Material 12 Spines as Ageing Material 12 Anal Fin Spines as Ageing Material 14 Assessment of Ageing Techniques: 15 Reader Precision 15 Hardpart Cross-Comparisons 15 Mean Length at Age from Anal Spines 16

DISCUSSION 18 Length Frequencies 18 Morphometric Relationships 19 Blue Marlin Hardparts as Ageing Materials 19 Otoliths as Ageing Material 20 Vertebrae as Ageing Material 21 Fin Spines as Ageing Material 22 Verification and Validation of Age Estimates in Blue Marlin 23

VI TABLE OF CONTENTS (continued)

DISCUSSION (Cont'd) Growth of Pacific Blue Marlin 24 Recommendations 26

LITERATURE CITED 28

TABLES 33

FIGURES 42

APPENDICES 94

v 11 LIST OFTABLES

Page

1. Summary of numbers and size ranges of Makaira nigricans from which skeletal hardpans and measurements were collected. 33

2a. Analysis of reader comparisons for otoliths. 34

2b. Analysis of reader comparisons for vertebrae. 34

2c. Analysis of reader comparisons for dorsal fin spines. 34

2d. Analysis of reader comparisons for anal fin spines. 34

3. Mean ( ± 95% confidence intervals) dorsal spine band diameters (mm) for Makaira nigricans. Calculated cumulative mean band diameter values were used to fill in those areas where these bands were destroyed. 35

4. Mean ( ± 95% confidence intervals) anal spine band diameters (mm) for Makaira nigricans. Calculated cumulative mean band diameter values were used to fill in those areas where these bands were destroyed. 36

5. Calculated mean length (EFL) at age as determined by otoliths, dorsal spines, and anal spines, for male Makaira nigricans. 37

6. Calculated mean length (EFL) at age as determined by otoliths, dorsal spines, and anal spines, for female Makaira nigricans. 38

7. Analysis of corresponding hardpart age assignments by Paired T-test. 39

8. Calculated mean lengths (EFL) at age as determined by anal spine bands counts for male Makaira nigricans. 40

9. Calculated mean lengths (EFL) at age as determined by anal spine bands counts for female Makaira nigricans. 41

viii LIST OF FIGURES

1. Measurements and skeletal hardparts.taken for Makaira nigricans. Fish outline adapted from Mather (1976). 42

2. Sagitta from a 220 kg blue marlin, Makaira nigricans , which shows morphological features (from Radtke, 1981). 44 3. Caudal vertebra number 23 from a specimen of Makaira nigricans which shows morphological features and orientation of the conically shaped centra. 46

4. Second anal spine from Makaira nigricans which shows basic morpho­ logical features and area of cross sectioning for age determination (Diagram adapted from Prince eta!., 1984). 48

5. Size frequencies [Eye to Fork Length (EFL) in em] for male (n=213) and female (n=106) Makaira nigricans sampled from four billfishing tournaments held during 1982, 1983, and 1984 iri Kailua-Kana, Hawaii. 50

6. Relationship between otolith weight (OW in mg) and EFL(cm) for male and female Makaira nigricans. 52

7a. Left sagitta from a 189.0 em EFL (80.1 kg) male Makaira nigricans. 54

7b. Left sagitta from a 299.2 em EFL (496.7 kg) female M. nigricans. 54

Sa. Left sagitta fro a 221.2 em EFL (161.5 kg) female M. nigricans. This sample could not be used for age estimation due to the mottled appearance of the rostral ridges. 56

8b. Right sagitta from a 141.0 em EFL (35.2 kg) female M. nigricans. 56

9a. Thin cross section through the core region of a sagitta from a 258.0 em EFL (27 5.8 kg) M. nigricans as seen by light microsc0py. 58

9b. Thin cross section through the rostrum of a 179.0 em EFL (74.4 kg) M. nigricans. 58

lOa. Relationship between centrum cone depth (CCD in mm) and EFL (em) for male Makaira nigricans. 60

lOb. Relationship between CCD (mm) and EFL (em) for female M. nigricans. 60

11. Longitudinal cross section of the anterior centrum from the 23rd vertebrae from a 156.0 em EFL (50.3 kg) male M. nigricans. 62

IX 12a. Relationship between dorsal spine width (DW in mm) and EFL (em) for male Makaira ni gricans. 64

12b. Relationship between DW (mm) and EFL (em) for female Makaira nigricans. 64

13a. Thin tranverse cross section of the 6th dorsal spine from a 162.6 em EFL (52.4 kg) male M. nigricans as viewed.by a binocular dissecting microscope at 63X magnification with transmitted light. 66

13b. Thin tranverse cross section of the 6th dorsal spine from a 162.6 em EFL (52.4 kg) male M. nigricans (same as 13a.) as viewed by a binocular dissecting microscope at 63X magnification with reflected light and a black background. 66

14. Thin tranverse cross section of the 6th dorsal spine from a 222.0 em EFL (138.7 kg) male M. nigricans as viewed by a binocular dissecting microscope at 63X magnification with reflected light and a black back- ground. 68

15. Thin tranverse cross section of the 6th dorsal spine from a 282.4 em EFL (419.1 kg) female M. nigricans as viewed by a binocular. dissecting microscope at 63X magnification with transmitted light. 68

16. Mean (and 95% confidence interval) dorsal spine band measurements (nun) for Makaira ni:.Dcans. (Refer also to Table 3) 70

17 a. Relationship between anal spine width (A W in mm) and EFL (em) for male Makaira nigricans. 72

17b. Relationship between A W (nun) and EFL (em) for female Makaira nigricans. 72

18. Thin tranverse cross section of the 2nd anal spine from a 208.8 em EFL (138.3 kg) female M. nigricans as viewed by a binocular dissecting microscope at 63X magnification with transmitted light. 7 4 ·

19. Thin tranverse cross section of the 2nd anal spine from a 95.4 em EFL (19.1 kg) female M. nigricans as viewed by a binocular dissecting microscope at 63X magnification with transmitted light. 74

20. Mean (and 95% confidence interval) anal spine band measurements (nun) for Makaira nigricans (Refer also to Table 4) 76

21. Relationship between total estimated otolith ridge counts (OC) and vertebral increment counts (VC) for Makaira nigricans. 78

22. Relationship between total estimated dorsal spine band counts (DC) and otolith ridge counts (OC) for Makaira nigricans. 80

23. Relationship between total estimated dorsal spine band counts (DC) and vertebral increment counts (VC) for Makaira ni :.Deans. 82

X 24. Relationship between total estimated anal spine band counts (A C) and otolith ridge counts (OC) for Makaira nigricans. 84

25. Relationship between total estimated anal spine band counts (AC) and vertebral increment counts (VC) for Makaira nigricans. 86

26. Relationship between total estimated anal spine band counts (A C) and dorsal spine band counts (DC) for Makaira nigricans. 88

27. Mean EFL at age for male Makaira nigricans based on anal spine age estimates (Refer also to Table 8) 90

28. Mean EFL at age for male Makaira nigricans based on anal spine age estimates (Refer also to Table 9) 92

Xl LIST OF APPENDICES

Page

la. Relationship betwee~ Eye Fork Length (EFL in ern) and Total Fork Length (TFL in em) for male Makaira nigrieans. 94

lb. Relationship between EFL (ern) and TFL (em) for female M. niweans 94

2a. Relationship between EFL (em) and Lower Jaw to Fork Length (LJFL in ern) for male M. nigrieans. 96

2b. Relationship between EFL (em) and LJFL (ern) for female M. niweans. 96

3a. Relationship between EFL (ern) and Eye to Anal Fin (EAFL in em) for male M. niweans. 98

3b. Relationship between EFL (ern) and EAFL (ern) for female M. niweans. 98

4a. Relationship between TFL (em) and LJFL (ern) for male M. nigrieans. 100

4b. Relationship between TFL (ern) and LJFL (em) for female M. niweans. 100

5a. Relationship between TFL (ern) and EAFL (ern) for male M. ni weans. 102

5b. Relationship between TFL (ern) and EAFL (em) for female M. niweans. 102

6a. Relationship between LJFL (em) and EAFL (em) for male M. nigrieans. 104

6b. Relationship between LJFL (em) and EAFL (em) for female M. niweans. 104

7a. Relationship between EFL (em) and Weight rN in kg) for male M. nigrieans. 106

7b. Relationship between EFL (ern) and W (kg) for female M. niweans. 106

Xi i 1

INTRODUCTION

Billfishes are among the most important oceanic pelagic fishes today as they are sought after by both sport and commerci~ fisherman on a world-wide basis (Joseph and Greenough, 1979; Joseph, 1980). In 1975 alone, the world commercial catch was 83.7 thousand metric tons, with a catch value close to $100,000,000 (Joseph and Greenough, 1979). There is no reliable record of sport-fishing catches, but anglers are known to spend $1,000 to $3,000 per day to catch a billfish (Joseph, 1980). Hundreds more angle for these fish each year and are undoubtedly having an increasing economic impact on coastal communities, especially those developing internationally acclaimed tournaments (Wilson, 1984). The rapid and steady expansion of longline fisheries for and since the mid-1950's has resulted in the increased utilization of billfishes as target for Korean and Japanese fishing industries (Yoshida, 1981). An increased level of fishing effort and an expansion to improved fishing methods, such as the use of purse seines, has resulted in a decrease in effective fishing effort for various billfish species (Yuen and Miyake, 1980; WPFMC, 1985). Many questions and concerns about the viability of billfish stocks remain unanswered, but current trends in catch data for several species, including the Pacific blue marlin, indicate "substantial" (Yeo, 1978; Yuen and Miyake, 1980; Yoshida, 1981). The billfish fishery is extremely difficult to manage. Billfish have an active migratory behavior (Joseph, 1980; Squire, 1985) and are found in all tropical and subtropical seas. A major problem faced at the fisheries management level is that the majority (90%) ofbillfishes are the supplemental catch of the tuna long-line fishery (Joseph and Greenough, 1979). As a result, any management actions by the Fisheries Conservation and Management Act (FMCA) regulating the harvesting of billfishes could directly affect tuna catches, which are under the jurisdiction of the International Commission for the Conservation of Tuna (ICCAT) and the Inter-American Tropical Tuna Commission (IA TTC)(Joseph, 1980). The billfish management plan of the Western Pacific Fishery Management Council (WPFMC) has recently been completed and implemented (WPFMC, 1985). The conclusion of the plan was that there is still a paucity of basic life history information for ~ost billfish species, and that only cursory data.were available for drafting important management decisions (WPFMC, 1985). 2

Increased know ledge of the age and growth rates of billfishes is essential for sensible management of this fishery. Prince and Pulos (1983) outlined the various problems related to age estimation of billfishes in comparison to other fish species. Acquisition of an adequate sample of a COll}plete size range is difficult due to their migratory habits, loose aggregations, and large size. In addition, the life cycle and large size of these fish makes rearing and tank culture experiments logistically difficult (Prince and Pulos, 1983). Size class modal analysis for age determination has been attempted for the Atlantic (de Sylva, 1957), (Koto and Kodama, 1962), and the striped and Pacific blue (Skillman and Yong, 1976). These length-frequency studies have been, for the most part, inconclusive due to the difficulty involved in distinguishing cohorts of fish which spawn throughout the year (Majkowski and Hampton, 1983). Also, most samples are caught by long-line or recreational fishing and these methods of sampling often result in problems with sampling gear bias (Ueyanagi et al., 1970). The most reliable means of estimating the age and growth rates of fishes is by examination of calcified hardparts for growth patterns (Bagenal, 1974) and tag/mark-rec1pture studies for validation of age estimates (Beamish and McFarlane, 1983). Jolley (1974) was the first to describe the internal growth patterns in the dorsal spines of the (Istiophorus plal:y]Jterus), and later provided growth data for this species (Jolley, 1977; Hedgepeth and Jolley, 1983). Dorsal spines have been shown to have similar patterns in the Atlantic white marlin (Tetrapterus albidus) and the (Makaira nigricans)(Prince et al., 1984). Radtke and Dean (1981) first described the morphology of sailfish otoliths and their potential for use as ageing structures. Otoliths have now been described as potentially useful structures for ageing most billfish species (Radtke, 1981; Radtke eta!., 1982; Radtke, 1983; Wilson and Dean, 1983). Growth data have now been published for Atlantic sailfish (Radtke and Dean, 1981), Atlantic (Radtke and Hurley, 1983; Wilson, 1984); white marlin and Pacific blue marlin (Wilson, 1984). Scales and vertebrae have been described as being useless for ageing billfishes (Prince et al., 1984). However, Jolley (1974) states that sailfish vertebrae have distinct and numerous circuli which increase in number with the size of the fish. Jolley (1974) chose not to further investigate the vertebrae because the dorsal spines were more readily accessible. Conically shaped vertebrae have been found to be useful for ageing closely 3

related scombrid species such as the (Lee, et al., 1983; Prince et al., 1985) and (Johnson, 1983). Age estimation of the Pacific blue marlin is still in the developmental stages, and most age data have resulted from using otoliths, with little effort on other skeletal structures. The purpose of this study was to examine, interpret and quantitatively compare patterns found in the otoliths, vertebrae, and dorsal and anal fin spines collected from blue marlin at various billfishing tournaments at Kana, Hawaii. Each hardpart was evaluated for usefulness in terms of legibility of growth patterns, ease of collection and processing, and the comparative precision and accuracy of the resulting age data. Once the most suitable structure was chosen, mean size-at-estimated age was plotted in an attempt to approximate the growth and longevity of this species. These results, along with information on population size, migratory patterns, reproductive biology, and natural and fishing mortality rates, will help fisheries biologists to evaluate the resilience of blue marlin populations and their ability to withstand the possible effects of overfishing. 4

MATERIALS AND METHODS

Pacific blue marlin were sampled from a total of four billfishing tournaments held in Kailua-Kana, Hawaii. The majority of the samples were obtained at the Hawaiian Billfishing Tournaments in Augusts of 1982, 1983, and 1984, and the Kana Gold Jackpot tournament in May of 1983. Additional spine samples were obtained from the Pacific Gamefish Research Foundation and from the National Marine Fisheries Service, Southeast Fisheries Center. Initially, only otoliths and vertebrae were collected at the first two tournaments attended. Upon thorough examination of the incremental growth marks of the vertebrae it was decided to collect additional skeletal structures (dorsal and anal fin spines). A complete array of supplemental data was collected for each fish, including: total fork length (TFL), eye to fork length (EFL), lower jaw to fork length (LJFL), eye to anal fin length (EAFL), round weight CN), sex, and date of capture (Figure 1). All measurements were to the nearest 0.1 em and weights were originally taken in pounds, then converted to kilograms (kg). Although four body length measurements were taken, EFL is the dimension most frequently used by Japanese fishery biologists as it is useful for specimens which have had their bills, and sometimes lower jaws, removed on the boats or in the market (Nakamura, 1985), and this dimension is used hereafter. Morphometric Analyses Cross-comparisons were made between the four body measurements (TFL, EFL, LJFL, EAFL) as well as between EFLand W. All morphometric data were processed with a program, "CURVE", written for the Hewlett Packard model 9825-A computer. The "CURVE" program calculated correlation coefficients (r) and coefficients of determination (il) values for twelve different linear and curvilinear equations, thus allowing a choice of the formulae which best described the relationship between the two variables. Comparisons were made for both separate and combined sexes. Significance (two-tailed test) of? values was tested using the methods of Scheller (1979). Otoliths Otoliths were collected following procedures described by Radtke (1983). Dissected otoliths were placed in glycerol in 2 dram vials for temporary storage. Otoliths were removed from tissues, cleaned with 5.25% sodium hypochlorite (bleach), and dehydrated with 95% ethanol. All otoliths were weighed to 0.001 gram. 5

For examination of external features (Figure 2), sagittae were mounted with nailpolish on Scanning Electron Microscope (SEM) stubs. Mounted otoliths were gold coated with a Polaron model E5100 cool sputter coater and observed and photographed at low magnifications (30X to 40X) with an International Scientific Instruments model SX-30 SEM. External features of the sagi.ttae were quantified with the aid of a compound stereoscope. I have found that gold coated sagittae can be viewed with much greater speed and simplicity using a compound stereoscope. Modelling clay was used to support the SEM stub in the various viewing positions. The prominences of the anterior rostrum edge, as well as the ridges of the lateral-dorsal surface of the rostrum were quantified for age determination. For internal examination of growth features, otoliths were embedded in epoxy resin and sectioned to 0.5 mm thickness using a Buehler ISOMET low speed saw with high concentration diamond grit blades. Polished sections were examined at 400 to 1000X magnification with a compound light microscope. Age estimates from internal features were based on the combined counts of features in the lateral and medial growth zones. The use of age estimates based on external ridge counts and internal annuli was assessed. A subsample of 10 sagittae was randomly selected and read twice with no knowledge of sex, fish length or weight, or previous counts. The number of annuli based on internal and external features were estimated and compared using a Paired T-test (Zar, 1984). All otoliths were read twice by the author. A subsample of 10 otoliths was read independently 2 times by the author and a second reader. Age estimates were compared for consensus within and between readers using a paired T-test (Zar 1984). Vertebrae Caudal vertebrae numbers 22 and 23 were removed from the area between the posterior portion of the second dorsal fin and the base of the caudal fin at the paired lateral keels (Figure 1), placed in labeled plastic bags, and frozen. Thawed vertebrae were cleaned of muscle tissue and simmered in hot water for approximately 3 hours to remove connective tissues. Vertebrae were air dried for at least 72 hours. Neural and haemal spines and arches were removed using a Dremel high speed hobby saw (Figure 3). Anterior and posterior centra were separated and bisected longitudinally along the dorsal-ventral plane. Samples were stored in 95% Isopropyl alcohol in labeled jars. 6

Preliminary observations of the vertebrae revealed numerous concentric growth marks which are superficial topographical features on the face of the centrum. These growth marks ranged from 0.05 to 0.1 mm in width. Several techniques were applied in attempt to further illucidate the growth rings, including X-radiography (Cailliet et al., 1983), silver nitrate staining (Stevens, 1975; Cai!liet et al., 1981), Alizarin red staining (Berry et al., 1977), and observations using a SEM. The best and simplest technique for examining the growth rings was to carefully peel away the thin layer of cartilaginous tissue which covers the bony face of the centra. The increments were most obvious at the moment the alcohol has evaporated from the surface of the bone. A sizable portion of the vertebrae had concentric rings which were visible in only a minor sector of their circumference. However, most vertebrae had at least partially visible rings along the entire length of the vertebra. This condition made the quantification of every single increment almost impossible. Since most rings were consistent iri width, it was proposed that the following technique would be feasible for estimating the approximate number of rings present: 1) Vertebra cone depth (as defined by Johnson 1983)was measured to the nearest 0.05 mm using calipers. 2) Vertebra cone width was measured to the nearest 0.05 mm. 3) Cartilage was carefully peeled away from the centrum face to enhance increments. Cartilage was saved in the sample jar. 4) Centrum length, from focus to outside edge, was marked and divided into approximate 5 mm long sections. 5) The average number of rings per millimeter was calculated for each section by counting three -1 mm portions in each section. The calculated average was multiplied by the actual length of the section. Section totals were summed to give the total increment count for the centrum. Small sub-sections of 10 of the vertebrae were read twice by the author and a second reader to test for consensus of counts. Increment counts were compared both within and between readers using a paired T-test (Zar, 1984). Dorsal and Anal Fin Spines Anterior dorsal fin spines 1-7 and anal fin spines I and 2 were collected at dockside. Excised spines were labeled, placed in plastic bags and frozen. Spines were later thawed, separated, cleaned of all extraneous tissue, and allowed to air dry for at 7

least 72 hours. The sixth dorsal spines and second anal spines were chosen to be utilized for age analysis. These particular spines were chosen for this study because they were the thickest of the spine complex. In addition, sections taken from spines anterior to these had more prominent core matrices developed. The following methodology was employed to ensure that thin sections were taken from the same relative position in each spine. Spine length was measured to the nearest millimeter (mm) with a flexible measuring tape. Spine length was defined as the distance from the hole at the center of the condyle base to the spine tip (Figure 4). The location to be sectioned on anal spines was marked at the point which was I 0% of the spine length from the condyle hole. Because dorsal spines are longer relative to their width, they were marked at 5% of the length from the condyle hole. The width of the spine at the sectioning mark was measured with calipers to the nearest 0.05 mm. When a series of thin sections was taken along the base of the spine, and it was found that sections taken closer to the base of the spine had a larger internal core matrix and greater destruction of growth bands. Sections taken closer to the tip of the spine had smaller matrices, however, there was a greater possibility of loss of early growth material due to the nature of the deposition of bands on the vertebrae. Therefore, it was felt that this was the best area of the spines selected for examination of growth patterns and quantification of age. Portions of the spines containing the area to ·be sectioned were removed with a saw and glued to wooden blocks with marine epoxy. A Buehler ISO MET saw was used to take 2-3 sections of 0.3 to 0.5 mm thickness. Thin sections were placed into labeled and sealed petri dishes with 95% Isopropyl alcohol for extraction of oil and for storage. Spine sections were exarrtined using a compound stereoscope at 63X and 120X magnification. Sections were exarrtined with either transmitted light or with reflected light and a black background, depending on the characteristics and legibility of the growth marks. Growth bands were counted and measured if the bands were visible around the entire circumference of the section. An optical micrometer was used to measure the radius of each concentric band from the focus of the spine section to the outside edge of the translucent zone. The ability to count and measure all growth bands was limited due to the gradual formation of a vascularized core from the center of the spine. This core varied in diameter depending directly upon the size of the fish as well as the relative position in the 8

spine. The core was widest at the condyle base, and narrowed toward the spine tip. The problem of assigning age estimates to larger fishes in which these early growth bands have been destroyed was solved by the statistical replacement of these early bands. The main assumption of this. technique is that there is a predictable number of growth bands per millimeter of radius in the inner core of the spine sections. Growth band radii were statistically compiled from those spine samples in which the first growth bands had not yet been destroyed. Radius data from all samples missing early bands were then compared numerically and visually to the means and 95% confidence intervals of these data. Ages were assigned to the remaining spine samples by comparing the radius of their first three to four visible bands to the radius statistics of these samples. All spine samples were read twice by the author. A subsample of 10 spine sections were read independently 2 times by the author and a second reader. Age estimates were compared for consensus within and between readers using a paired T-test (Zar 1984). Hardpart Growth One of the basic criteria for using a bony structure for ageing studies is that the structure grows in proportion to the growth of the (Bagenal1974). For this study, the relationship between hardpart size [otolith weight (mg), vertebra cone depth (mm), spine width(mm)] and EFL (em) was determined using the previously described "CURVE" program. Significance (two-tailed test) of z2 values was tested (Shefler, 1979). Assessment of Ageing Techniques Several criteria were used to assess the usefulness of each hardpart for estimating age in the blue marlin. These criteria included the precision of age estimates, legibility of each hardpart, and ease of collection. The precision of age estimates included measurements of the consistency of age estimates within and between readers, variability of ages between corresponding hardparts, and variability of mean length at age data for specific age categories for each hardpart. To test the consistency of age estimates within and between readers, a subsample of 10 of each hardpart was read two times each by two readers. Readings were compared using a paired T-test (Zar, 1984). Direct comparisons of the age estimates from corresponding hardparts were made. The relationship between counts of corresponding hardparts was tested using a 9

paired T-test (Zar, 1984). Regression analyses were also used to examine these comparisons. In order to test the null hypothesis that counts in otoliths, and dorsal and anal spines were equal, the slopes of the regressions were tested to see if they varied

: significantly from parity (H0 beta= l)(Zar, 1984). This was tested by subtracting the predicted slope (beta= 1) from the observed slope and dividing this difference by the standard error of the observed slope to give the t- statistic (Zar, 1984). In addition, the significance of correlation coefficients (r) of the comparisons was tested using methods outlined by Schefler (1979). Age -Length Relationship The relationship between length an<;! age was described from data of the chosen hard part. The choice of which hardpart to use was based on several criteria, including the number of samples available for each hardpart and the relative ease of interpretation. A computer program developed for the HP 9825 computer was utilized to calculate von Bertalanffy growth parameters according to four different methods, as described by Everhart et al. (1975), Walford (1946), Gulland (1965), and Allen (1966). The mean square error was calculated to determine which, if any, of the four methods provided the best fit to the von Bertalanffy growth curve. 10

RESULTS

Summary of Fishes Sampled Hardpans and morphometric data were taken from a total of 213 male and 106 female blue marlin. Males ranged in size from 95.4 em EFL (19.1 kg) to 222.0 em EFL (138.8 kg). Females ranged from 125.7 em EFL (20.9 kg) to 398.8 em EFL (748.0 kg)(Table 1). The 222.0 em EFL male and the 398.8 em EFL female are the largest specimens of blue marlin from which any biological data have ever been collected. The mean length of females (231.9 em EFL) was significantly greater than that of males (178.6 em EFL)(Student's T-test P<0.05). Length frequency histograms (Figure 5) demonstrate well-defmed unimodal distributions for each sex, and no indication of size or age classes. Morphometries There were statistically significant linear correlations between EFL (em) and the other three body measurements for males, females, and combined sexes (Appendices 1-6)(P< 0.001 for all correlations). The relationships between TFL, LJFL, and EAFL were linear and statistically significant as well (P< 0.001 for all correlations). Coefficients of determination were extremely strong for all length comparisorts, ranging from r2 = 0.916 for TFL vs. EAFL (Appendix Sa; males), to?= 0.997 for EFL vs. LJFL (Appendix 2b; females). In all cases, morphometric relationships were slightly stronger for female marlin than for males. The curvilinear relationship between EFL (em) and Weight (kg) was also statistically significant and was best expressed by the equation: .W(kg)= 4.354 x e

(r-2 = 0.596; P< 0.001) than for females (r-2= 0.450; P< 0.001). Otoliths ranged in weight from 0.414 to 4.986 mg for males and from 0.799 to 7.70 mg for females. Sagittae had external and internal morphologies and features which were similar to those described by Radtke et al. (1982), Radtke (1983), and Wilson (1984). External features included a deep, well defined sulcus on the medial surface of the sagittae, and ridges or layerings on the convex external surface of the rostrum (Figure 7a). These ridges were parallel to the direction of rostral growth and increased in number with otolith size. The legibility of rostral ridges was often affected by overgrowths of calcified material which created the "finger-like" patterns described by Wilson (1984)(Figures 7a,b), or over the entire surface of the rostrum creating a mottled appearence (Figure Sa). In addition to those features previously described by others, ridges or prominences were observed along the anterior and occasionally the posterior edges of the rostrum (Figure 8b) in the area described by Wilson (1984) as having no obvious external growth features. These prominences of the anterior rostrum edge, as well as the ridges of the lateral-dorsal surface of the rostrum were quantified for age determination. Thin transverse cross sections of resin-embedded sagittae contained features similar to those described by Wilson (1984) when observed by light microscope. The concentric internal increments closest to the core were evenly spaced centrally (Figure 9a) and were analogous in appearance and number to the initial broad increments of the caudal vertebrae. Beyond these initial increments, the increments varied in width and density resulting in the appearance of opaque and translucent zones. These irregularly spaced zones in the region of lateral growth were interpreted as annuli, based on the works of Radtke and Dean (1982) and Wilson (1984). Beyond the site of change in direction of rostral growth, the opaque and translucent zones were less apparent and annuli were seen as points of convergence along the outside edge of the rostrum (Figure 9b). These convergences corresponded, for the most part, to the ridges of growth seen on the external rostral surface of whole sagittae. Results of the analysis between external ridge counts and internal annuli indicated that there was no significant difference between paired external counts, paired internal counts, or between external and internal counts (Paired T -test, alpha=0.05; Table 2a). When the precision of age estimates based on external features only was assessed between readers for a subsample of 37 otoliths, results revealed no significant 12

differences either within or between readers (alpha=0.05; Table 2a). Standard deviations (SD) of paired otolith counts provided an index of the variability of these counts and were higher, in general, than those reported for other hardparts in this study (Table 2a). Paired otolith count standard deviations ro.nged from 1.37 ridges (N = 10) for internal counts to 4.23 ridges (N = 37) for counts between readers. Vertebrae as Ageing Material Vertebrae were collected from 123 male and 78 female marlin. Males ranged in size from 146.0 em EFL (58.1 kg) to 222.0 em EFL (138.8 kg) and females ranged from 125.7 em EFL (20.9 kg) to 300.0 em EFL (447.7 kg)(Table 1). There was a significant positive linear relationship between EFLand centrum cone depth (Figures 10a,b). The relationship was much weaker for males (r2=0.155; P< 0.001) than for females (r2=0.468; P< 0.001). Cone depth ranged from 17.45 to 30.35 mm for males and from 18.30 to 42.15 mm for females. Concentric incremental rings were observed in all caudal vertebrae examined. These increments increased in number with the size of the vertebra. The growth marks were superficial topographical features on the face of the centrum (Figure 11). Attempts to enhance these features through bleaching for short periods resulted in the destruction of these rings leaving only the porous matrix of the bone below. The face of the centrum containing these rings was between 0.2 and 0.5 mm in thickness. The growth rings ranged from 0.05 to 0.1 mm in width. A sizeable portion of the vertebrae had an area with 25 to 30 larger growth increments starting from the focus of the centrum (Figure 11). Upon close examination of these larger increments it was observed that they were also composed of 3-5 of the smaller increments previous! y described. There were no obvious areas on the centra with changes in density of ring counts which would be suggestive of slower or faster rates of deposition. There were no prominent 3-dirnensional features on the centra which might be indicative of annular events. Comparison of reader counts of small sections of the vertebrae revealed no significant difference either within or between readers (Paired T-test, alpha=0.05; Table 2b) Standard deviations of these counts ranged from 0.99 rings (N = 10) between readers to 1.20 rings (N = 10) for reader 1 (Table 2b). Dorsal Fin Spines as Ageing Material Dorsal spines samples were taken from 65 male and 29 female marlin. Males ranged in size from 153.0 em EFL (46.3 kg) to 222.0 em EFL (138.8 kg) and females 13

ranged from 141.0 em EFL (35.2 kg) to 398.8 em EFL (748.0 kg)(Table 1). There was a significant positive linear relationship between EFL and dorsal spine width for males and females (Figures 12a,b). The relationship was weaker for males (~=0.386; P< 0.001) than for females(~= 0.805; P< 0.001), which is probably a reflection of the smaller size range of males sampled. Dorsal spine width ranged from 15.15 mm (162.2 EFL) to 22.65mm (203.8 em EFL) for males and from 13.2mm (141.0 em EFL) to 49.0 (398.8 em EFL) for females. Growth bands similar to those described by Jolley (1977) and Prince eta!. (1984) were present in all spine sections examined and increased in number with the width of the spine and the size of the fish. Growth bands viewed with transmitted light consisted of pairs of dark opaque and light translucent zones (Figure 13a), and were the opposite for sections viewed with a black background and reflected light (Figure 13b). Many growth bands observed were composed of smaller rings that were most obvious at the widest lateral portion of the spine section (Figure 14 ). Since band radii were measured from the focus outward along the widest portion of the spine, excessive numbers of rings along this axis made delineation of the outside edge of the translucent zone difficult at times, especially in exceptionally large spine samples. In such cases, it was necessary to refer to the dorsal and ventral areas of the section where the dark and light portions of the bands where more compressed and could be more clearly delineated (Figure 15). A small number of spine samples (2%) were encountered which were excessively opaque in all areas, and required some sanding, polishing, or alternate light sources, such as a dark background with reflected light. The use of reflected as opposed to transmitted light served only to enhance the existing growth patterns, and in no way altered the way in which they were interpreted (Figures 13 a, b). Results of the comparisons of the precision of age estimates based on counts of dorsal spine bands within and between readers revealed no significant differences in dorsal spine counts either within or between readers (alpha=0.05;Table 2c). Standard deviations of paired dorsal spine band counts ranged from 0.97 bands for reader 2 (N = 10) to 1.18 bands between readers 1 and 2 (N = 10)(Table 2c). From the spine band radii measured for all dorsal spine samples, 11 of the 94 spines (12%) had a visible first annulus that had not yet been destroyed by inner matrix expansion. Mean radius measurements for these eleven samples (Table 3) were 14

compared to the radius measurements of the remaining 82 samples. Twelve of these 82 samples had radii that matched well within the 95% confidence limits for the 2nd through the 6th band radii for the original 11 samples. It was therefore determined that these samples had a visible 2nd annulus. The raqius measurements of these 12 samples were compiled with the radius data from the original 11 samples. The remaining 70 spine samples were assigned ages by comparing the radii of their first several visible bands to the radius statistics of these 23 samples (Table 3; Figure 16). Anal Fin Spines as Ageing Material Anal spine samples were collected from 144 male and 47 female blue marlin. Males ranged from 95.4 em EFL (19.1 kg) to 222.0 em EFL (138.8 kg) and females ranged from 141.0 em EFL (35.2 kg) to 300.0 em EFL (447.7 kg)(Table 1). There was a significant positive linear relationship between EFL and anal spine width for both males and females (Figures 17a,b). The relationship was weaker for males (r2=0.445; P< 0.001) than for females (?=0.785; P< 0.001). As for dorsal spines, this was probably a reflection of the smaller size range of males sampled relative to females. Anal spine width ranged from 8.35 mm (95.4 em EFL) for males and from 11.45 mm (141.0 em EFL) to 32.2 mm (282.4 em EFL) for females. Growth bands were visible in all anal spine sections examined, and were similar in characteristic to those found in dorsal spine sections in this study as well as anal spine sections described by Wilson (1984)(Figures 18,19). Growth bands of anal spine sections contained smaller rings similar to those found in dorsal spine sections. Anal spines, however, were less compressed dorso-ventrally than dorsal spines, and there were fewer problems with the lateral broadening of the bands and the diffusion of the border of the border between translucent and opaque zones (Figure 18). A small percentage of anal spine samples (4%) were difficult to interpret because of excessive opaqueness of the bone. This problem was overcome by using similar techniques as were used for dorsal spine sections. Paired T -tests revealed no significant differences of age estimates either within or between readers (Table 2d). Standard deviations of paired anal spine band counts ranged from 1.03 bands for reader 1 (N = 10) to 1.83 bands for reader 2 (N = 10; Table 2d). For the spine band radii which were measured for all anal spine samples, 19 of the 191 (10%) anal spines had a visible 1st annulus which was not yet obscured by the 15

inner matrix. Mean radius measurements were numerically compared to the radii of the remaining 172 samples. Twenty-three of those 172 samples had radii that matched well within the 95% confidence intervals of the 2nd to 5th or 6th band radii of these samples. Radius measurements of these 23 samples were compiled to the data of the originall9 samples. The remaining 143 samples were assigned ages by comparing the radii of their first several visible bands to the band radius statistics (Table 4; Figure 20). Assessment of Ageing Techniques Reader Precision : Paired T-tests of paired hardpan counts revealed no significant differences either within or between readers for all hardpans, nor did they reveal differences within or between external and internal counts of otoliths (Tables 2a-d). The standard deviations of these paired readings indicated the variability of these counts, and provided an index of the relative interpretability of each hardpan. Otoliths had the highest standard deviation (SO = 4.23 ridges) of all paired counts in this study (reader 1 vs. 2; Table 2a) relative to all other hardpans. Dorsal and anal fin spines had similar paired count variabilities ranging from SO = 0.97 bands for dorsal spines (reader 2; Table 2c) to SO= 1.83 bands for anal spines (reader 2; Table 2d). Counts of vertebral increments were of a different dimension and could not be compared directly to the SO's of other hardparts. Hardpan Cross-Comparisons: Attempts to statistically compare distributions of size in specific age classes between hardparts using an ANOVA were not feasible due to the differences in sample size in each age class category for each hardpan (Tables 5,6). In the majority of cases, however, mean length at age for a particular hardpan fell within the 95% confidence limits of the other two hardpans (Tables 5,6). All dorsal and anal spine age estimates refered to in the following age comparisons were corrected for missing internal bands using the previously described techniques. A direct comparison of counts between corresponding dorsal spines and otoliths revealed a positive linear relationship between these two hardpans (Figure 21). The regression of the comparison was a significant one (r = 0.861; P< 0.001), and the slope of the regression did not differ significantly from parity (P< 0.05). The greatest difference between corresponding counts was 7, where the dorsal spine band count was 9 and the otolith ridge count was 16 (Figure 21). A comparison of counts between corresponding anal spines and otoliths also revealed a significant positive linear relationship between the two hardparts (r = 0.820; 1 6

P< 0.001) (Figure 22). The slope of the regression did not significantly differ from parity (P < 0.01). The greatest difference in counts between corresponding parts was 9, where the anal spine band count was estimated at 7 and the otolith ridge count was 16 (Figure 22). Similarly, the comparison of count between corresponding anal spines and dorsal spines revealed a significant positive linear relationship which was stronger than the comparisons of these two hardparts to otoliths (r = 0.950; P< 0.00 !)(Figure 23). The slope of this regression did not differ significantly from parity (P< 0.01). The greatest difference in corresponding counts was 5 bands, where the anal spine was estimated at 11 bands and the dorsal spine at 16 bands (Figure 23). The comparisons of vertebral increment counts to corresponding otolith, dorsal spine and anal spine counts revealed positive linear relationships between the hardparts, but these relationships were more variable compared to those previously discussed in this section (Figures 24-26). Nonetheless, the coefficients of determination were statistically significant for each comparison. The relationship between otoliths and vertebrae had an r value of 0.510 (P< 0.001; Figure 24), between dorsal spine and vertebrae it was r = 0.760 (P< 0.001; Figure 25), and between anal spines and vertebrae it was r = 0.660 (P< 0.001; Figure 26). They-intercepts of these three linear regressions were similar, ranging from 318 (dorsal spine vs. vertebrae, Figure 25) to 357 (otoliths vs. vertebrae, Figure 24). The slopes of these regressions were similar as well, ranging from 15.17 (Figure 24) to 18.17 (Figure 25). Thus, for every otolith ridge or spine band count there were between 15 and 18 vertebral increment counts. Paired T-tests (two-tailed) between counts of corresponding otoliths, dorsal spines, and anal spines provided additional information about their relationships (Table 7). There were no significant differences in counts between these three hardparts (P< 0.05; Table 7). The comparison between corresponding dorsal spines and otoliths had a mean difference of 0. 78 counts, with a standard deviation of 2.62 counts (N = 39). The comparison between anal spines and otoliths had a mean difference of0.14 counts and a standard deviation of 2.55 counts (N =53). The test between anal spines and dorsal spines had a mean difference of 0.47 bands and a standard deviation of 2.41 bands. Mean Length at Age from Anal Spines Because otoliths, dorsal spines and anal spines appear to yield similar growth data, growth of the blue marlin was described from ages derived from anal spine 17

sections since there were the most data available for this hardpart. An additional datum from the record female dorsal spine was added to this data set. Attempts to model the growth of males and females using various methods for the von Bertalanffy equation resulted in hi_ghly questionable estimates of the t0 and L= parameters and high mean square error values for each parameter (or age class). For example, t0 values for females ranged from -3.5 to 2.0 years, depending upon which model was being used. These are unreasonable values considering the fact that marlin are oviparous fishes, and theoretically should have a t0 value close to zero. In addition, L= estimates for females were well below the L maximum value of 398.8 em EFL reported in this study. An attempt to force-fit more reasonable values of these two parameters (t = 0, L= = 425 em EFL) to obtain an estimate of the growth coefficient, 0 K, resulted in extremely high mean square error values. For these reasons, the growth of male and female marlin were simply described by plotting mean EFL-at-estimated age (Figures 27,28). There was a pronounced difference in growth between male and female marlin. Males appear to grow steadily to an average size of 177 em EFL at an estimated age of 6 years, after which their growth levels off rapidly (Figure 27; Table 8). The largest male sampled, which was also the largest male on record, was estimated to be 9 years old. The oldest male , 18 years, was no larger than the average size males sampled. Growth of female marlin is steady and does not level off as rapidly as for males (Figures 27,28; Table 9). The largest female sampled (398.8 em EFL) was also one of the two oldest at 26 years. Mean length at age of females was much more variable than that of males (Figure 28). 18

DISCUSSION

Length Frequencies Size frequencies (EFL em) of blue lJlarlin sampled for this study were similar in several respects to size frequencies presented by Kume and Joseph (1969), who sampled close to 3,600 blue marlin from the commercial fishery in the eastern Pacific region. Size frequencies were similar in terms of size range for both sexes, modes of size distributions, and differences in size between sexes. Similarities in the size frequencies of this study and that of Kume and Joseph (1969) suggests a similar bias in the selectivity of sport and commercial fishing gears employed for collections. In this study, there was a rarity of male blue marlin below 150 em EFLand above 210 em EFLand a rarity of female blue marlin below 170 em EFL and and above 270 em EFL. This paucity of small marlin may be due to the size selectivity of fishing gear, or may be an indication of a size related migratory or schooling pattern which segregates younger individuals from the rest of the population. The largest male collected in this study (222.0 em EFL, 138.8 kg) was probably the largest male blue marlin sampled to date. Kume and Joseph (1969) did report a few rare male individuals measuring approximately 300 em EFL, however, the true sex of these specimens is questionable considering the fact that these males would be at least 250 kg over the generally accepted maximum weight for males (Skillman and Young, 197 6). The largest female sampled in this study (398.8 em EFL, 748.0 kg) was close in weight to the largest female reported in the literature (817.3 kg; Mather, 1976), but was light in weight for her length, according to the length-weight relationship of this study (Appendix 7b). Nevertheless, considering the difficulties and constraints involved in the collection of these large pelagic fishes, it was felt that the size range of specimens collected for this study was representative of what could have been collected on a larger scale (Kume and Joseph, 1969). Data from this study, as well as from others, support the theory that ultimate size in blue marlin is determined by sex (Yeo, 1978; Wilson, 1984). The obvious differences in both average and ultimate size between male and female blue marlin in this study were consistent with the findings of others (Kume and Joseph, 1969; Ovchinnikov, 1970; Yeo, 1978; Wilson, 1984). These differences in size between the sexes has been theorized by deSylva (1974) to indicate protandric hermaphroditism. The occurrence of small, young females in this and other studies 19

(Wilson; 1984) lends evidence against this theory. Mowhometric Relationships Morphometric analyses revealed an extremely strong relationship between pairs of the four body measurements taken. This information will be useful for standardizing length data among various studies through conversion equations. It may also be utilized for future studies attempting to differentiate stocks and substocks by the minute differences in allome.tric relationships (Bowering and Misra, 1982). Analysis of the length-weight relationship of blue marlin demonstrated that power functions best described the data Wilson (1984) found a statistically significant difference in these weight-length regressions for both sexes between the Atlantic and Pacific blue marlin, thus supporting Morrow's (1959) and Nakamura et al.'s (1968) reports of osteological and lateral-line differences between these two populations. Similarly, differences in morphometric relationships may provide supportive evidence for the division of Atlantic and Pacific substocks in future studies. Blue Marlin Hardparts as Ageing Stmctures One of the main criteria which must be met in order for any hardpart to be utilized for age and growth studies is that the growth of the structure is proportional to and representative of the growth of the fish (Bagenal, 1974). The significant positive relationships between EFL and the size parameters (otolith weight, vertebral cone depth, and spine width) of each hardpart supported the use of each of these structures for age estimation. Coefficients of determination for the regressions of these comparisons were higher for females in the cases of fm spines and vertebrae. The fact that these relationships were not as strong for males may be due to the smaller size range of males sampled compared to females, or may be due to the fact that the growth of males stops at a much earlier time than females. The relationship between otolith weight and EFL was, on the other hand, stronger for males than females in this study. It is possible that the factors governing the rate of otolith growth, as opposed to skeletal growth, may be more variable for female marlin. The variability of blue marlin otolith morphologies observed in this study will be discussed further in the next section. Another possible explanation for the variabilities of hardpart growth in relation to EFL growth is that these relationships may fit some mathematical model other than those tested with the CURVE pro gram in this study. Information on growth was available from each of the hard parts (otoliths, 20

vertebrae, and fin spines) studied. Each of these hardparts had growth patterns that were, to varying degrees, recognizable and quantifiable. These growth patterns observed increased in number with the size of the fish, thus meeting another important criteria for ageing studies. Although each·hardpart had recognizable growth patterns, the details of the nature of their depositions are not well understood. The advantages and disadvantages of using each hardpart for age estimation will be discussed in the following sections. Otoliths as Ageing Material Otoliths are tedious to collect and process. Good bilateral dissections of large marlin heads can be difficult. Furthermore, once bisected, the removal of the inner ear, especially the sacculus, can present problems. Much time, expertise, and expense is involved in the processing of otolith materials. They require a great deal of care in handling as they are extremely fragile. If age estimates are to be made by external features only, they must be mounted and gold-coated to observe the surface topographies. If internal features are to used for estimating age, the specimen must be embedded in resin and thin sectioned using the jeweler's saw which is not always successful. Embedding and sectioning causes permanent destruction of the otolith, so they must be photographed before doing so. The only way to photograph the external features with the entire otolith in focus is by using SEM. Inspection of daily and other internal incremental patterns requires skilled sample preparation and the use of SEM. Otoliths were used in this study only to estimate age by counting what were interpreted to be annual features, and no detailed study of their incremental features was attempted. Partial evidence for the annual nature of these ridges was provided by the tag-recapture of a sailfish which was at large for almost II years and was estimated to be 13 years of age based on features in the otoliths which are analogous to those found in blue marlin sagittae (Prince et a!. 1986). However, it was found that the external and internal growth features found in these calcified structures were difficult to interpret. Even with specialized training in otolith morphology and development, interpreting the growth zones was perhaps more subjective than for the fin spine sections. There were problems involved with calcium overlayering, and the overlapping of successive ridges. Sometimes large distinct ridges had smaller ridges within them. Otoliths were variable in size and shape from fish to fish. Wilson ( 1984) reported a similar individual variability in the general morphology and clarity of growth features of blue marlin otoliths. 21

Wilson (1984) concluded that the total count of the external ridges on the rostrum of blue marlin sagittae did not provide an accurate estimate of age, because up to 5 years may pass before the onset of external ridge formation. This early growth was obscured by the calcium overgrowths that had developed on the convex external surface of the rostrum. In this study, however, features were observed on the anterior edge of the rostrum which corresponded well with the internal counts of the same region of the sagittae. Comparisons of age estimates based on external vs. internal counts using Paired T-tests statistically supported the use of external counts alone for gross age estimation by sagittae. The exact processes governing the formation of rostral ridges in billfish otoliths is yet undetermined, but it is reasonable to assume that they are a reflection of some change in the rate of deposition of organic and inorganic materials throughout the life of the fish. This change in the rate or disruption of material deposition would result in regions of slower and faster growth on the surface of the otolith, observed as ridges. The standard deviations of paired otolith readings served as an index of variability of counts, and gave an indication of the interpretability of this structure relative to other hardparts discussed in this study. The highest standard deviation was that reported between readers. Standard deviations within reader counts were higher relative to those for other hardparts as well. This provides additional evidence for the subjectiveness of this hardpart to individual interpretations. Even considering the problems encountered, marlin otoliths still hold promise in providing detailed information of age from their incremental patterns, which are theorized in other tropical fishes to be deposited on a daily, weekly, and monthly schedule. The true nature of the growth patterns observed in the otoliths of blue marlin remains to be solved. Vertebrae as Ageing Material Comparison of vertebral increment counts to age data from the other three hardparts revealed relatively high variability. This variability may be accounted for, in part, by the error incurred by the application of the described vertebral increment counting technique. Another source of error is the assignment of discrete ages of annual intervals to the other hardparts, when the specimens were actually some interval of that age. Vertebral incremental markings similar in appearance have been described for the 22

conical vertebrae of Yellowfin (Seriola) (Munekiyo et al., 1982) as well as other fish species. The vertebrae of yellowfin were different in that they had regions of ring density change which were counted as annual markings. No such regions were visible in the vertebrae of the blue marlin. The vt;.rtebrae of Squatina californica have been described as having incremental growth patterns which have no apparent predictable chronological periodicity, but rather are deposited at intervals which are random from any particular time scale (Natanson, 1984; Natanson et al., 1984). Such may also be the case for the vertebrae of the Pacific blue marlin, and until validation studies can be accomplished, it will be difficult to interpret the true periodicity of these increments. The deposition rate of the vertebral increments of blue marlin may vary with age. This is evidenced in the cross-comparisons of vertebral increment counts with other hardparts in which they-intercepts of each comparison ranged from 318 to 357 increment counts. One possible explanation for this observation is that the vertebrae are growing and depositing increments before the other skeletal structures have developed significantly. Young marlin are known to grow in length more than weight initially (deSylva and Ueyanagi, 1974), and this would imply an early rapid growth in the length of their vertebrae. The meaning of the incremental features contained in the surface of the caudal vertebra has yet to be determined. Direct comparisons of vertebral increment counts to corresponding hardpart counts gave a ratio of between 15-18 vertebral increments per year. Assuming the counts of these other hard parts to be annual representations, it is possible that the vertebral increments are deposited on either a bi-weekly or monthly basis. Lunar influences on the deposition of growth rings have been proposed to explain patterns observed in the otoliths of a number of fish species, including juvenile Starry flounder (Campana, 1984) and sailfish (Radtke and Dean, 1981). The possible effects of lunar rhythmicities on the variable deposition of growth markings may include changes in light, changes in activity rates associated with feeding or spawning behaviors, variability in diet, or a concurrence of several of these factors (Campana, 1984; Gibson, 1978). Until work can be accomplished to validate the increments of blue marlin vertebrae, the question of whether they are deposited in relation to some set of external stimuli or simply as the result of growth independent of time will remain unanswered. 23

Fin Spines as Ageing Material Dorsal and anal fm spines appear to be the simplest and most consistently reliable material for the gross age estimation of the Pacific blue marlin. Fin spines are relatively simple to collect, process, interpret, and store compared to otoliths. Spines were also easier to process in the laboratory as they reqUired little in the way of equipment and supplies. The growth bands found in the fm spines were a consistent feature from specimen to specimen. Interpretation of growth bands in blue marlin fm spines is a simple procedure to learn and is, perhaps, less subjective than the interpretation of the gross anatomical growth features of their otoliths. This was evidenced by the lower standard deviations of paired reader counts and counts between readers for both dorsal and anal fin spines. Reader 2 of the spines had relatively little experience in the interpretation of skeletal hardparts, but had little trouble in deriving age estimates from the spines subsamples observed for the first time. The main disadvantage to the use of fm spines is the presence of the internal matrix which obscures the early growth bands in larger fishes (Prince et al., 1986). This problem, however, can be at least partially overcome through the statistical replacement of these inner bands. Problems encountered with the doubling and tripling of growth bands were similar to those described for sailfish dorsal spines (Jolley, 1977; Hedgepeth and Jolley 1983), and swordfish anal fin spines (Berkeley and Houde, 1983). They attributed this multiple banding to the actual splitting of the annulus, which was observed ventral or dorsal to the core of the spine. The actual cause of band splitting in fin spines is speculative at this point, but may reflect life history events such as minor migrations or multiple spawnings throughout the year. Double and triple banding can sometimes pose problems to the reader, but interpretation can be resolved by referring to the dorsal and ventral portions of the spine. Verification and Validation of Age Estimates in Blue Marlin Verification and validation of age estimates are now considered to be essential, yet often overlooked, elements in age and growth studies (Beamish and MacFarlane, 1983). Wilson et al. (1983) defined verification as the "confirmation of a numerical interpretation" (or, the determination of precision) and validation as "the confirmation of the temporal meaning of a growth increment" (or, the determination of accuracy). Age estimations in blue marlin were at least partially verified in this study when comparing 24

analogous features in the otoliths, dorsal and anal fm spines. The application of paired T-tests and other tests between readers and between hardparts provided corroborative evidence of similar age determination. The inherent danger of using this type of comparison is that the results are meaningless if both hardpart interpretations are biased in the same way. The variability in counts between otoliths and their corresponding spines was greater than between the counts of corresponding anal and dorsal fin spines. There were several isolated cases in which the ages of the otoliths were as much as 5-10 counts above or below those of the corresponding fin spine estimates (Figures 22,24). Similarly, Prince et al. (1984) found discrepancies of up to 6 counts between the otoliths and dorsal spines of the Atlantic blue marlin, and up to 9 count differences between these two hardparts from the Atlantic white marlin (Tetrapterus albidus). This higher variability of counts between otoliths and their corresponding spines is probably an artifact of the variability in sagitta structural features which, in turn, affected their legibility. These count discrepancies may be clarified through finer structural analysis of the otoliths. In general, there was good agreement in age estimates between corresponding otoliths, dorsal spines, and anal spines. This provided corroborative evidence that the growth features in each hard part quantified for age estimation resulted from similar growth stimuli. Other studies on the age and growth of billfishes have revealed comparable results. Sailfish spines (Hedgepeth and Jolley, 1983) and otoliths (Radtke and Dean, 1981) appear to yield similar growth data. The same situation is true when swordfish anal spines (Berkeley and Houde, 1983) and otoliths (Radtke and Hurley, 1983) are compared. Possible factors influencing the deposition of variable growth patterns in this tropical species may include light variation throughout the year, yearly patterns of migration, seasonal variability of diet, hormonal changes, or a combination of these factors. Although verification of the observed growth patterns is available by these comparisons, these results do not provide direct evidence of the meaning of their true periodicity. Obtaining age validation evidence for the Pacific blue marlin has been and will continue to be logistically difficult, and was not within the scope this study. Billfishes have never been reared in captivity and tag returns have been extremely rare. An additional problem has been obtaining accurate estimation of fish size at the time of 25

tagging (Squire and Nielson, 1983). Growth of Pacific Blue Marlin Based on evidence presented in this study, Pacific blue marlin males appear to have a longevity of at least 18 years, and females appear to live to at 26 years of age. The largest female sampled was also the oldest fish and weighed 748 kg. There are unofficial records oflarger Pacific blue marlin, including "Choy's Monster"(817.4 kg; Mather, 1976), and unconfrimed reports of commercially caught specimens exceeding 900 kg, so females probably live to exceed an age of 30 years. The largest male sampled in this study (138.8 kg) was only 9 years of age. The oldest male sampled (aged 18) was only of an average size, so it is probable that males may attain an age of 25 years or more. Visual comparison of age data from this study to Wilson's (1984) age estimates for the same species revealed similar results in terms of relative longevity and ultiinate size for each sex, but rates of growth were difficult to compare as Weight (kg) was compared to age in his study. Wilson (1984) reported Pacific blue marlin males as old as 17 years and females as old as 21 years. None of the von Bertalanffy growth model techniques applied to the anal spine data gave reasonable estimates of the t , K, and L= parameters. This may be due, in 0 part, to a lack of data at both extremes of the size range. It may also be explained by the variability in size and age from individual to individual, especially in the case of females. Similarly, Wilson's (1984) attempts to apply von Bertalanffy growth models to describe blue marlin growth had variable results. For these reasons the von Bertalanffy growth models were not further investigated in this study, but will be reinvestigated once a broader range of sample sizes has been collected. It should be remembered, however, that the von Bertalanffy growth models are not a panacea for describing the growth of all fish species, and that better models may apply for the blue marlin. Blue marlin in this study had a sex-related difference in size which was independent of age. Males appear to grow steadily to an average length of 177 em EFL at an age of 6 years, after which their growth rapidly levels off for the remainder of their lifespan. Growth of female marlin is more steady for a greater portion of their lifespan, however, this growth is quite variable from individual to individual. Females probably continue to grow, especially in girth, for the remainder of their lifespan. These results concur with the conclusions of Wilson (1984) and lend additional evidence against deSylva's (1974) suggestion that blue marlin are protandrous hermaphrodites. 26

Age data from this study provided further evidence of the inherent problems in using length-frequency data for the delineation of age-class cohorts. Age was quite variable with fish length for any one size class in this study. For example, a 200 em EFL female could range from 6 to 16 years of age, and a "175 em EFL male could range from 6 to 18 years of age. Furthermore, the fish size distributions of this study and that of Kume and Joseph (1969) were unimodal for each sex, showing no evidence for division of age groups by size classes. Further inconsistencies are revealed when growth data in the literature are compared to length-frequency studies for the same species. Studies on the growth of sailfish using otoliths (Radtke and Dean, 1981) and dorsal fin spines (Jolley 1977; Hedgepeth and Jolley, 1983) have indicated rates of growth that are approximately half of those estimated from length-frequencies (de Sylva, 1957; Koto and Kodama, 1962). Similarly, a visual comparison of the mean-length-at-age data (using anal fm spines) from this study to the von Bertalanffy growth curves developed by Skillman and Y ong (1976) indicates a growth rate that is approximately half of the rate predicted by their use of length-frequency data. Based on existing reproduction data, blue marlin males are sexually mature between 130 and 140 em EFL (Nakamura, 1985) which corresponds to an age of approximately 4 years. Sexually mature females as small as 155 em EFL (5-6 years) have been reported, but the majority of ripe females are o'>(er 200 em EFL (Kume and Joseph, 1969), which corresponds to an age of between 7 and 11 years. Recommendations Based on the findings of this study, dorsal and anal fin spines are the most practical and useful tools for age estimation of the Pacific blue marlin. They hold the most promise for use by fisheries biologists as they are easy to collect, prepare, interpret, and store in comparison to otoliths. Relatively little investment is required in the way of equipment or training. Problems encountered with the expansion of the spine core can be overcome through a collection of spines from younger fishes as well as a comparison to the spine band diameter data presented in this study. Otoliths should not be discounted as useful structures for age determination, but more practical means of interpreting the major and minor growth features of the sagittae should be developed. The higher variability of counts between the otoliths and their corresponding spines indicates either a greater difficulty in the interpretation of the otolith features, or a 27

variability in the factors which determine the deposition of growth features of spines and otoliths. This question remains unresolved. Future investigations on the growth of blue marlin and other billfish species are needed to define the true meaning of the growth patterns observed in this study. These studies should attempt to validate these periodicities through improved mark-recapture techniques. The problem of obtaining accurate estimations of size at the time of marking may be improved through the use of large measurement scales on the side of the fishing vessel and photographic records from an observer vessel. Attempts should also be made to spawn and rear larval and juvenile marlin in culture systems. Such work has been successfully been accomplished with other oceanic pelagic fishes such as dolphinfish (Coryphaena hippurus)(Beardsley, 1967; Hassler and Hogarth, 1977), and Euthynnus affinis (Kaya et al., 1981), and with the refinement of these techniques should hold great promise for billfishes as well. These studies would enable researchers to better define the true meaning of incremental depositions found in the otoliths and vertebrae, and over a longer term would allow for validation of major markings observed in their otoliths and fm spines. Further attempts to verify the growth patterns observed in the otoliths and fm spines should include detailed studies of marginal increment depositions and detailed chemical analyses in these hardparts. Electron microprobe analyses for Phosphorus and Calcium have proven useful for delineating changes in hardparts for numerous other species (Casselman, 1983; Cailliet et al., 1986). In addition, analyses for ratios of Strontium and Calcium along a cross section of the otolith may reveal minute changes in temperature throughout the life history of the fish (Radtke, 1984; Radtke and Targett, 1984), and may be useful for defming migrations of these large fishes to colder and warmer bodies of water. 28

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Table I. Summary of numbers and size ranges of Makaira nigricans from which skeletal hardparts and rneasure1pents were collected. "M = Male, F = Female.

Size Range OfMakaira nigricans

Eye-Fork Length (ern) Weight (kg) Hardpart N Min. Max. Min. Max.

Otoliths M 54 146.0 222.0 58.1 138.8 F 45 141.0 398.8 35.2 748.0

Vertebrae M 123 146.0 222.0 58.1 138.8 F 78 125.7 300.0 20.9 447.7

Dors. Spines M 65 153.0 222.0 46.3 138.8 F 30 141.0 398.8 35.2 748.0

Anal Spines M 144 95.4 222.0 19.1 138.8 F 48 141.0 300.0 35.2 447.7

All Fishes M 213 95.4 222.0 19.1 138.8 F 106 125.7 398.8 20.9 748.0 34

Table 2. Analysis of reader comparisons for otoliths, vertebrae, and dorsal and anal fin spines by paired T-test. N = number of individuals aged by each reader. Mean D = the mean difference between successive readings or between readers. SD =Standard deviation of Mean D. df =degrees of freedom. t calc.= calculated value oft. t crit. =critical value oft.

Source N MeanD Sl2 df t calc. t cri t. a) Otoliths Reader 1 10 0.700 2.41 9 0.920 2.262 Reader2 10 0.641 2.93 9 0.996 2.262 1 vs. 2 38 0.237 4.23 37 0.346 2.042 Internal 10 0.100 1.37 9 0.231 2.262 External 10 0.700 2.41 9 0.920 2.262 Int./Ext. 10 0.400 3.63 9 0.126 2.262 b) Vertebrae Reader 1 10 0.100 1.20 9 0.264 2.262 Reader 2 10 -0.100 1.10 9 0.287 2.262 1 vs. 2 10 -0.100 0.99 9 0.319 2.262 c) Dorsal Spines Reader 1 10 <0.001 1.16 9 <0.001 2.262 Reader 2 10 0.500 0.97 9 1.629 2.262 1 vs. 2 10 0.250 1.18 9 0.667 2.262 d) Anal Spines Reader 1 10 0.200 1.03 9 0.613 2.262 Reader2 10 1.000 1.83 9 1.733 2.262 1 VS. 2 10 0.900 1.35 9 2.110 2.262 35

Table 3. Mean ( ± 95% confidence intervals) dorsal spine band diameters (mm) for Makaira nigricans. Calculated cumulative mean band diameter values used for "replacement" of bands in which these bands have been destroyed. N = number.(Refer also to Figure 16.)

Range Band# Mean + 95% C.I. Low High N I 1 2.40 ± .118 2.04 2.60 11

II 1 3.21 ± .188 2.81 3.83 11 2 3.23±.118 2.75 3.83 12 Cum. 3.22± .123 2.75 3.83 23

III 1 4.30±.284 3.52 4.90 11 2 4.03 ±.090 3.67 5.20 12 Cum. 4.12 ± .096 3.52 5.20 23

IV 1 5.24 ± .274 4.44 5.81 11 2 5.12 ± .120 4.59 5.97 12 Cum. 5.16±.099 4.44 5.97 23

v 1 6.31 ± .387 5.14 7.34 11 2 6.09 ± .200 5.20 7.19 12 Cum. 6.15 ± .157 5.14 7.34 23

VI 1 7.40 ± .484 6.43 8.72 10 2 7.08 ± .321 5.81 8.42 12 Cum. 7.15 ± .232 5.81 8.72 22

VII 1 8.57 ± .517 7.57 9.79 9 2 7.97 ± .363 6.27 9.49 11 Cum. 8.09 ± .267 6.27 9.79 20 36

Table 4. Mean ( ± 95% confidence intervals) anal spine band diameters (mm) for Makaira nigricans. Calculated cumulative mean band diameter values used for "replacement" of bands in which these bands have been destroyed. (Refer also to Figure 20).

Range Band# Mean+ 95% C. I. Low High N

I 1 2.41 ± .096 1.99 2.75 19

II 1 3.29 ± .107 2.75 3.83 19 2 3.31 ± .084 2.75 3.83 23 Cum. 3.30± .071 2.75 3.83 42

III 1 4.12 ± .105 3.67 4.44 19 2 4.11 ± .084 3.52 5.05 23 Cum. 4.14±.059 3.52 5.05 42

IV 1 5.00±.135 4.44 5.66 18 2 4.96±.090 4.28 5.81 23 Cum. 4.97 ± .068 4.28 5.81 41

v 1 5.61 ± .203 5.20 6.43 12 2 5.69 ± .147 4.90 7.04 23 Cum. 5.69 ± .106 4.90 7.04 35

VI 1 6.12±.120 5.51 6.58 9 2 6.45 ± .170 5.20 8.11 23 Cum. 6.41 ± .127 5.20 8.11 32

VII 1 6.66± .205 6.12 7.04 8 2 7.08 ± .437 5.66 9.03 20 Cum. 7.01±.167 5.66 9.03 28

VIII 1 7.11 ± .275 6.73 7.65 6 2 7.58 ± .279 5.81 9.33 13 Cum. 7.46 ± .198 5.81 9.33 19

IX 1 7.61 ± .259 7.34 7.96 4 2 8.22± .446 6.12 9.95 6 Cum. 7.98 ± .338 6.12 9.95 10

X 1 2 8.52± .663 6.43 11.2 6 Cum. 8.52 ± .663 6.43 11.2 6

XI 1 2 9.42 ± .911 7.80 12.2 4 Cum. 9.42 ± .911 7.80 12.2 4 37

Table 5. Calculated mean length (EFL) at age as determined by otoliths, dorsal spines, and anal spines, for male Makaira nigricans. [Mean length (em) with 95% confidence intervals. N =number of individuals in each age category.]

Age Otoliths N Dorsal Sj]ines N Anal SJlines N 0 1 2 3 95.4 1 4 131.3±11.5 6 5 174.9±33.1 2 155.9±5.68 2 6 162.7±13.7 4 164.2±22.0 2 176.6±6.66 10 7 176.3±7.17 4 171.1±6.08 6 176.2±3.14 25 8 168.6±8.93 7 178.0±5.29 14 178.7±3.14 36 9 181.8±7.99 9 181.8±5.19 20 185.9±3.53 37 10 176.0±14.5 9 185.7±3.16 15 185.9±3.53 17 11 186.4±8.48 6 192.1±14.9 6 190.7±7.74 7 12 196.4±5.10 2 189.6±14.9 2 184.5±10.8 2 13 195.9±4.76 4 182.2±16.1 2 14 194.2±11.1 4 15 16 184.0±9.80 2 17 18 193.8 1 38

Table 6. Calculated mean length (EFL) at age as determined by otoliths, dorsal spines, and anal spines, for female Makaira nigricans. [Mean length (em) with 95% confidence intervals. N =number of individuals in each age category.]

Age Otoliths N Dorsal Spines N Anal Spines N 0 1 2 3 4 151.4 1 5 141.0 1 141.0 1 141.0 1 6 193.1±28.8 2 178.4 1 7 193.6 1 206.7 1 8 208.1±1.37 2 203.9±10.3 3 9 230.5±18.0 3 197.0 1 191.7±18.7 3 10 223.0±24.9 5 191.6 1 211.8±16.5 5 11 216.4±15.3 3 206.0±7.83 3 217.3±22.6 5 12 247.1±28.4 3 217.8±20.1 5 216.3±17:8 4 13 218.7±28.0 3 208.8 1 227.3±16.8 5 14 237.6±10.2 5 297.0 1 248.9±31.4 6 15 251.5±20.6 6 233.4±14.0 5 234.7±14.8 5 16 255.5±14.2 6 240.5±10.5 5 223.4±44.5 2 17 270.8 1 246.2 1 252.4 1 18 256.9 1 275.5±48.0 2 262.8±2.88 2 19 269.2±35.7 2 20 287.4 1 21 22 398.8 1 23 323.5 1 24 299.2 1 299.2 1 25 290.8 ± 16.5 2 26 361.2 + 73.8 2 323.5 1 39

Table 7. Analysis of corresponding hardpan age assignments by paired T-test. N =number, Mean D =calculated mean difference in age estimates between the two hardpans, SD = standard deviation of calculated Mean D, df = degrees of freedom, t calc. =calculated t value, t crit. =critical value oft at a 0.05 level of significance.

Source MeanD .@ df t calc. tcrit.

Otoliths vs. 39 0.777 2.62 38 1.781 2.201 Dorsal Spines

Otoliths vs. 53 -0.137 2.55 52 0.384 2.021 Anal Spines

Dorsal vs. 94 -0.467 2.41 93 1.860 2.000 Anal Spines 40

Table 8. Calculated mean lengths (EFL) at age as determined by anal spine bands counts for male Makaira nigricans. S.E. =standard error, N =number. (Refer also to Figure 27 .).

Range Age EFLCcml+95% C I. S.E. Min. Max. N

0 1 2 3 95.4 1 4 131.6 ± 11.47 5.86 119.0 158.0 6 5 155.9 ± 5.87 2.90 153.0 158.8 2 6 176.6± 6.73 3.43 159.6 196.5 10 7 176.2± 3.24 1.65 161.0 191.8 25 8 178.7 ± 3.07 1.57 159.8 197.6 36 9 185.5 ± 3.59 1.83 160.2 222.0 38 10 185.9 ± 7.45 1.81 169.0 198.4 17 11 190.3 ± 6.76 3.45 174.0 203.6 8 12 184.5 ± 10.8 5.50 179.0 190.0 2 13 182.2± 16.1 8.20 174.0 190.4 2 14 15 16 17 18 193.8 1 41

Table 9. Calculated mean lengths (EFL) at age as determined by anal spine bands counts for female Makaira nigricans. S.E. =standard error, N =number. (Refer also to Figure 28.).

Range ~ EFLCcm)+95% C.I. s.E. Min. Max. N 0 1 2 3 4 5 141.0 1 6 178.4 1 7 206.7 1 8 203.9 ± 10.31 5.26 197.0 214.2 3 9 191.7 ± 18.74 9.56 174.8 207.9 3 10 211.8 ± 16.49 8.42 191.6 236.7 5 11 217.3 ± 22.60 11.54 174.6 240.0 5 12 216.3 ± 17.80 9.08 199.4 241.4 4 13 227.3 ± 16.78 8.56 204.2 249.2 5 14 248.9 ± 31.44 16.04 208.8 300.0 6 15 234.7 ± 14.83 7.57 208.8 248.8 5 16 223.5 ± 44.49 22.70 200.8 246.2 2 17 252.4 1 18 262.8 ± 2.88 1.47 261.4 264.2 2 19 269.2 ± 35.67 18.20 251.0 287.4 2 20 21 22 23 24 25 290.8 ± 16.46 8.40 282.4 299.2 2 26 361.2 ± 73.79 37.65 323.5 398.8 2 42

Figure 1. Measurements and skeletal hardparts taken for Makaira nigricans [fish outline adapted from Mather (1976)]. Dorsal Fin Spines

Otolit

!E'---EYE-ANAL FIN LENGTH---;.j

~--EYE -FORK LENGTH ------>J

~------L 0 WE R JAW-FORK LENGTH ------7{

~------~OTAL FORK LENGTH------~ 44

Figure 2. Left sagitta from a 220 kg blue marlin, Makaira nigricans,which shows morphological features (from Radtke, 1981; drawn by Reggie Kawamoto). 45 46

Figure 3. Caudal vertebra number 23 from Makaira nigricans which shows morphological features and orientation of the conically shaped centra. 47

w ~· z -ll. -ll. (/) (/) Ill: ::I! -I ~ giii!

:5 c. Cll Q Cll r:: 8 E .5 ar:: 48

Figure 4. Second anal spine from Makaira nigricans which shows basic morpho­ logical features and area of cross sectioning for age determination (Spine outline adapted from Prince et al. 1984). 49

SPINE LENGTH

SPINE

AREA OF CROSS SECTION

Condyle Base 50

Figure 5. Size frequencies for male (n=213) and female (n=106) Makaira nigricans sampled from four billfishing tournaments held during 1982, 1983, and 1984 in Kailua-Kana, Hawaii. Males are represented by open cells, females by hatch-marked cells. 51

0 "'

~

Q ~ "' 0 n 0 "'N - c ,..., ~ c• "' E ~ .. 0 .. (;I"' ~ (;I E J: "' 1- :::1: ...."' (!) z w D ~ 0 -1 "' :.:: "' a: 0 u. wI >- w 0 0 "'

0 '-----rt:t' ~

0 -0 z 52

Figure 6. Relationship between otolith weight (OW in mg) and EFL (em) for male and female Makaira nigricans.

Functional mean regression equation for males: EFL = 6.724 (OW)+ 163.46, n =54, ? = 0.596, P< 0.001 for two-tailed test. Functional mean regression equation for females: EFL = 7.705 (OW)+ 200.87, n = 43, ? = 0.450, P< 0.001 for two-tailed test. 53

.,... .,... 0

0 0 01 0 - 0 "'"E -.... :::1: 0 0 0 co~ -w 0 s: 0 • IOJ: oo .... 0 • 0 • -...J • 0 Oo • "'!!"I- oo • 0 ;t o8 • 0 • cP 0 • ., O'o • 0 0'1 71.. :- 71.. e • :::e .. •• . ': •o ""r • • 0 • • 0 ••• Q • •

0 0 0 0 0 0 0 0 0 10 0 1.0 0 1.0 0 10 ('f) ('f) C\1 C\1 ,... ,.. (WO) HJ.E>N31 )U:::IO.:I-3A3 54

Fie:ure 7a. Left sagitta from a 189.0 em EFL (80.1 kg) male Makaira nigricans. R =rostrum; A = antirostrum; C =core region; o =calcium carbonate overlayer obscuring ridges; arrows indicate rostral ridges quantified for age estimation (Bar= 0.5 mm).

Figure 7b. Left sagitta from a 299.2 em EFL (496.7 kg) female M. nigricans. R =rostrum; o =calcium carbonate overlayer obscuring ridges; arrows indicate rostral ridges quantified for age estimation (Bar= 0.5 mm). 55 57 59 60

Figure lOa. Relationship between centrum cone depth (CCD) and EFL for male Makaira nirncans. Functional mean regression equation: EFL = 1.288 (CCD) + 143.99, n = 123, r-2 =0.155, P< 0.001 for two-tailed test.

Figure lOb. Relationship between centrum cone depth (CCD) and EFL for female Makaira nigricans. Functional mean regression equation: EFL =3.784 (CCD) + 100.10, n =77, r-2 =0.468, P< 0.001 for two-tailed test. 61

35il

Males 3ilil

~ E (j 25il ~ _c ~ 2ilil ..• c m .. ·.. :·.<:~/.~ .' '· ...,. - ....J .. ..>::: 15il - L 0 LL I ,....m lilil w

5il

il il lil 2il 3il 4il 5il

35il

females 3ilil

~ E (j 25il ~ _c ~ 2ilil c m ....J ..>::: L 15il 0 LL I ,....m lilil w

5il

il il lil 2il 3il 40 50 Centrum Cone Depth Cmm) 62

Figure 11. Longitudinal cross section of the anterior centrum from the 23rd vertebrae from a 156.0 em EFL (50.3 kg) male M. nigricans. a= anterior end; d =dorsal; v =ventral; f = centrum focus. Arrow indicates area of change in growth (Bar= 5 mm). 63 64

Figure 12a. Relationship between dorsal spine width (DW) and EFL for male Makaira ni gricans. Functional mean regression equation: EFL = 4.084 (DW) + 105.13, n = 65, f2 = 0.386, P< 0.001 for two-tailed test.

Figure 12b. Relationship between dorsal spine width (DW) and EFL for female Makaira ni!!ricans. Functional mean rer.ession equation: EFL = 6.152 (DW) + 62.69, n = 28, r = 0.805, P< 0.001 for two-tailed test. 65

T I I I I I T T I

41ilfil - Males - ~ E 0 ~ 31ilfil - - ..r:_,_, r:n c - - m ---' ->::: 21ilfil 1-- - L . 0 LL. ~... I 1-- - m >-.. w 11ilfil 1-- -

1-- -

liJ I I I I I I I I I I liJ 5 11il 15 21il 25 31il 35 4fil 45 51il 55

41ilfil females

fiJL---L---L-~L-~L-~--~--~--~--~--~---J liJ 5 11il 15 21il 25 35 45 51il 55 Dorsal Spine Width (ot Si.L) 66

Figure 13a. Thin tranverse cross section of the 6th dorsal spine from a 162.6 em EFL (52.4 kg) male M. nigricans as viewed by a binocular dissecting microscope at 63X magnification with transmitted light. Arrows indicate bands quantified for age estimation; F =spine focus; M =matrix (Bar= 2mm).

Figure 13b. Thin tranverse cross section of the 6th dorsal spine from a 162.6 em EFL (52.4 kg) male M. nigricans (same as 13a) as viewed by a binocular dissecting microscope at 63X magnification with reflected light and a black background. Arrows indicate same bands quantified for age estimation. (Bar= 2 rom). 67 68

Figure 14. Thin tranverse cross section of the 6th dorsal spine from a 222.0 em EFL (138.7 kg) male M. nigricans as viewed by a binocular dissecting microscope at 63X magnification with reflected light and a black back­ ground. Arrows indicate bands quantified for age estimation; F =focus; M =matrix (Bar= 3 mm).

Figure 15. Thin tranverse cross section of the 6th dorsal spine from a 282.4 em EFL (419.1 kg) female M. nigricans as viewed by a binocular dissecting microscope at 63X magnification with transmitted light. Arrows indicate growth bands; M =matrix (Bar= 3 mm). 69 70

Figure 16. Mean (and 95% confidence interval) dorsal spine band measurements for Makaira nigricans (Refer also to Table 3). Wide horizontal lines represent mean band radius values. Vertical lines terminated by narrow horizontal lines represent 95% confidence intervals. 71

... 1+1 Ll'l .cCl.l E z::J "0 HI '

N

,...

0 CQ M ,... 0 72

Figure 17a. Relationship between anal spine width (AW) and EFL for male Makaira nigricans. Functional mean regression equation: EFL = 5.671(A W) + 93.22, n = 144, r' = 0.445, P< 0.001 for two-tailed test.

Figure 17b. Relationship between anal spine width (A W) and EFL for female Makaira nigricans. Functional mean re:f:ession equation: EFL = 6.867(AW) + 77.33, n = 46, r = 0.785, P< 0.001 for two-tailed test. 73

350

Males 31illil

~ E 251il 0 ~ ...c. &, 21illil c Ill _J ..>:: 151il 0'- LL. I ~ 11illil w

5[ll

Iii Iii 5 11il 15 21il 25 30 35

350

31illil females

~ E 251il 0 ~ ...c. &, 21illil c Ill _J ..>:: '- 151il 0 LL. I ~ 11illil w

51il

Iii Iii 5 11il 15 21il 25 31il 35 Anal Spine Width (at 107.U 74

Figure 18. Thin tranverse cross section of the 2nd anal spine from a 208.8 em EFL (138.3 kg) female M. nigricans as viewed by a binocular dissecting microscope at 63X magnification with transmitted light. Arrows indicate growth bands; F =focus; M =matrix (Bar= 3 mm).

Figure 19. Thin tranverse cross section of the 2nd anal spine from a 95.4 em EFL (19.1 kg) female M. nigricans as viewed by a binocular dissecting microscope at 63X magnification with transmitted light. Arrows indicate growth bands; F =focus; (Bar= 2 mm). 75 76

Figure 20. Mean (and 95% confidence interval) anal spine band measurements for Makaira nigricans (Refer also to Table 4). Wide horizontal lines represent mean band radius values. V erticallines terminated by narrow horizontal lines represent 95% confidence intervals. 77

T'" T'"

0 T'"

en

co ... Q) .0 E 1+1 r-. :::I :z '"0s:: CD m III Q) s::

It) ·-c. HI en «i s:: '¢ <

Ill

HI

0 T'" 0 0 T'" T'" 78

Figure 21. Relationship between total estimated dorsal spine band counts (DC) and otolith ridge counts (OC) for Makaira nirncans. Functional regression equation: OC = 0.826(DC) + 1.232, n = 39, r = 0.861, P< 0.001 for two-tailed test. Slope does not significantly differ from 1 (P< 0.05). 79

30 28 26 24 22 r: -:::l 20 0 (.) 18 (I) 0') :s! 16 a: ..r: 14 =0 12 0- 10 8 6 4 2

0 I I I I I I I I I I 0 2 4 6 8 1 0 1 2 1 4 1 6 1 8 2 0 2 2 2 4 2 6 2·8 3 0 Dorsal Spine Band Count 80

Figure 22. Relationship between total estimated anal spine band counts (A C) and otolith ridge counts (OC) for Makaira nigricans. Functional regression equation: OC = 0.765(AC) + 2.591, n =53, r = 0.820, P< 0.001 for two-tailed test. Slope does not significantly differ from 1 (P< 0.01). 81

30 28 26 24 22

l: 20 -::::1 u0 18 Q) C) 16 't:) a: 14 .c .:= 12 0 0- 10 8 6 4 2 0 0 2 4 6 8 10 1214 1618 20 22 24 26 28 30 Anai Spine Band Count 82

Figure 23. Relationship between total estimated anal spine band counts (A C) and dorsal spine band counts (DC) for Malcaira nigricans. Functional regression equation: DC= 0.905(AC) + 1.435, n = 92, r = 0.950, P< 0.001 for two-tailed test. Slope does not significantly differ from 1 (P< 0.01). 83

30 28 26 24 r:: 22 -:I 0 u 20 "C r:: Cl3 18 CD Q) 16 .5 enc.. 14 12 caen ... 10 c0 8 6 4 2 0 0 2 1 6 8 10 12 14 16 18 20 22 24 26 28 30 Anal Spine Band Count 84

Figure 24. Relationship between total estimated otolith ridge counts (OC) and vertebral increment counts (VC) for Makaira nig;ricans. Functional regression equation: VC = 15.17 (OC) + 357.5, n = 90, > = 0.510, P< 0.001 for two-tailed test. 85

900

BOD • • • -c • • :I 700 0 (.) -c Ql 600 E ...Ql 0 .5 500 f! .0 Ql 400 t: Ql > 300 • • •

200 0 2 4 6 8 10 12 14 16 1 8· 20 22 Otolith Ridge Count 86

Figure 25. Relationship between total estimated dorsal spine band counts (DC) and vertebral increment counts (VC) for Makaira nigricans. Functional regression equation: VC = 18.172(DC) + 318.02, n = 45, r= 0.760, P< 0.001 for two-tailed test. 87

700 c: -:I 0 (.) c: 600 -Q) E ::! CJ .5 500 iii... ..Q Q) t:: Q) > 400

0 2 4 6 a 10 12 14 16 1a 20 22 24 26 2a ao Dorsal Spine Band Count 88

Figure 26. Relationship between total estimated anal spine band counts (A C) and vertebral increment counts (VC) for Makaira nigricans. Functional regression equation: VC = 17.587(AC) + 323.0, n = 100, r= 0.660, P< 0.001 for two-tailed test. 89

BOO • • 700 • • c • -::1 • 0 • () • • c 600 • • -Cll • • • E • ...Cll • • • (,) • • I • • .5 500 • • • • • -m I • • ... I J:l • Cll • • t: • • • Cll 400 • • > • • • • • • • 300 0 2 4 6 8 10 12 1 4 1 6 1 8 20 22 24 26 28 30 Anal Spine Band Count 90

Figure 27. Mean EFL at age for male Makaira ni!!ricans based on anal spine age estimates. Wide horizontal lines represent mean EFL values. Vertical lines terminated by narrow horizontal lines represent 95% confidence intervals (n = 148)(Refer also to Table 8). 250

Males

200 -E I I + 0 I -J:: I -s::0'1 150 Q) ..J ..::t:... f 0 1..1.. 100 + Q)' w>­

50

N = 1 6 2 1 0 25 36 38 17 8 2 2 1

0 2 4 6 8 10 12 14 16 18 Anal Spine Band Count 92

Figure 28. Mean EFL at age for male Makaira ni rncans based on anal spine age estimates. Wide horizontal lines represent mean EFL values. Vertical lines terminated by narrow horizontal lines represent 95% confidence intervals (n = 48)(Refer also to Table 9). 450 Females 400

350 -E 0 -J: 300 -0'1 I t:: _.Q) 250 .. I .!11::.... I 200 + II d u.0 I I I I + Q) >- 150 w +

100

50

N= 1 1 1 3 3 5 5 4 5 6 5 2 1 2 2 2 2 0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 Anal Spine Band Count

<0 w 94

Appendix la. Relationship between Eye Fork Length (EFL in ern) and Total Fork Length (TFL in ern) for male Makaira nigricans. Functional mean regression equation: TFL = 1.299(EFL) + 4.105; N = 120, r 2 = 0.929, P< 0.001 for two-tailed test.

Appendix lb. Relationship between EFL (ern) and TFL (ern) for female M. nicricans. Functional mean regression equation: TFL = 1.353(EFL)- 4.836, N =52, r 2 = 0.987, P< 0.001 for two-tailed test. 95

61illil

551il Males 51illil

~ E 451il 0 ~ 41illil L +' Ulc 351!1 _j"' 31illil _y L 0 251il '-'--

~ 21illil 0 +' 0 151il 1- 11illil

51il

lil lil 51il 11illil 151il 21illil 250 31il0 351il 41illil -150

61il111

55111 Females 5111111

~ E 45111 0 ~ 41110 J: ~c 35111 _j"' 31illil X L 0 251'1 LL..

~ 21il111 0 +' 0 151'1 1- 1111111

51'1 lil 111 5111 1111111 151'1 21il111 251'1 31il111 351il 41111'1 45111 Eye to Fork Length Com) 96

Appendix 2a. Relationship between EFL (em) and Lower Jaw to Fork Length (LJFL in em) for male M. nigricans. Functional mean regression equation: LJFL = 1.080(EFL) + 11.780, N = 120, r 2 = 0.975, P< 0.001 for two-tailed test.

Appendix 2b. Relationship between EFL (em) and LJFL (em) for female M. nicricans. Functional mean regression equation: LJFL = l.094(EFL) + 9.512, N =52, r 2 = 0.997, P< 0.001 for two-tailed test. 97

500 450 Males

~ E u ~ 400

..!: "" 350 Clc ID _J 300

-"'L 0 250 LL. 0 200 "";o .,0 150 L ID ;o Hlil 0 _J 50 0 0 50 100 15il 200 250 300 350 400 150 500

450 females ~ E u ~ 400

..!: "" 350 cCl ID _J 300 -"' L 0 250 LL. 0 200 "";o .,0 150 L ID ;o 100 0 _J 50

0 0 50 100 150 200 250 300 350 40~ 45~ Eye to Fork Length (em) .98

Appendix 3a. Relationship between EFL (em) and Eye to Anal Fin (EAFL in em) for male M. nigricans. Functional mean regression equation: EAFL = 0.775(EFL)- 6.615, N = 115, r 2 = 0.963, P< 0.001 for two-tailed test.

Appendix 3b. Relationship between EFL (em) and EAFL (em) for female M. nigricans. Functional mean regression equation: EAFL = 0.736(EFL) + 0.645, N =50, r 2 = 0.980, P< 0 ..001 for two-tailed test. 99

250

Males

200

~ E 0 ~ c -~ 150 LJ._

~ 0 c < 100 _,_,0 m >-. w 50

0 0 50 100 150 20H 250 300 350

250'

females 200 ..

~ E 0 ~ c -~ 150 LJ._

~ 0 c < 100 _,_,0 m >-. w 50

50 100 150 211llil 250 300 350 Eye to Fork length (em) 100

Appendix 4a. Relationship between TFL (em) and LJFL (em) for male M. nigricans. Functional mean regression equation: LJFL = 0.786(TFL) + 18.951, N = 119, r 2 = 0.940, P< 0.001 for two-tailed test.

Appendix 4b. Relationship between TFL (em) and LJFL (em) for female M. nimcans. Functional mean regression equation: LJFL = 0.800(TFL) + 15.964, N =52, r 2 = 0.989, P< 0.001 for two-tailed test. 1 01

500

450 Males ~ E 0 ~ 400 ...c ..., 350 01c (I) _J 300

-"'{. 0 250 LL. ...,0 ., 200 ...... ,0 !50

{. .,(I) 100 0 _J 50 0 0 50 100 !50 200 250 300 350 400 450 500 550 6110 500

450 Fer,;tales

~ E 0 ~ 400 -:5 350 01c (I) _J 300

-"'{. 0 25~ LL. ...,0 ., 200 ...... ,0 !50

{. .,(I) 100 0 _J 50 0 0 50 100 !50 200 250 300 350 400 450 500 550 6110 Total Fork Length Ccm) 102

Appendix Sa. Relationship between TFL (em) and EAFL (em) for male M. nigricans. Functional mean regression equation: EAFL = 0.554(TFL) + 0.977, N = 114, r 2 = 0.916, P< 0.001 for two-tailed test.

Appendix 5b. Relationship between 1FL (em) and EAFL (em) for female M. nimcans. Functional mean regression equation: EAFL = 0.533(TFL) + 6.563, N =51, r 2 = 0.962, P< 0.001 for two-tailed test. 103

250

Males

200

~ E u ~ c ·~ 151il LL ...... IJc < llil0 ....0 -- 51il

lil lil 51il llillil !50 21illil 251il 31illil 350 41'llil 451il

251il

Females

21illil

~ E u ~ c . ·. ·~ 151il LL ...... IJc < llillil ....0 -- 51il

51il llillil 151il 21illil 251il 31illil 351il 41illil 451il Total Fork Length (em) 104

Appendix 6a. Relationship between LJFL (em) and EAFL (em) for male M. nimcans. Functional mean regression equation: EAFL =0.670(LJFL)- 11.301, N = 115, r 2 =0.955, P< 0.001 for two-tailed test.

AppendLx 6b. Relationship between LJFL (em) and EAFL (em) for female M. nimcans. Functional mean regression equation: EAFL =0.67l(LJFL)- 5.418, N =50, r 2 =0.979, P< 0.001 for two-tailed test. 105

250

Males

200

~ E 0 ~ c ·~ !50 LL.

~ 0 c < 100 ..,0 !D >-. w 50

0 0 50 1011 !50 200 251'l 300 351! 401'1 250

females 200

~ E 0 ~ .· c ·~ !50 LL.

~ 0 c < 100 ..,0 !D >-. w 50

0~~~--~~~--~-L~L-~-L--L-~~--~~~ 0 50 !50 250 350 400 Lower J ow to Fark Length (ern) 106

Appendix 7a. Relationship between EFL (em) and Weight (Win kg) for male M. ni gricans. Functional mean regression equation:

W = 4.354 x e<0·016 x EFLJ, N = 213, r 2 = 0.884.

Appendix 7b. Relationship between EFL (em) and W (kg) for female M. nigricans. Functional mean regression equation:

W = 7.129 x e(O.Ol3 x EFLJ, N = 105, r 2 = 0.872. 107

800 750 Males 700 650 600 550 ~m 500 ..>::: ~ 450 +> 400 ...c .~ 350 3o'" 300 250 200 150 100 50 0 0 50 100 150 200 250 300 350 400 800 750 700 Females 650 600 550 ~m 500 . ..>::: ~ 450 +> 400 ...c .~ 350 ~'" 300 250 ..... 200 ·. .. 150 • • •• • ... & ·.: •• 100 ·' 50 0 0 50 100 !50 200 250 300 350 400 Eye-Fork Length (em)