AGE, GROWTH, AND SEXUAL MATURITY OF THE DEEPSEA SKATE, BATHYRAJA

ABYSSICOLA

A Thesis

Presented to the Faculty of

Alaska Pacific University

In Partial Fulfillment of the Requirements

For the Degree of

Master of Science in Environmental Science

by

Cameron Murray Provost

April 2016

Pro Q u est Nu m b er: 10104548

All rig hts reserv e d

INF O RM ATI O N T O ALL USERS Th e q u a lity of this re pro d u ctio n is d e p e n d e nt u p o n th e q u a lity of th e c o p y su b mitt e d.

In th e unlik e ly e v e nt th a t th e a uth or did n ot se n d a c o m ple t e m a nuscript a n d th ere are missin g p a g es, th ese will b e n ot e d. Also, if m a t eria l h a d to b e re m o v e d, a n ot e will in dic a t e th e d e le tio n.

Pro Q u est 10104548

Pu blish e d b y Pro Q u est LL C (2016). C o p yrig ht of th e Dissert a tio n is h e ld b y th e A uth or.

All rig hts reserv e d. This w ork is prot e ct e d a g a inst un a uth orize d c o p yin g un d er Title 17, Unit e d St a t es C o d e Microform Editio n © Pro Q u est LL C .

Pro Q u est LL C . 789 E ast Eise nh o w er P arkw a y P. O . Box 1346 A nn Arb or, MI 48106 - 1346 ACKNOWLEDGEMENTS

This project would not have been possible without the cooperation and dedication of several individuals. I would like to thank my committee chair and advisor Bradley P. Harris for his tireless support and guidance throughout the entirety of this project. I would also like to thank my committee: David A. Ebert, Kenneth J. Goldman, and Cindy A. Tribuzio for their invaluable assistance and mentoring and Lisa J. Natanson for teaching and aiding me with the histological aspects of this project. Sarah Webster, Nathan Wolf, Aileen Nimick, T. Scott Smeltz, Kelsey

James, and James Knuckey your help with this project was greatly appreciated. Last but not least, thank you At-Sea Processors Association for funding my project and education at Alaska

Pacific University as part of the Fisheries, Aquatic Science & Technology Laboratory.

ii

ABSTRACT

Research into the age, growth, and reproductive characteristics of chondrichthyan fishes has increased substantially over the past couple of decades. This study set out to estimate age deepsea skate, Bathyraja abyssicola using vertebral centra and caudal thorns, estimate length at age, and determine length at maturity. Sixty-three specimens of B. abyssicola (n=29 males; n=34 females) were taken on National Marine Fisheries Service bottom trawl surveys between 2001 and 2012. Information derived and structures collected from these samples included sex, maturity class, total length, caudal thorns and, vertebrae. Ageing methods attempted include histology and gross sectioning (vertebral centra) and surface staining (caudal thorns). Moderate success with centra sectioned using the histological method allowed some inference to be made into life history characteristics. Deepsea skates appear to have slow average growth (26 mm yr-1

±5.41, 95% c.i.) and mature at a large size (males: TL50 = 1175.4 mm, females: TL50 = 1267.3 mm). Band pair counts were not validated as true ages. Males from which growth bands could be enumerated were smaller (� = 10, �̅ = 718 mm, SD = 209 mm) on average than that for females (n = 7, �̅ = 990 mm, SD = 319 mm). This study provides the first attempt to assess abyssicola age, growth rate, and sexual maturity traits; information needed for informed skate management.

iii TABLE OF CONTENTS

ACKNOWLEDGEMENTS...... ii

ABSTRACT...... iii

TABLE OF CONTENTS...... iv

LIST OF TABLES...... v

LIST OF FIGURES...... vi

GENERAL INTRODUCTION...... 1

ASSESSMENT OF AGE, GROWTH AND MATURITY OF THE DEEPSEA SKATE,

BATHYRAJA ABYSSICOLA

1.1 INTRODUCTION...... 6

1.2 METHODS…………………………………………………………………………………...8

1.3 RESULTS………………………………………………………………………………...…13

1.4 DISCUSSION………………………………………………………………………………16

REFERENCES...... 22

TABLES...... 28

FIGURES...... 31

APPENDICES...... 39

iv LIST OF TABLES

Table 1…………………………………………………………………………………………...28 Procedure for histology embedding.

Table 2……………………………………………………………………………………...……29 Procedure for histology staining.

Table 3……………………………………………………………………………………...……30 Maturity classes and definitions.

v LIST OF FIGURES

Figure 1…………………………………………………………………………………………..31 Vertebral centra and caudal thorn sampling areas.

Figure 2…………………………………………………………………………………………..31 Photographs of gross sectioned (A) and histological sectioned (B) vertebral centra and alizarin red stained caudal thorn (C) from Bathyraja abyssicola.

Figure 3…………………………………………………………………………………………..32 Bathyraja abyssicola total length (mm) at band pair count as determined by histology with fit linear regression line.

Figure 4…………………………………………………………………………………………..33 Female Bathyraja abyssicola total length (mm) at band pair count as determined by histology with fit linear regression line.

Figure 5…………………………………………………………………………………………..34 Male Bathyraja abyssicola total length (mm) at band pair count as determined by histology with fit linear regression line.

Figure 6…………………………………………………………………………………………..35 Bathyraja abyssicola total length (mm) at band pair count as determined by caudal thorns with fit linear regression line.

Figure 7…………………………………………………………………………………………..36 Bathyraja abyssicola total length (mm) at band pair count as determined by histology and caudal thorns.

vi Figure 8…………………………………………………………………………………………37 Bathyraja abyssicola total length (mm) at band pair count as determined by histology separated by maturity class for males and females.

Figure 9…………………………………………………………………………………………38 Estimated probability of maturity at size based on an ogive for male and female samples of B. abyssicola.

vii GENERAL INTRODUCTION

The class Chondrichthyes (derived from the Greek prefix khondros meaning “cartilage” and suffix ikhthus meaning “fish”) is a species diverse group of fishes with simplified internal cartilaginous skeletons, lacking true bone (Ebert 2003, Klimley 2013). Other distinguishing characteristics of this class include fins without bony rays, true upper and lower jaws, nostrils on the underside of the head, and transverse rows of teeth or fused tooth plates that are constantly replaced from inside the mouth (Ebert 2003). Cartilaginous fishes also lack bony plates on their heads, have placoid scales (toothlike dermal denticles) and fertilization takes place internally

(Ebert 2003).

Two subclasses comprise the class Chondrichthyes, the small subclass Holocephali

(chimaeras) and the large subclass Elasmobranchii (sharks and rays) (Ebert 2003, Klimley 2013).

The Holocephali have a single gill opening on each side of their heads and are given the common name chimaeras because of their unique body shape, which vaguely resembles the mythic Greek monster of the same name (Klimley 2013). The subclass Elasmobranchii is a large taxonomic group comprised of the cylindrical-bodied sharks and flat-bodied rays (Klimley 2013). These species have multiple gill openings on each side of their heads numbering from five to seven

(Klimley 2013).

Over the past few decades, the sensitivity of global chondrichthyan populations to fishing mortality has been an area of focus because they typically possess life history characteristics such as slow growth rates, late maturation, and low fecundity which make them particularly vulnerable to exploitation (Holden 1974, Brander 1981, Dulvy et al. 2000, Stevens et al. 2000,

Dulvy and Reynolds 2002). The order Rajiformes (skates) are a species-rich, but relatively undiverse group of batoids receiving increased attention as once common species are becoming

1 increasingly scarce or locally extinct (Brander 1981, Dulvey et al. 2000). In the northwest

Atlantic, the winter skate, Leucoraja ocellata, barndoor skate, Dipturus laevis, and blue skate,

Dipturus batis have all demonstrated population declines due in part to fishing mortality

(Johnson 1979, Brander 1981, Casey and Myers 1998). Research efforts are relatively high for targeted or frequently encountered commercial fishery bycatch species. However, research efforts are generally lacking for non-target and rarely encountered skate species (Winton et al.

2013).

Deeper-dwelling, cooler water species generally have slower growth rates, later maturation and lower productivity leading to increased sensitivity to fishing mortality and risk of overexploitation (Cailliet et al. 2001, Garcia et al. 2008, Simpfendorfer and Kyne 2009).

Additionally, there is strong evidence that suggests intra-species, latitudinal variation in life history characteristics of deep-water skates with expansive geographic distributions (Winton et al. 2013). The expansion of bathybenthic fishing efforts by commercial fisheries (Koslow et al.2000, Haedrich et al. 2001, Roberts 2002, Morato et al. 2006, Simpfendorfer and Kyne 2009) in combination with well documented declines of skate stocks and the life history characteristic trends described for a few deep-water skate species highlight the need for continued research effort towards the description of their life history characteristics.

The collection and analysis of basic life history characteristics like age, growth rate and sexual maturity enables fisheries scientists to incorporate biologically relevant variables into population dynamics models and better predict how a given population may respond to changes in mortality (Ricker 1975, Campana 2001, Cailliet et al. 1986a, b, Cailliet and Goldman 2004,

Cotton et al. 2011). Anatomical structures are used to assess age in many fish species in the form of calcified deposits with potentially varying densities that grow in proportion with an individual

2 (James et al. 2014). Unlike teleost fishes that are aged using banding patterns on otoliths or scales, chondrichthyan fishes have diminutive otoliths and their dermal denticles increase in number, and not size, with age (Applegate 1967, Cailliet et al 1986a, b, Cailliet 1990, Gallagher and Nolan 1999, McFarlane et al. 2002). This requires other structures be used to determine age.

Growth bands suitable for ageing may be found in a variety of hard structures in chondrichthyans including, but not limited to, vertebral centra, caudal thorns, dorsal spines, and neural arches (Cailliet and Goldman 2004). A band pair usually consists of one translucent

(light) band and one opaque (dark) and can sometimes be correlated with summer and winter seasons respectively (Ebert 2003). Unfortunately, these hard parts are sometimes not calcified enough to provide viable information on age (especially in deeper dwelling species) or the deposition of the calcified growth bands within the hard structures may not be related to growth in any tangible way (Cailliet et al. 1986a, b, Cailliet 1990, Natanson and Cailliet 1990,

McFarlane et al. 2002, Cailliet and Goldman 2004, Natanson et al. 2008). If banding is assumed to be annual (one band pair indicating a year of growth), structures must be validated for accurate estimations of age (Campana 2001, Cailliet and Goldman 2004).

Reproductive parameters are important in establishing population estimates and predicting how a population may respond to different stressors (James et al. 2014).

Determination of maturity in chondrichthyans is typically done by visual inspection of the primary and secondary reproductive organs. Development of reproductive organs may be gradual with subtle changes occurring as the animal progresses through life. These developmental changes can be categorized into maturity stages (Ebert 2005).

The basic life history characteristics of age, growth rate and sexual maturity have yet to be described for one of the deepest dwelling skates on record, Bathyraja abyssicola, the deepsea

3 skate. Although this species is abundant at depth and prefers substrates unfavorable for trawling,

(David A. Ebert, Pacific Shark Research Center, Moss Landing Marine Laboratories, pers. comm.) they are occasionally taken as bycatch in National Marine Fisheries Service trawl surveys. This, in conjunction with the general life history characteristics trends of other deep- water species of skate, makes inquiry into B. abyssicola life history relevant. This study provides the first attempt to assess age, growth rate and sexual maturity life history characteristics for B. abyssicola, information vital for informed management.

4

ASSESSMENT OF AGE, GROWTH AND MATURITY OF THE DEEPSEA SKATE, BATHYRAJA ABYSSICOLA

5 1.1 INTRODUCTION

The genus Bathyraja (Rajiformes, Arhynchobatidae) is a diverse group of elasmobranchs with approximately 50 species and at least half residing in the North Pacific Ocean where many are frequently encountered by fisheries as bycatch (Orr 2011). These skates are distinguished by their short, flexible snouts and are typically found in deep water around the outer continental shelf and upper slopes (Ebert 2003). Bathyraja abyssicola is one of many species that is part of this large genus that lacks description of basic life history characteristics, further, it is a deep dwelling species and as a result relatively few are encountered and subsequently available to study. Still, as fishing gear evolves and the capacity to fish deeper grows, the need to study deep- sea organisms, particularly deep-sea chondrichthyans, becomes increasingly relevant. Also, skates that dwell in cooler, deeper waters typically exhibit slower growth rates and later maturation, traits that drastically increase susceptibility to overfishing (Cailliet et al. 2001,

Garcia et al. 2008).

Bathyraja abyssicola have a moderately triangular anterior disc margin, broadly rounded posterior disc margin, and a disc width slightly greater than its length. The dorsal surface of the disc has 1-5 nuchal thorns separate from a row of 21 to 31 continuous median tail thorns and an interdorsal thorn (Zorzi and Anderson 1988, Zorzi and Martin 1994, Ebert 2003). The two dorsal fins are closely set and very similar in size. B. abyssicola are usually a uniform purplish gray coloration dorsally, with occasional dark blotches, and the ventral surface is similar in color with the exception of whitish areas around the mouth and anterior pelvic fins. It is a relatively large species of skate, with females reaching at least 1.6 m in total length (tip of snout to tip of tail in a straight line) and males reaching at least 1.35 m in total length (Zorzi and Anderson 1988, Zorzi and Martin 1994, Ebert 2003).

6 This species ranges from northern Baja California, from off Coronado Island and Cortez

Bank continuing north to the Bering Sea and as far west as Japan. Although considered uncommon, video footage from remotely operated vehicles has shown the skate to be quite abundant at depth (Ebert 2003; D.A. Ebert, pers. comm.). The preferred habitat of these skates appears to be on deepsea cobble and rocky reef substrates (D.A. Ebert, pers. comm.). This species is one of the deepest dwelling known skate species with a depth range from 350 m to over 3,000 m (Zorzi and Anderson 1988, Zorzi and Martin 1994, Ebert 2003). The deepsea skate feeds on benthic invertebrates and teleost fishes. Larger individuals feed on a higher proportion of teleost fishes than smaller individuals that prefer invertebrate prey items (Ebert 2003).

Bathyraja abyssicola, like all other skates, have internal fertilization and are oviparous or egg laying. Females are estimated to mature at about 1.4 m total length and males around 1.1 to

1.2 m total length (Ebert 2003). Egg cases are large and longitudinally striated with an olive green coloration (Ebert and Davis 2007). At birth, B. abyssicola are estimated to be quite large measuring approximately 200 mm in total length with the smallest observed free-swimming specimens measuring 340-360 mm (Ebert 2003, 2009). The lack of basic life history information about B. abyssicola make it important to assess and describe their life history characteristics.

The objectives of this research were to: 1) determine which hard structure (vertebral centra and caudal thorns) and procedure (gross sectioning and histology for vertebral centra and surface reads for caudal thorns) may be suitable for age assessment and if one can be found to then determine length at age, 2) determine trends in total length, age, and sexual maturity, 3) examine mathematical models to estimate the growth rate based on the length at age information.

7 1.2 METHODS

Sample Collection

Bathyraja abyssicola specimens (n = 63) were collected on northeast Pacific Ocean

National Marine Fisheries Service (NMFS) cruise trawls for the West Coast Groundfish Survey off the coast of California, Oregon, and Washington (32º 01’ N, 118 º 02’ W to 47º 49’ N, 125 º

52’ W) between the years of 2001 and 2012. Haul depths ranged from 780 m to 1430 m and all sampling of specimens was opportunistic. Sex, maturity, and total length were assessed and caudal thorns and vertebrae were collected at sea or the samples were shipped frozen, whole to the Pacific Shark Research Center (PSRC), Moss Landing Marine Laboratories (MLML) for later assessment. Caudal thorn samples were taken just in front of the tail insertion (Figure 1). A series of 4-6 vertebral centra were taken from the spinal column above the thoracic cavity

(Figure 1). Excess tissues were removed to facilitate processing. Caudal thorns and vertebrae samples were taken at MLML and frozen for travel and later assessment.

Ageing: Histology

Decalcification

Histology preparation and execution were adapted from the procedures of Natanson et al.

(2007). The vertebral centra were separated and stored in 70% ethanol solution prior to histological preparation. All vertebral centra were placed in histological cassettes. If too large for the cassettes, the centra were cut sagittal keeping the focus intact. Rapid decalcifier (RDO®) solution (Thermo-Fisher Scientific L.L.C.) was constantly stirred using a mixing plate and magnetic stir-bar, and cassettes were placed in solution at set times dependent on size (Natanson et al. 2007). Larger specimens soaked for ~30 minutes, smaller ones for ~20 minutes.

Decalcification of centra was complete when centra felt soft to the touch and no white coloration

8 remained (indicative of calcium dissolution). All centra were returned to fresh 70% ethanol solution for storage after the decalcification process.

Embedding and Sectioning

Samples were transferred to 1000 mL beakers containing 70% ethanol solution in which they were stored, and placed in an autotechnicon, a machine that successively advances samples through several chemical baths over a given time, with nine successively increasing alcohol based solutions. All solutions increased in alcohol concentration, lessening the concentration of distilled water and ethanol and increasing the concentration of tert-butyl alcohol until samples reached the Paraplast Plus® baths (a paraffin wax) (Table 1). Following the various soaks in the autotechnicon, samples were embedded in paraffin wax (Paraplast Plus®) blocks. Embedded samples were sectioned into 50-80 µm sections from the center of the centra using a sledge microtome. Anywhere from 3-8 sections were retained in histological cassettes for staining.

Staining

The vertebral centra sections were placed in 100% xylene baths in order to remove all paraffin wax from each section. Sections were stained with Harris Hematoxylin and moved through several successively increasing glycerin baths until reaching 100% glycerin solution

(Table 2). After soaking, the sections were mounted to slides and sealed using clear nail polish.

Ageing: Gross Sectioning

Vertebral centra were kept frozen until sectioning was performed. Using a Buehler

Isomet Low Speed Saw, a sagittal cut was made, slightly to the side of the centra focus. If a centrum was too small to be cut with the saw, a scalpel blade was used. After initial cuts were made, centra were adhered to slides using Crystal Bond 509 clear adhesive (Aremco Products

Inc.) with the cut side facing down. The adhesive was activated by crushing it into a powder,

9 placing it on a slide, then melting it to a liquid over a hot plate by gradually increasing the temperature (between 71°C -121°C) to avoid slide breakage. Once all centra were mounted the adhesive was allowed to set overnight.

When the adhesive set, excess centrum was cut away (only larger specimens) and sanded down using wet 200 grit sandpaper. For finer tissue removal, wet 400 grit sandpaper was used.

Once at the desired thickness (light easily shines through under a microscope at center of vertebral centrum), wet 600 grit sandpaper was used to polish the sample. Mineral oil was brushed over the top to enhance growth bands.

Ageing: Thorn Staining

Caudal thorns were thawed and cleaned further with warm water and a small brush if there were residual clinging tissues. Initial assessment of the caudal thorns under a microscope indicated there were ridges present, however ridges were difficult to discern. Alizarin red was selected as a stain solution to enhance the ridges on the caudal thorns. The stain solution was mixed at a ratio of 0.25g alizarin red powder to 1mL distilled water.

Each thorn was submerged in the stain solution of alizarin red for one, 30 second soak and then submerged in a distilled water bath briefly to remove excess stain from the thorn.

Thorns were then placed in unsealed labeled whirl-packs and were allowed to air dry before sealing the packs. Thorns were viewed under a microscope using low magnification and varying degrees of transmitted light from the microscope and reflected external lighting. The larger thorns were cut sagittaly through the apex using a razor blade if transmitted light was unable to pass through the entire thorn. Clay was used to position and hold samples when viewing with reflected light.

10 Ageing: Reads and Bias

A Leica Microsystems® M60 Microscope equipped with an IC80 HD camera was used in conjunction with Leica Application Suite (LAS) Version 4.1 software to photograph all centra and caudal thorns. ImageJ® software was then used to assess and, if growth bands were discernable, process images and count growth bands in centra and caudal thorns (Figure 2).

Slight manipulation of the saturation, brightness, and contrast of the images in ImageJ® helped to distinguish growth banding further. Two reads of vertebral centra band pairs and one read of caudal thorn band pairs were made by a single reader using the high resolution images. A contingency table analyzed by a chi-squared test of symmetry (Bowker 1948) was constructed to determine if reader bias existed between vertebral centra reads.

Growth Models

A least squares linear regression was used to model B. abyssicola growth (Figure 3). The regression line was calculated as follows:

� = �� + � where y is the predicted total length value based on a given x (b) with

� ∑�−�(�� − �̅)(�� − �̅) � = � � ∑�−�(�� − �̅)

and

� = �̅ − ��̅

where n is the total number of samples, xi is a given band pair count, �̅ is the mean of all band pair counts, yi is a given total length and �̅ is the mean of all total lengths. The coefficient of determination is defined as:

11 ��� �� = � − ��� where R2 is the coefficient of determination, RSS is the residual sum of squares and TSS is the total sum of squares. Regressions were constructed in Excel® (Microsoft® Office 2016, version

15.19.1).

Maturity

Sexual maturity classes for B. abyssicola specimens used in this study were determined by researchers at the PSRC following the methods of Ebert (2005). Maturity classes included: 1-

Embryo, 2-Juvenile, 3-Adolescent, 4-Adult, 5-Gravid Adult (females only) and were based on the development of reproductive organs. The reproductive morphology criteria for males and females respectively are presented in Table 3.

Maturity determination in male specimens was done by inspection of the external reproductive organs, claspers. These are located on the median edges of the pelvic fins and are tube-like copulatory organs. The claspers of juveniles are small and flexible, but as they mature the claspers become increasingly calcified, harden and elongate, and form articulations with the pelvic fin base (Carrier et al. 2004, Ebert 2005, Winton et al. 2013, James et al. 2014). The testes can also be assessed to determine maturity in male individuals. In individuals that are not sexually mature, the testes are poorly developed and appear as inconspicuous white tissue. When mature, this tissue is a distinct organ and fluctuates in size throughout the year, swelling during the breeding season and shrinking during other parts of the year (Carrier et al. 2004, Ebert 2005).

Increased coiling of the epididymis and testes is also observed in individuals as they mature

(Ebert 2005, Winton et al. 2013, James et al. 2014).

12 Female specimens lack external secondary reproductive structures; ovaries are the most common reproductive structure used for maturity determination. Sexually immature individuals have ovaries that appear as thin granulated tissue. Once mature, females’ ovaries become much larger and often gain a yellow coloration (Carrier et al. 2004, Ebert 2005, Winton et al. 2013,

James et al. 2014). The differentiation of the oviducal gland from the uterus can also be used to assess sexual maturity of females. If there is little or no differentiation, the female is not sexually mature. If differentiation between the oviducal gland and uterus is obvious, the female is sexually mature (Ebert 2005, Winton et al. 2013, James et al. 2014).

Size-at-maturity was estimated for male and female specimens using maturity ogives fit to binomial maturity data (0-Juvenile/Adolescent, 1-Adult/Gravid Adult). The median size (total length) at which 50% of the sampled animals were mature was calculated using a logistic regression model with a logit link function. The regressions were constructed using the “stats” package in the R statistical programming software (R Core Team 2016; version 3.2.3) as:

���� = � + ��� � ��

where TL50 represents the logistic function at 0.5 mature (proportion), I is the intercept coefficient, TLc is the total length coefficient, and TL is the median total length at which 50% of the sampled animals are mature.

1.3 RESULTS

Ageing

Three ageing methods were explored (vertebrae histology and gross sectioning, and caudal thorn surface reads), with the histology and caudal thorn surface read techniques yielding discernable growth bands for 17 of 52 (~38%) specimens. The histological process either did not

13 work or destroyed many of the samples and only associated caudal thorns could be used for comparison. Gross sections failed to yield discernable bands even with the application of mineral oil. Caudal thorns provided readable growth bands with the application of alizarin red as a stain.

The two independent reads that were made for each histological sample with discernable band pairs differed on average by ±2 band pair counts. Bowker’s contingency table chi-square test of symmetry for both reads indicated differences between the reads were due to random error

(X2= 13, p = 0.53, df = 14). Histological sample band counts were reevaluated and an agreed band count was used for further analysis.

Growth Models

Males from which growth bands could be assessed were smaller (� = 10, �̅ =

718 mm, SD = 209 mm) on average than that for females (n = 7, �̅ = 990 mm, SD =

319 mm), however; this difference in size was not significant (n = 17, df = 9, p = 0.10,

Independent Samples T-Test). The inclusion of specimens lacking band pair counts (i.e. vertebra damaged) with length data to this analysis also supported this trend with sampled males remaining smaller (� = 28, �̅ = 878 mm, SD = 298 mm) on average than sampled females

(� = 31, �̅ = 1016 mm, SD = 296 mm). The difference in size between males and females remained not significant with the inclusion of samples lacking band pair counts (n = 59, df = 56, p = 0.08, Independent Samples T-Test).

Length at age data for histological samples and caudal thorn samples were comprised of mostly juveniles and adolescent individuals. Due to the low sample size, the data were linear, and were not fitted well to a typical growth model (i.e. slowed growth rate with increased length and age); as such, a growth rate could not be estimated. A least squares regression was used to

14 examine average growth (in length) per year. Skates sampled grew an average of 26 mm yr-1

(±5.41, 95% c.i.) and vertebrae-derived band counts and total length were strongly associated (R2

= 0.87, Adj. R2 = 0.86, n = 17, p < 0.01) (Figure 3). Similar trends were seen when samples were separated by sex. For females, a least squares regression suggests that the skates sampled grew an average of 26 mm yr-1 (±12.29, 95% c.i.) and that vertebrae-derived band counts and total length were strongly associated (R2 = 0.85, Adj. R2 = 0.82, n = 7, p < 0.01) (Figure 4), and for males that the skates sampled grew an average of 22 mm yr-1 (±4.42, 95% c.i.) and vertebrae- derived band counts and total length were strongly associated (R2 = 0.94, Adj. R2 = 0.94, n = 10, p < 0.01) (Figure 5). Length at birth was estimated to be 205.05mm (±144.07 mm, 95% c.i.)

(Figure 3). Length at birth estimated by the linear regression was similar for females and males with female size at birth (264.19 mm ±373.40 mm, 95% c.i.) slightly larger than males (237.24 mm ±104.24 mm, 95% c.i.) (Figures 4 & 5).

Caudal thorn length at age data also showed linear growth although a least squares linear regression was not as good of a fit for these data as for the histologically derived length at age data (R2 = 0.63, Adj. R2 = 0.59, n = 12, p < 0.01) (Figure 6). The average growth per year estimated by caudal thorns was greater than what was estimated by histological data (32 mm yr-1

±18.57 mm yr-1, 95% c.i.) as well as a larger length at birth (300.50 mm ±295.43 mm, 95% c.i.)

(Figure 6). Differences in age estimates between histological samples and caudal thorns increased with age and were significantly different (n =12, df = 11, p < 0.01, Paired Samples T- test) and thus were discarded as a viable structure for age determination (Figure 7).

Maturity

Males (~1000 mm) appear to begin to mature at a smaller size than females (~1100mm,

Figure 10). There was only one adult female individual with size at age information, making it

15 not possible to determine if there is an age difference at which sampled males and females mature (Figure 8). There were no adolescent females and no adult males with discernable growth bands (Figure 8). The median total length at which 50% of sampled males were mature (TL50 =

1175.4 mm) was smaller than that of females (TL50 = 1267.3 mm, Figure 9).

1.4 DISCUSSION

Age and Growth

Pending validation that band pair counts are indeed annual as well as a larger sample size,

B. abyssicola may be among the longest living skates on record as well as one of the slowest growing. The highest growth band count tabulated for B. abyssicola was a 1375 mm adult female

(45 growth band pairs). If band counts for this species were validated to be annual, this one female would exceed the maximum age estimates of other long lived skate species by just under

10 years (Davis et al. 2007, Ebert et al. 2007 and 2009, Gburski et al. 2007, Ainsley et al. 2011,

Winton et al. 2013).

Bathyraja abyssicola growth may also be shown to be slow with the validation of age.

Size at assumed age (growth band counts) did not display typical growth signals and were best represented by a linear regression. This resulted in an estimate for average growth per year as opposed to an overall growth rate. While these data suggest the slow growth associated with other deep-water bathyrajid species (Davis et al. 2007, Ainsley et al. 2011, Winton et al. 2013), a more comprehensive sample (i.e. larger sample size, more adult individuals) is necessary to make this determination. It is likely, considering juvenile and adolescent individuals largely made up this dataset, that this analysis captured early life history characteristics for the species.

As animals grow, reach maturity and attain a larger size, a reduction in growth (i.e. an asymptotic signal) is usually observed.

16 Growth banding patterns in B. abyssicola were relatively difficult to discern due to poor vertebral calcification, typical of deep dwelling elasmobranchs (Cailliet et al. 2001, Nybakken and Bertness, 2005, Gennari and Scacco 2007, Winton et al. 2013). In vertebral centra prepared using the gross sectioning method slight banding was observed on the edges of the corpus calcareum arms. With a lack of strong banding patterns, the application of an enhancing oil or stain to read clearly and more accurately is recommended (Licandeo et al. 2006, McFarlane and

King 2006, Ainsley et al. 2011, Winton et al. 2013, James et al. 2014). Mineral oil was applied in an attempt to enhance the inner growth bands but failed to do so. A reduction in growth band clarity in gross sectioned vertebral centra approaching the focus or edges has been observed in other studies employing similar methods (Casey et al. 1985, Gallagher and Nolan 1999,

Licandeo et al. 2006, McFarlane and King 2006, Ainsley et al. 2011). In a similar study by

Ainsley et al. (2011), it was recommended that studies encountering this “cloudiness” issue use the histological method for sample preparation. If gross sectioning is attempted for a second time, it may be best to explore alternate procedures for sectioning. For example, embedding in a hard resin and sectioning with a high speed saw or using different stains may prove useful.

Vertebral centra prepared using the histological method expressed much stronger bands in B. abyssicola vertebra although the process itself destroyed many samples (tearing or stain adhering issues). The gross sectioning method is far simpler and less expensive yet many turn to the more expensive and complex histological method for improved accuracy and precision of age estimates (Natanson et al. 2007, Ainsley et al. 2011, Winton et al. 2013, James et al. 2014). It may be beneficial to modify decalcification and stain soak times to compensate for the lack of calcification in vertebrae of deep dwelling skates. These modifications may reduce the quantity of samples torn during sectioning with the microtome and help with hematoxylin stain adhesion

17 although these issues are not uncommon in the histological process (James et al. 2014). Vertebral section quality may also be a contributing factor to high failure rates in histological samples.

Tribuzio et al. (2015) found that success in vertebral centra preparation in S. suckleyi can vary from animal to animal but may also be attributed to the size or thickness of the sample or the freshness of the hematoxylin stain. These factors should be taken into account for future studies of B. abyssicola, although a larger sample size would be required to reveal individual variability

(several animals of the same size and age). Lastly, vertebral centra quality may have been impacted by freezing and thawing samples. The expansion and contraction of fluids when freezing and thawing samples may have been a contributing factor to the high number of torn samples in this study. Many of the samples were held in freezers for long periods of time and were frozen and thawed a few times before being processed which could compromise the structure of the already soft vertebral centra. In future studies, it is recommended that samples of

B. abyssicola and other deep-water species of skate be sampled and kept with minimal or no freezing and rather be kept in ethanol when possible.

Caudal thorns are frequently explored as an alternative non-lethal ageing structure for skates. Age estimates in bathyrajid species using caudal thorns have proven successful with some species (Gallagher and Nolan 1999, Henderson et al. 2004, Matta and Gunderson, 2007, Serra-

Pereira et al. 2008). However, other studies have deemed caudal thorns inappropriate ageing structures for several skate species (Gallagher 2000, Gallagher et al. 2005, Davis et al. 2007,

Ebert et al. 2009, Perez et al. 2011, Ainsley et al. 2011, Winton et al. 2013, James et al. 2014).

Bathyraja abyssicola caudal thorns showed defined surface structure (ridges), but counts did not fully match up with those from histologically prepared vertebral samples. Gallagher et al. (2005) suggest that skates from deeper waters show more surface structure on the caudal thorns resultant

18 of a drastic slowing of somatic growth in a seasonal cycle. Although the ridges on B. abyssicola thorns were apparent with the application of alizarin red stain, ridge counts were not consistent with the growth band counts found in histologically prepared vertebral centra. Additionally, growth band counts from caudal thorns match vertebral centra counts for the first few bands

(approximately the first 5 bands) then appear to show no relationship to vertebral centra counts

(considerably lower total growth band counts). This is in agreement with findings from several studies involving deep water skate species in the eastern North Pacific Ocean (Davis et al. 2007,

Ebert et al. 2007, Ainsley et al. 2011, Perez et al. 2011). Caudal thorn age estimate results tightly aligned with those found in other deep-water skate studies, therefore caudal thorns may serve as a viable ageing structure in some species, but not for B. abyssicola.

The evidence of latitudinal variation of growth in deep water skate species requires further investigation of B. abyssicola life history characteristics, as this species has a very large range. Regional growth differences were documented for B. trachura (Winton et al. 2013), a congener of B. abyssicola, with northernmost representatives of the species growing more slowly and reaching higher maximum ages than their more southern counterparts. While B. abyssicola are considered well protected from fishing mortality due to their deep water habitat (water depth and substrate preference) off the coast of California, Oregon and Washington, regional differences in habitat may be of concern in some regions of Alaska. In southeast Alaska and the

Aleutian Islands B. abyssicola have been taken at depths of 440-500 m, far shallower than specimens captured in Bering Sea (950-1400 m) (Stevenson et al. 2007). While these depths of capture, even at the shallowest recorded depth, are quite deep and documented encounters with these animals are limited, it warrants at least basic exploration of potential fishing pressures and

19 life history characteristics of B. abyssicola in Alaskan waters to see if latitudinal variation is present in the species.

The deposition of growth bands in B. abyssicola may vary with shallower dwelling individuals laying down more prominent growth banding patterns than individuals that are deeper dwelling. Increasing pressure with depth leads to a decrease in calcification of hard structures and increasing depth leads to less dynamic seasonal shifts (i.e. temperature, water quality, and biomass) (Nybakken and Bertness 2005). Given the generally isothermic patterns of the deep-sea, it is likely that temperature does not influence the deposition of growth bands in B. abyssicola. Food availability and deep-water currents may have more of an effect on growth band deposition in the deep-sea; however, information regarding prey availability and deep- water currents where these animals dwell remains largely unknown. Furthermore, the B. abyssicola samples assessed in this study only represented a small portion of the depth range of the species (780 m- 1430 m) and sample size was limited, so there were no trends suggesting differences in growth band deposition between shallower and deeper dwelling B. abyssicola.

Low sample size is a frequently encountered obstacle to age and growth studies of chondrichthyan fishes, especially rarely encountered species like B. abyssicola (Stevenson and

Orr 2005, Smart et al. 2012). Age and growth studies typically require at least 200 samples covering the full length range of a species to obtain unbiased estimates of parameters (Kritzer et al. 2001, Thorson and Simpfendorfer 2009). To resolve this issue, Smart et al. (2012) established back calculation procedures that produced reasonable estimates of age and growth for five species of shark. These back calculation procedures could be invaluable to the study of rarely encountered chondrichthyan species and should be considered for future study of B. abyssicola age and growth.

20 Maturity

The data derived from this sample suggest B. abyssicola mature at a large size (males:

TL50 = 1175.4 mm, females: TL50 = 1267.3 mm). Female B. abyssicola appear to begin maturing at a larger size than males, a characteristic found in some chondrichthyans (Cortes 2000, Ainsley et al. 2011). However, other studies have shown exceptions in several skate species (Ebert 2005,

Ruocco et al. 2006, Ebert et al. 2008b). Research suggests B. abyssicola begin to transition to maturity around 63-70% of their maximum total length and are 50% mature at about 73-79% of their maximum total length which is lower than most skate species (78-82%) and similar to most shark species (75-77%) (Holden 1973, Cortes 2000, Ebert 2005, Gedamke et al. 2005, Ebert et al. 2008a & b). However, it is important to note that a 1600 mm (approximate maximum size) animal was not observed in this study and the percentages are based on an approximated maximum total length, which may change in future study with a greater sample size and more adult animals. Additionally, with more ageable adult individuals the metric of 50% maturity can be calculated at age providing more robust population based estimates and descriptions for sexual maturity.

Bathyraja abyssicola, was postulated to have a long lifespan, slow growth and late maturation; life history characteristics notorious for increasing the vulnerability of a species to exploitation. Results of this study support these inferences but do not confirm them. Given the life history characteristic trends observed in this study and the potential for latitudinal variations of these characteristics and trends, further study of B. abyssicola age, growth and sexual maturity is necessary.

21 REFERENCES

Ainsley, S.M., D.A. Ebert, G.M. Cailliet. 2011. Age, growth, and maturity of the whitebrow skate, Bathyraja minispinosa, from the eastern Bering Sea. ICES Journal of Marine Science. 68(7), 1426-1434.

Applegate, S.P. 1967. A survey of shark hardparts. P.W. Gilbert, R.F. Mathewson and D.P. Rall, eds. Sharks, Skates and Rays. Johns Hopkins Press, Maryland. 37-67.

Bowker, A.H. 1948. A test for symmetry in contingency tables. American Statistical Association. 43(244): 572-574.

Brander, K. 1981. Disappearance of common skate Raja batis from the Irish Sea. Nature. 5801, 48-49.

Cailliet, G.M., M.S. Love, and A.W. Ebeling. 1986a. Fishes: A Field and Laboratory Manual on Their Structure, Identification, and Natural History. Original publisher: Wadsworth Publishing Company, Belmont, CA; present publisher: Waveland Press, Prospect Heights, IL.

Cailliet, G.M., R.L. Radtke, and B.A. Welden. 1986b. Elasmobranch age determination and verification: areview, in Indo-Pacific Fish Biology: Proceedings of the Second International Conference on Indo-Pacific Fishes, T. Uyeno, R. Arai, T. Taniuchi, and K. Matsuura, Eds., Ichthyological Society of Japan, Tokyo. 345–359.

Cailliet, G.M. 1990. Elasmobranch age determination and verification: an updated review, in Elasmobranchs as Living Resources: Advances in the Biology, Ecology, Systematics, and the Status of the Fisheries, W. S. Pratt, Jr., S. H. Gruber, and T. Taniuchi, Eds., NOAA Tech. Rep. 90:157–165.

Cailliet, G.M., A.H. Andrews, E.J. Burton, D.L. Watters, D.E. Kline & L.A. Ferry-Graham. 2001. Age determination and validation studies of marine fishes, do deep dwellers live longer? Experimental Gerontology. 36, 739-764.

Cailliet, G. M., & K.J. Goldman. 2004. Age Determination and Validation in Chondrichthyan Fishes. In J. C. Carrier, J. A. Musick, & M. R. Heithaus (Eds.), Biology of Sharks and Their Relatives. New York, NY: CRC Press. 399-447.

Campana, S.E. 2001. Accuracy, precision and quality control in age determination, including a review of the use and abuse of age validation methods. Journal of Fish and Biology. 59, 197- 242.

22 Carrier, J.F., H.L. Pratt Jr. & J.I. Castro. 2004. Reproductive Biology of Elasmobranchs. In J. C. Carrier, J. A. Musick, & M. R. Heithaus (Eds.), Biology of Sharks and Their Relatives. New York, NY: CRC Press. 399-447.

Casey, J.G., H.L. Pratt Jr. & C.E. Stillwell. 1985. Age and growth of the sandbar shark (Carcharhinus plumbeus) from the western North Atlantic. Canadian Journal of Fisheries and Aquatic Science 42:963-975.

Casey, J.M. & R.A. Myers. 1998. Near extinction of a large, widely distributed fish. Science. 281, 690-691.

Cortés, E. 2000. Life history patterns and correlations in sharks. Reviews in Fishery Science 8: 299-344.

Davis, C.D., G.M. Cailliet & D.A. Ebert. 2007. Age and growth of the roughtail skate Bathyraja trachura (Gilbert 1892) from the eastern North Pacific. Environmental Biology of Fishes 80: 325-336.

Dulvy, N.K., J.D. Metcalfe, J. Glanville, M.G. Pawson & J.D. Reynolds. 2000. Fishery stability, local extinctions and shifts in community structure in skates. Conservation Biology 14(1), 283- 293.

Dulvy, N.K & J.D. Reynolds. 2002. Predicting extinction vulnerability in skates. Conservation Biology. 16, 440-450.

Ebert, D.A. 2003. Sharks, rays and chimaeras of California. Barkeley, CA. University of California Press.

Ebert, D.A. 2005. Reproductive biology of skates, Bathyraja (Ishiyama) along the eastern Bering Sea continental slope. Journal of Fish Biology 66: 618-649.

Ebert, D.A. & C.D. Davis. 2007. Descriptions of skate egg cases (Chondrichthyes: Rajiformes: Rajoidei) from the eastern North Pacific. Zootaxa. 1393: 1-18

Ebert, D.A., W.D. Smith, D.L. Haas, S.M. Ainsley & G.M. Cailliet. 2007. Life history and population dynamics of Alaskan skates providing essential biological information for effective management of bycatch and target species. North Pacific Research Board Project 510 Final Report, 124 pp.

Ebert, D.A., L.J.V. Compagno & P.D. Cowley. 2008a. Aspects of the reproductive biology of skates (Chondrichthyes: Rajiformes: Rajoidei) from southern Africa. ICES Journal of Marine Science 65, 81-102.

23 Ebert, D.A., W.D. Smith & G.M. Cailliet. 2008b. Reproductive biology of two commercially exploited skates, Raja binoculata and R. rhina, in the western Gulf of Alaska. Fisheries Research 94, 48-57.

Ebert, D.A., J.R. Maurer, S.M. Ainsley, L.A.K. Barnett & G.M. Cailliet. 2009. Life history and population dynamics of four endemic Alaskan skates: determining essential biological information for effective management of bycatch and target species. North Pacific Research Board Final Report 715. 120pp.

Ebert, D.A. 2009. Featured Elasmobranch: Deepsea Skate. www.psrc.mlml.calstate.edu. May 15, 2013.

Gallagher M.J. & C.P. Nolan. 1999. A novel method for the estimation of age and growth in rajids using caudal thorns. Canadian Journal of Fisheries and Aquatic Sciences 56, 1590–1599.

Gallagher, M.J. 2000. The fisheries biology of commercial ray species from two geographically distinct regions. Ph.D. Thesis, Department of Zoology, University of Dublin, Trinity College, Dublin 2, Ireland.

Gallagher, M.J., C.P. Nolan & F. Jeal. 2005. The structure and growth processes of caudal thorns. Journal of Northwest Atlantic Fisheries Science 35, 125-129.

Garcia, V.B., L.O. Lucifora & R.A. Myers. 2008. The importance of habitat and life history to extinction risk in sharks, skates, rays and chimaeras. Proceedings of the Royal Society. B 275, 83-89.

Gburski, C.M., S.K. Gaichas & D.K. Kimura. 2007. Age and growth of the big skate (Raja binoculata) and longnose skate (R. rhina) in the Gulf of Alaska. Environmental Biology of Fishes. 80, 337-349.

Gedamke, T., W.D. DuPaul & J.A. Musick. 2005. Observations on the Life History of the Barndoor Skate, Dipturus laevis, on Georges Bank (Western North Atlantic). Journal of Northwest Atlantic Fisheries Science 35, 67-78.

Gennari, E. & U. Scacco. 2007. First age and growth estimates in the deep water shark Etmopterus spinax (Linnaeus, 1758), by deep coned vertebral analysis. Marine Biology. 152, 1207-1214.

Haedrich, R.L., N.R. Merret & N.R. O’Dea. 2001. Can ecological knowledge catch up with deep-water fishing? A north Atlantic perspective. Fisheries Research. 51, 113-122.

Henderson, A.C., A.I. Arkhipkin & J.N. Chtcherbich. 2004. Distribution, growth and reproduction of the white-spotted skate, Bathyraja albomaculata (Norman, 1937) around the Falkland Islands. E-journal of the Northwest Atlantic Fishery Science. 35, article 1.

24

Holden, M. J. 1974. Problems in the rational exploitation of elasmobranch populations and some suggested solutions, in Sea Fisheries Research, F.R. Jones, Ed., Halstead Press/John Wiley & Sons, New York. 117–138.

James, K.C., D.A. Ebert, L.J. Natanson & G.M. Cailliet. 2014. Age and growth characteristics of the Starry Skate, Raja Stellulata, with a description of life history and habitat trends of the central California, U.S.A., skate assemblage. Environmental Biology of Fishes. 97, 435-448.

Johnson, A.G. 1979. A simple method for staining the centra of teleost vertebrae. Northeast Gulf Science. 3, 113-115.

Klimley, A.P. 2013. The Biology of Sharks and Rays. Chicago, IL. The University of Chicago Press.

Koslow, J.A., G.W. Boehlert, J.D.M. Gordon, R.L. Haedrich, P. Lorance & N. Parin. 2000. Continental slope and deep-sea fisheries: implications for a fragile ecosystem. ICES Journal of Marine Science. 57, 548-557.

Kritzer, J.P., C.R. Davies & B.D. Mapstone. 2001. Characterizing fish populations: effects of sample size and population structure on the precision of demographic parameter estimates. Canadian Journal of Fisheries and Aquatic Sciences. 58, 1557-1568.

Licandeo, R.R., J.G. Lamilla, P.G. Rubilar & R.M. Vega. 2006. Age, growth, and sexual maturity of the yellownose skate Dipturus chilensis in the southeastern Pacific. Journal of Fish Biology 68: 488-506.

Matta, M.E. & D.R. Gunderson. 2007. Age, growth, maturity, and mortality of the Alaska skate, Bathyraja parmifera, in the eastern Bering Sea. Environmental Biology of Fishes. 80, 309-323.

McFarlane, G.A., J.R. King & M.W. Saunders. 2002. Preliminary study on the use of neural arches in the age determination of bluntnose sixgill sharks (Hexanchus griseus). Fish. Bull. 100, 861-864

McFarlane, G.A. & J.R. King. 2006. Age and growth of big skate (Raja binoculata) and longnose skate (Raja rhina) in British Columbia waters. Fisheries Research 78, 169–178

Morato, T., R. Watson, T.J. Pitcher & D. Pauly. 2006. Fishing down the deep. Fish and Fisheries. 7, 24-34.

Natanson, L. J. & G. M. Cailliet. 1990. Vertebral growth zone deposition in angel sharks. Copeia 1990, 1133–1145.

25 Natanson, L.J., J.A. Sulikowski, J.R. Kneebone & P.C. Tsang. 2007. Age and growth estimates for the smooth skate, Malacoraja senta, in the Gulf of Maine. Environmental Biology of Fishes 80(2-3), 293-308.

Natanson, L.J., S.P. Wintner, F. Johansson, A. Piercy, P. Campbell, A. De Maddalena, S.J.B. Gulak, B. Human, F.C. Fulgosi, D.A. Ebert, F. Hemida, F.H. Mollen, S. Vanni, G.H. Burgess, L.J.V. Compagno & A. Wedderburn-Maxwell. 2008. Ontogenetic vertebral growth patterns in the basking shark Cetorhinus maximus. Marine Ecology Progress Series. 361, 267-278.

Nybakken, J. W., & M.D. Bertness. 2005. Marine Biology: An Ecological Approach (Sixth ed.). San Francisco, CA: Benjamin Cummings. 115.

Orr, J.W., D.E. Stevenson, G.R. Hoff, I. Spies & J.D. McEachran. 2011. Bathyraja panthera, a new species of skate (Rajidae: Arhynchobatinae) from the western Aleutian Islands, and resurrection of the subgenus Arctoraja Ishiyama. NOAA Professional Paper NMFS 11.

Perez, C.R, G.M. Cailliet & D.A. Ebert. 2011. Age and growth of the Sandpaper Skate, Bathyraja kincaidii, using vertebral centra, with an investigation of caudal thorns. Journal of the Marine Biological Association of the United Kingdom 91(6), 1149-1156.

Ricker, W. E. 1975. Computation and interpretation of biological statistics of fish populations. Bull. Fish. Res.Board Can. 191, 1–382.

Roberts, C.M. 2002. Deep impact: the rising toll of fishing in the deep sea. Trends in Ecology and Evolution. 17, 242-245.

Ruocco, N.L, L.O. Lucifora, J.M. Díaz de Astarloa & O. Wöhler. 2006. Reproductive biology and abundance of the white-dotted skate, Bathyraja albomaculata, in the Southwest Atlantic. ICES Journal of Marine Science 63, 105-116.

Serra-Pereira, B., I. Figueiredo, I. Farias, T. Moura & L.S. Gordo. 2008. Description of dermal denticles from the caudal region of Raja clavata and their use for estimation of age and growth. ICES Journal of Marine Science. 65, 1701-1709.

Simpfendorfer, C.A. & P.M. Kyne. 2009. Limited potential to recover from overfishing raises concerns for deep-sea sharks, rays and chimaeras. Environmental Conservation. 36 (2), 97–103.

Smart, J.J., A.V. Harry, A.J. Tobin & C.A. Simpfendorfer. 2012. Overcoming constraints of low sample sizes to produce age and growth data for rare or threatened sharks. Aquatic Conservation: Marine and Freshwater Ecosystems. 23, 124-134.

Stevens, J.D., R. Bonfil, N.K. Dulvy & P.A. Walker. 2000. The effects of fishing on sharks, rays, and chimaeras (chondrichthyans) and the implications for marine ecosystems. ICES Journal of Marine Science. 57, 476-494.

26

Stevenson, D.E. & J.W. Orr. 2005. New records of two different deepwater skate species from the eastern Bering Sea. Northwestern Naturalist. 86, 71-81.

Stevenson, D.E., J.W. Orr, G.R. Hoff & J.D. McEachran. 2007. Sharks, skates, and ratfishes of Alaska. Fairbanks, Alaska. Alaska Sea Grant College Program.

Thorson, J.T. & C.A. Simpfendorfer. 2009. Gear selectivity and sample size effects on growth curve selection in shark age and growth studies. Fisheries Research. 98, 75-84.

Tribuzio, C.A., G.H. Kruse. & W.R. Bechtol. In Press. Improved Ageing Techniques and Growth Models for Spiny Dogfish (Squalus suckleyi) in the Gulf of Alaska. North Pacific Research Board Final Report. Project 1106/1106B.

Winton, M.V., L.J. Natanson, J. Kneebone, G.M. Cailliet & D.A. Ebert. 2013. Life history of Bathyraja trachura from the eastern Bering Sea, with evidence of latitudinal variation in a deep- sea skate species. Journal of the Marine Biological Association of the United Kingdom. 94(2), 411-422.

Zorzi, G.D. & M.E. Anderson. 1988. Records of the deep-sea skates, Raja (Ambyraja) badia (Garman, 1899) and Bathyraja abyssicola (Gilbert, 1896) in the eastern North Pacific, with a new key to California skates. California Fish and Game. 74(2), 87-105.

Zorzi, G.D. & L.K. Martin. 1995. The batoid fishes of California-part 1: The skates. Chondro. 5(2), 1-6.

27 TABLES

Step Percent Formula Time Temperature Alcohol (h) 1 70% 500 ml ethanol 2 Room 300 ml distilled water 200 ml tert-butyl alcohol 2 85% 500 ml ethanol 2 Room 150 ml distilled water 350 ml tert-butyl alcohol 3 100% 450 ml ethanol 2 Room 550 ml tert-butyl alcohol 4 100% 250 ml ethanol 2 Room 750 ml tert-butyl alcohol 5 100% 1000 ml tert-butyl 2 Room (warm enough to be alcohol liquid) 6 100% 1000 ml tert-butyl 2 Room (warm enough to be alcohol liquid) 7 50% 500 ml tert-butyl 2 60F alcohol 500 ml Paraplast Plus 8 1000 ml Paraplast 2 60F Plus 9 1000 ml Paraplast 2 60F Plus

Table 1: Step-by-step procedure for histology embedding from Natanson et al. 2007.

28

Step Formula Time (min.) Notes 1 100% xylene 10 Time in xylene depends on amount of Paraplast Plus® in tissue and size of tissue. Increase time for Paraplast Plus® and tissue size. 2 100% xylene 10 3 100% xylene 10 4 100% ethanol 5 5 100% ethanol 5 6 100% ethanol 5 7 95% ethanol 5 5% distilled water 8 80% ethanol 5 20% distilled water 9 100% distilled water 5 Process can be stopped here for overnight. 10 Harris Hematoxylin 10 Sections should be checked to ensure proper staining. 11 water rinse until clear 12 acid alcohol* 2 This can be adjusted depending on staining strength. 13 water rinse 1 Use agitation 14 lithium carbonate** 5 Or until the tissue turns blue 15 running water 10 16 distilled water 2 17 25% glycerin 10 18 50% glycerin 10 19 75% glycerin 10 20 100% glycerin 10 Tissues can be stored for longer periods at this step.

Table 2: Step-by-step procedure for histology staining from Natanson et al. 2007.

29

Maturity Class Male Female (1) Embryo In egg case In egg case

(2) Juvenile Claspers small and flexible Undifferentiated ovaries with and do not extend beyond no visible follicles pelvic fins No differentiation between Undifferentiated testes oviducal gland and uterus

No coiling of epididymis Uterus thin (width, 1% of total length) and transparent

(3) Adolescent Claspers at or beyond pelvic Slightly differentiated ovaries fins and partially calcified with small whitish follicles

Testes differentiated but not Some differentiation between vascularized oviducal gland and uterus

Somewhat coiled epididymis Oviducal gland not well defined in shape or color

(4) Adult Claspers extend past pelvic Vascularized ovaries with fins (20% of total length) and yellow, vitellogenic follicles highly calcified (12 mm in diameter)

Testes differentiated, lobular Oviducal gland opaque, and vascularized kidney-shaped, and well differentiated (5% of total Highly coiled epididymis length) from uterus

Uterus extended

(5) Gravid Adult N/A Egg case(s) present

Table 3: Determination of maturity stages by visual inspection from Winton et al. (2013), derived from Ebert (2005).

30 FIGURES

Figure 1: Photograph of Bathyraja abyssicola and indicated areas of vertebral centra and caudal thorn sampling areas. Vertebral centra taken from area just above the thoracic cavity indicated by solid box, caudal thorns taken from area where tail merges to body indicated by dashed box.

Figure 2: Photographs of gross sectioned (A) and histological sectioned (B) vertebral centra and alizarin red stained caudal thorn (C) from Bathyraja abyssicola.

31

Figure 3: B. abyssicola B. abyssicola total length (mm) at band pair count as determined by histology with fit linear regression line. The least squares regression suggests that the skates sampled grew on average 26 mm yr-1 (±5.41, 95% c.i.) , (R2 = 0.87 Adj. R2 = 0.86, n = 17, p < 0.01). It also suggests that size at birth is “large” (205.05mm ±144.07 mm, 95% c.i ).

32

Figure 4: Female B. abyssicola total length (mm) at band pair count as determined by histology with fit linear regression line. The least squares regression suggests that the skates sampled grew on average 26 mm yr-1 (±12.29, 95% c.i.) , (R2 = 0.85, Adj. R2 = 0.82, n = 7, p <0 .01). It also suggests that size at birth is “large” (264.19 mm ±373.40 mm, 95% c.i.).

33

Figure 5: Male B. abyssicola total length (mm) at band pair count as determined by histology with fit linear regression line. The least squares regression suggests that the skates sampled grew on average 22 mm yr-1 (±4.42, 95% c.i.) , (R2 = 0.94, Adj. R2 = 0.94, n = 10, p < 0.01). It also suggests that size at birth is “large” (237.24 mm ±104.24 mm, 95% c.i.).

34

Figure 6: B. abyssicola total length (mm) at band pair count as determined by caudal thorns with fit linear regression line. The least squares regression suggests that the skates sampled grew on average 32 mm yr-1 (±18.57 mm yr-1 , 95% c.i.) , (R2 = 0.63, Adj. R2 = 0.59, n = 12, p < 0.01). It also suggests that size at birth is “large” (300.50 mm ±295.43 mm, 95% c.i.).

35

Figure 7: B. abyssicola total length (mm) at band pair count as determined by histology and caudal thorns. Caudal thorn and histology band pair counts are close within the first 5 bands then appear to have no relationship. Band counts were significantly different (n =12, df = 11, p < 0.01, paired samples T-test).

36

Figure 8: B. abyssicola total length (mm) at band pair count as determined by histology separated by maturity class for males (n = 29) and females (n = 34) separately. Individuals with undiscernible band pairs are indicated on the x-axis as “U”. No adolescent individuals in female dataset.

37

Figure 9: Estimated probability of maturity at size based on an ogive for male (top) and female (bottom) samples of B. abyssicola. Points indicate individual skates, median size at maturity (TL50) is indicated.

38

APPENDICES

300

200

100 s l a u d i 0 s e 0 10 20 30 40 50 R -100

-200

-300 BandPairCount

Appendix I: Residuals Figure 3.

300

200

100 s l a u d i 0 s e 0 10 20 30 40 50 R

-100

-200

-300 BandPairCount

39 Appendix II: Residuals Figure 4.

300

200

100 s l a u d i 0 s e 0 10 20 30 40 50 R

-100

-200

-300 BandPairCount)

Appendix III: Residuals Figure 5.

300

200

100 s l a u d i 0 s e 0 10 20 30 40 50 R -100

-200

-300 BandPairCount

Appendix IV: Residuals Figure 6.

40