The Egg-Cases Of.The Swell Shark, Cephal0scyllium Ventriosum
THE EGG-CASES OF.THE SWELL SHARK, CEPHAL0SCYLLIUM VENTRIOSUM:
•FORMATION,- FUNCTION, AND POPULATION DIFFERENCES.
by
CHARLES A. GROVER
B.Sc. California State College at, Long Beach 1967
A THESIS SUBMITTED IN PARTIAL FULFILMENT OF
THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE
in the Department
of
Zoology
We accept this thesis as conforming to the required standard
The University of British Columbia
August, 1970. In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study.
I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission.
Department
The University of British Columbia Vancouver 8, Canada ii
ABSTRACT • ,
The swell shark, CephaloscyIlium ventriosum Garman
(Scyliorhinidae), is an inshore, reef-dwelling, nocturnal species of the Eastern Pacific rim.
Reproduction is oviparous. One ovary is developed. Ova are transported through the coelom by cilia, to a single ostium, which serves both oviducts. Egg formation is usually synchronous in both oviducts, and proceeds generally as in other elasmobranchs, but published and new data are combined in a new description of the egg-forming sequence. Photomicrographs show sperm stored in the shell-secreting tubules of the shell gland. This storage allows the production of fertile eggs in the absence of males for some months after mating.
A membrane surrounds the embryo and yolk during the early stages of development, contrary to prior descriptions of related species. A chalaza-like structure is attached to this membrane.
The young of this and several other oviparous species of sharks posess two dorso-lateral rows of enlarged denticles. In the swell shark, these appear to function in the emergence of the shark from the egg-case.
Eggs are preyed upon in nature, possibly by a Stenoglossid gastropod.
The sharks form at least two different populations, separated by as little as 30 km. The egg-cases of one population have no tendrils over 2cm.; the other population has long tendrils, to
2 m. Differences are also found in egg size and in the morpho• metries of the adult sharks. TABLE OF CONTENTS
PAGE ABSTRACT ii LIST OF TABLES iii LIST OF FIGURES iv ACKNOWLEDGEMENTS v INTRODUCTION 1 MATERIALS AND METHODS 4 RESULTS 8
1. Descriptive Anatomy of the Female Reproductive System. 8 a. Ovary, ova, and peritoneal ciliation functioning in ovum transport. b. Ostium and oviducts. 9 c. Circulatory system. 11 d. Shell gland structure, secretions, and sperm storage. 12 2. Egg-case Structure. 16 3. Mechanics and Sequence of Egg-case Formation. 26 4. Development of the Embryo and Egg-case Changes. 35 5. Emergence of the Shark from the Egg-case. 40 6. Predation on Egg-case. 43 7. Population Differences. 45 DISCUSSION 52 SUMMARY 61
REFERENCES 64 LIST OP TABLES
Page Comparisons by two-tailed t-test of measurements of egg- case samples from sharks captured at Isthmus Cove, Santa
CataJLina Island, and the mainland near Los Angeles. 49
Comparisons by two-tailed t-test of morphometric ratios of samples of sharks from Isthmus Cove, Santa
Catalina Island, and Point Dume, California. 51 iv
LIST OP FIGURES Page 1. Diagramatic section drawings of a shell gland. 15
2. Oblique section of a keratin secretory tubule. 17
3. Section at base of laamellae of a shell gland. 18
4. Egg-case showing tendrils. 20
5. Typical pair of egg-cases. 21
6. Typical quartet of egg-cases. 22
7. Egg-case containing an embryo. 23
8. Diagramatic sections of egg-cases. 24
9. Two incomplete eggs laid by a swell shark. 31
10. Active and inactive shell glands. 32
11 . Backlit photograph of an embryo in its case. , 38
12. A newly-hatched swell shark. 41
13. Source locations of egg-cases. 48 V
ACKNOWLEDGEMENTS
I wish to dedicate this work to the memory of the late Arthur S.Lockley, formerly of the Biology Department, Calif• ornia State College at Long Beach. He was a dedicated teacher and a good friend.
I wish to thank Dr. R. Fay, of Pacific Bio-Marine Supply, Messrs. F.Brocato, F.Calendrino, and B. Falcone, collectors at Marineland of the Pacific, andJ. Prescott, Curator? without their assistance, this work would not have begun.
Dr. S. Applegate, Los Angeles County Museum of Natural History, Mr. S. Springer, U.S. Bureau of Commercial Fisheries, Dr. C. Hubbs and Dr. R. Rosenblatt, Scripps Institution of Oceanography, and Mr. J. Fitch, California Department of Fish and Game, have all contributed assistance and valuable discus• sion of the work in progress. Drs. J. McPhail, N. VJilimovsky, and R. Liley, Depart• ment of Zoology, U.B.C, have provided helpful criticism of the manuscript. Mr. L. Sharman ably handled the preparation of the histological material. Finally, my thanks to Mr. and Mrs. I. Neish, for their assistance in preparation of figures, and typing the manuscipt. INTRODUCTION
The swell shark, Cephaloscyllium ventriosum Garman (= C_. uter) . of the family Scyliorhinidae, is common in the waters of California from Monterey Bay south. It is occasion• ally found as far south as Acapulco, Mexico, and is found in Chilean waters (Kato, Springer, and Wagner, 1967). It also inhabits the waters of at least some of the offshore islands. The natural history of this shark has not been described, so I have included some of my own observations. Around Santa Catalina Island, California, trapping data and direct observation using SCUBA diving gear indicate that the sharks are normally found in depths of twenty to forty meters, both day and night, and are less abundant in greater and shallower depths. Two specimens have been taken in deep trap sets; one from about 160 meters (pers. comm., Brocato), and one from about .560 meters (pers. comm., 3. Applegate). These sharks are predominantly nocturnal, and are found during the day in the crevices of rocky reefs. The members of this genus have the pe culiar ability to inflate their stomachs with water, greatly increasing their body size. The anatomy of this mechanism has been described (Clark, 1947). When disturb• ed, they use this ability to wedge themselves into the reef crevices generating an internal pressure sufficient to make it quite difficult for a diver (and, presumably, any other potential predator) to remove them from the rocks, once they - 2 - are fully inflated. V/hen removed from the water, the sharks will often inflate themselves with air, and, if returned to the water in this condition, they will float, upside down and helpless, for some time. However, this latter behavior plays no part in the natural ecology of the animal, as they are bottom dwellers (see Herald, 1962). The femaJ.es reach a maximum length of about 1.1 meters; the largest males found with them are slightly smaller.
Their feeding behavior has not been studied in detail, but fish are evidently an important part of their diet. Pish are among the best baits for trapping. Laboratory observa• tions of feedings of whole fish show that even a quite small, freshly caught she.rk will quickly manipulate a fish in its mouth so that it may be swallowed head first, no matter where the fish is initially grasped. The small teeth are not used to chew or bite pieces from the prey; if it is too large to swallow whole, it is rejected. However, the large mouth and gullet and the highly distensible stomach can accomodate fish which are quite large in proportion to the shark. A shark of about 40 cm. can eventually swallow a Pacit^ic mackerel some 30 cm. long. Vision plays little or no part in feeding. Pish juices introduced into lab tanks induce active swimming and snapping in quiescent sharks during the day (recall that they are noc• turnal J. When food is introduced, its detection seems hit- - 3 -
or-miss; dependant upon the shark actually running into the food, rather than orienting to it visually. This study is concerned with the egg-case of the swell shark. While the various sections of the thesis are all rel• ated • in this respect, they are also relatively independent of one another. Consequently, the pertinent historical background and the results of my own work have been grouped together within each section. The work falls into the following sec• tions.
1. Descriptive Anatomy of the female Reproductive System. a. Ovary, ova, and peritoneal ciliation functioning in ovum transport. b. Ostium and oviducts. c. Circulatory system. d. Shell gland structure, secretions, and sperm storage. 2. Egg-case structure. 3. Mechanics and Sequence of Egg-case Eormation. 4. Development of the Embryo and Egg-case Changes. 5. Emergence of the Shark from the Egg-case. 6. Predation on Egg-cases. 7. Population Differences. MATERIALS AND METHODS
Sharks were captured by cage trap and while SCUBA diving at various locations off the California coast in the vicinity of Los Angeles, and at Santa Catalina Island, California. Some of the sharks and eggs examined are in the Scripps Institution of Oceanography Museum, and are so designated.
With the exception of four live eggs taken while diving at Santa Catalina Island, and one from the mainland which was brought to the laboratory, all live eggs were obtained from sharks held in running sea water tanks at the Marineland of the Pacific laboratory. The eggs were removed to smaller tanks. No continuous temperature records were available. Because of fluctuations in water supply and room temperature, periodic temperature readings could not provide an accurate picture of the temperature fluctuations in the tanks, but I would estimate the absolute limits of seasonal and short-term fluctuations to be between 13° and 22° C.
Dead and empty cases obtained by diving and those brought to the laboratory by beachwalkers were also examined. The first three sections of the thesis - reproductive anatomy, egg-case structure, and egg-case formation - are based on gross dissections of fresh and preserved material, and on microscopic examination of fresh material and of stained histological sections. - 5 -
Portions of the oviducts were fixed in Bouin's, then changed to 70>£> isopropanol at 3-5 days. Sections were stained with Van G-ieson's to show collagen and muscle.
Shell, glands were fixed in the same manner. Sections were stained i^ith Heidenhain's iron hematoxylin, a chromatin stain, to demonstrate the presence of sperm.
Section four, concerned with the embryo in the egg-case, is based primarily upon observations made by shining a strong light through intact egg-cases, which are generally more trans• parent than those of other elasxnobranchs. In addition, several embryos were removed from their egg-cases and examined under a dissecting scope for comparison with published descriptions of the course of development of other species.
Section five describes the exit of the shark from the egg-case, and is based upon the experimental simulation of natural conditions. It was not practical to observe and film the natural emergence of a shark for several reasons; the time of hatching couldii't be predicted with any accuracy, hatching could not be induced at will, and all natural hatchings, at least of eggs held in aquaria, occurred at night. To circumvent these problems, a shark that had hatched the night before was re-inserted into the egg-case through an incision in the curve of the posterior end of the case, next to the ridge which forms the edge of the case (see figure 4). The structure and consistency of the egg-case is such that, - 6 -
if the experimental case was laterally compressed, the incision woxild open, but would spring closed when the pressure was rel• eased . After the shark was replaced in the egg-case, it was returned to the aquarium, and all air expelled; The "re- hatching" was recorded on 35 nun. color motion picture film, which was later examined both in normal projection and frame- by- frame.
Section six, on predation, resulted from the examination of a sample of egg-cases collected on one dive at Ship Rock, Santa Catalina Island, California.
Section seven, on population differences, started with the discovery of a consistent difference in the morphology of egg-cases in samples from various locations, described in the text, and led me to make morphometric comparisons of samples of the sharks themselves. Sharks measured for morphometric comparison came from the mainland side of Santa Catalina Island, and from the region of Point Dume, approximately 10 km. north of Los Angeles (see figure 13). Both samples were measured on the same day. Both had been fresh-frozen for storage, and were measured immediately after thawing. The dimensions ultimately chosen for comparison are given in appendix 1. Because of a very flexible body, such dimensions as jaw width, body depth, gill slit width, gill - 7 - slit interspace, etc., were not reproducible with any accuracy. The dorsal fin height measurements were found to be consistent• ly reproducible if the fin was spread flat on the table surface, one point of the caliper placed in the notch at the posterior fin base, and the other point swung in an arc at the fin tip. fa 9 The caudal width was taken in a similar manner, swing1'the caliper in an arc from a point on the edge of the lower caudal lobe at the widest point of the tail, to the dorsal edge of the tail. In addition, preserved specimens in the Scripps Institu• tion Museum Collection, from Isle Guadaloupe, Santa Catalina Island, and various points on the mainland, were compared for color pattern and gross morphological differences. Measure• ments of some of these specimens were taken also, but small differences between shark specimens measured when fresh and those measured when preserved are not to be relied upon, because of dimensional changes in the cartilaginous skeleton caused by formalin and alcohol preservation (pers. comm., S. Springer). The vertebral counts given in appendix 2 were obtained from whole-body X-ray plates which I made of preserved specimens at the Scripps Museum. Caudal vertebrae are ex• cluded from these counts; they vary greatly (pers. comm., S. Springer) and are in any case difficult to count accurat• ely, toward the tail tip. A pin was inserted into the spinal column through the center of the curve at the rise of the tail, - 8 - and the vertebrae counted between it and the chondrocranium. Because of variability in the insertion of this pin, I would estimate an error in these counts of - 1.
RESULTS
1. Descriptive Anatomy of the Female Reproductive System. a. Ovary, Ova, and Peritoneal Ciliation Functioning in Ovum Transport.
In many elasmobranchs, only one ovary develops as such and becomes functional (Daniel, 1934, Ghieffi, 1962). After ovulation, the large ova are transported in the coelom by ciliary action. Metten (1939) has described this process in great detail, as it occurs in another scyliorhinid, Scyliorhinue canicuius ( = Scylliorhinus caniculumj. He found that almost all the surfaces in the coelom - peritoneal wall, mesentaries, and organ surfaces - between the ovary and the ostium are evenly cilia.ted.
In C.ventriosum , only one ovary is functional. It is attached to the coelom wall by a mesovarium in the usual manner. In a large female, it has a volume of 600-700 cc. A detailed survey of ovarian development through the year ws.s not done, but all mature females dissected were found to have ova of all sizes. This is consistent with the year-round production of eggs, which will be discussed la. ter. C_. ventriosum is like 2>. canicuius in that nearly all surfaces in the coelom are ciliated, but differs in the pat• tern of ciliation. The cilia occur in discrete patches, rather than being distributed evenly.
b. Ostium and Oviducts.
The oviducal ostia of elasmobranchs are located at the anterior end of the coelom, and are supported by or incorpor• ated into the falciform ligament. They may be separate (Hobson, 1930), or combined, with a single common opening. Metten (1939) ha.s described this latter condition as it occurs in £>. cani cuius. In this species, the single opening lies between the two fimbrial septa of the falciform ligament. These septa are asymmetrical. The opening of the right ovi• duct forms the actual funnel which receives ova from the coelom, while the left oviduct opens into the wall of the right, just inside the ostium. Both oviducts are functional in almost all elasmo• branchs (Daniel, 193if) . From their opening(s), they curve laterally and caudally along the sides of the coelom to the cloaca, into which they empty. A shell (oviducal, nidimental, nidamentary, of other authors) gland forms a part of each oviduct, and is located anterior to the middle of the oviduct. It is in this.gland that the ovum receives its coatings of albumen and shell material. This process is the subject of - 10 - a later section of this thesis. That portion of the oviduct anterior to the shell gland is described as cranial, and the posterior portion as caudal. In C. ventriosum, I found the structure of the falci• form ligament and single ostium to be quite different from that described by Metten for £5. caniculus. The ostium lies between two septa of the falciform ligament, as in S. caniculus. but there the similarity ends; there is no asymmetry. The fimbrial septa are equal in size and in their lateral dis• placement from the body midline. The single ostium, of a size to just admit a single ovum when stretched, opens into a slightly larger sac. Prom this sac, two openings of equal size are symmetrically located on either side, and lead to the right and left oviducts. This entire structure, and the cranial oviducts, are thin, membranous, and ciliated on their inner surfaces. Little if any smooth muscle can be seen in the histological sections. The caudal oviducts are quite different in structure, though only slightly greater in diameter (about 1.5 cm.) when empty. Instead of thin-walled tubes, down which the contents are moved by ciliary action, the caudal oviducts are thick-walled and muscular. The interior walls are highly convoluted, and are lined with a smooth, unciliated, columnar epithelium. The wall is composed of an inner, annu• lar layer of smooth muscle, and an outer, longitudinal layer -11-
of smooth muscle. Both layers contain and are separated by fibrous connective tissue. Dissections of females with eggs in the caudal oviducts showed that the walls were tightly stretched around the egg-cases. This, the intestine-like structure, and the absence of cilia, leave little doubt that the transport of completed eggs down the caudal oviducts to the cloaca is accomplished by peristaltic contractions, c. Circulatory S.vstan
Daniel (l9j54) » in his definitive work on elasmobranch anatomy, describes the arterial system supplying the oviducts and shell glands of Scyliorhinus (= Scyllium) and other sharks, but makes no mention of "che vessels draining these organs. Nor have I found more than passing reference to this venous system by any of the authors who have investigated the reproduct• ive anatomy of sharks, who are cited later in this work. For example; "In preparing an oviducal gland for sectioning pur• poses, the ciliated peritoneal epithelium on the outside readily detatches itself, and it does not figure in the accounts of any of the authors mentioned". So wrote Metten (±939). I found a similar "ciliated peritoneal epithelium" in C. ventriosum; it is part of a large blood sinus surrounding the oviduct and shell gland. Blood is supplied to these organs via segmental arteries arising from the dorsal aorta, as described by Daniel. It drains to a tubular sinus which encloses the entire oviduct - 12 -
(including the shell gland), and is loosely attached to it by thin trabeculae. This sinus is enlarged in the area of the shell gland, and extends medially, where it empties into the postcardinal vein, via many anastomoses. The right and left postcardinals are confluent in this area, so the oviduct sinuses of both sides and the postcardinals form what is, in effect, one large blood sinus. In an egg-producing female, this sinus complex and the enclosed organs are engorged with blood; as much as 100 cc. Since only some 400 cc. can be drained from a heavily heparinized shark of near maximum* size, it is ap• parent that this large sinus complex contains a high propor• tion of the total blood volume in an egg-producing female.
d. Shell G-land Structure. Secretions, and Sperm Storage The shell gland structure of S. caniculus and Chilo- scvllium griseum have been described, respectively, by Metten (1939), and Nalini (1940), both of whom have reviewed the pert• inent previous work in detail. Both of these authors described four separate secretory portions of the gland. In anterior- posterior order, these are: albumen, anterior mucus, keratin, and posterior mucus.
Threadgold (l957) histochemically demonstrated five separate secretory zones in the shell gland of S.caniculus in addition to the posterior mucus zone, with which he did not deal. Hiz zone A corresponds to what is known as the albumen secreting portion. However, the "albumen" is non-protein; Please insert the following paragraph at the asterisk on p. 13.
Metten (1939) was the first to locate.the sperm storage site in the keratin secretory tubules of the shell gland. He published photomicrographs showing bundles of tubules' in the tubules. He found no sperm in either the albumen secretory por• tion of the gland (located anterior to the shell secretory por• tion), or in the cranial oviduct. Ima later paper (Metten, 1 944), he showed that some sperm are also found embedded in the walls of the caudal oviduct. - 13 -
Threadgold suggested that it is probably a mucopolysaccharide. Collenot (1966) confirmed this. Threadgold*s zone B equals the area described earlier as the anterior mucus tubules, and also secretes a carbohydrate. Zones C-E are different areas of the keratin or shell secreting portion. As this study is not concerned with the chemistry but with the mechanics of egg-case formation, I will continue to use the term albumen, with the understanding that its use is restricted to the traditional "egg-white" sense. In addition, I will consider the shell secreting portion as a homogeneous functional unit.
Clark (1922) suggested the occurrence of sperm storage in elasmobranchs. He described several instances of rays laying numerous fertile eggs after several weeks of isolation in aquaria. Libby (1959) described one instance of an isolat• ed ray which laid infertile eggs until a male was introduced into the aquarium for a short period, and then removed. The female then laid two fertile eggs about every four days for nearly nine months, with no fux-ther matings. Clark placed the site of sperm storage "in the upper reaches of the oviduct". Nalini (1940), upon reading Metten's earlier paper, re• examined her material from C. griseum. and found what might have been bundles of sperm in the shell tubules, but reserved judgement. Prasad (1945) described sperm in the shell secretory - 14 -
tubules of a spe cimen of G-alQocerdo cuvieri (— G. tigrinus), a live-bearing shark. It had ova in both the cranial oviducts and the uteri.
I found the shell gland structure of C. ventriosum to conform closely to the descriptions given by Metten and by Nalini, cited above. The following description may thus be taken as typical of a number"of oviparous sharks, as well as specific to the swell shark.
Figure 1 (a & b) shows diagramatic saggital (with res• pect to the gland) and cross sections of the shell gland, with the plane of each section indicated on the other. Notice that there are two separate secretory masses, which form the two faces of the lumen. The shell gland is oriented in the animal so that these two masses lie dorsal and ventral to the lumen. Three of the four secretory areas are easily different• iated in gross dissections; the albumen, keratin, and posterior mucus (see fig. 1). Notice that, though the area of keratin secretion into the lumen of the gland is but a small portion of the lumen's area, the keratin secretory tubules themselves form the major portion of the mass of the gland. The proximal ends of these keratin tubules empty into the lumen in rows, between successive rows of deeply-folded lamellae, which lie in a plane normal to the longitudinal axis of the gland. This may be seen in figure 1c, a diagramatic dorsal view of the ventral secretory mass, which forms the ventral wall of the lumen. -15-
Figure 1. Diagramatic section drawings of a shell gland.
A. Saggital.
B. Cross. C. Frontal.
Dashed lines a-a, b-b, c-c; planes of sections A,B, and C. al; albumen secreting areas. la; lamellae of the keratin secreting areas. k; keratin secreting tubules. mu; mucus secreting areas. l.g.; grooves at the edge of the shell gland lumen (the lumen is shown in fine lines in section A.).
- 16 -
The shell gland of oviparous elasmobranchs coats the ovum successively with albumen, a keratinous capsule, and finally, a mucus layer which facilitates the egg's passage down the caudal oviduct. There is good evidence, in the swell shark and at least two other species, that the shell gland also serves as a site of sperm storage.
Figure 2 is a slightly oblique section of a shell gland secretory tubule. Sperm (arrow) may be seen in the lumen of the tubule. Figure 3 is a portion of the area where the kera• tin tubules empty between the lamellae into the shell gland. The sperm are indicated hy the arrow .
The sperm of elasmobranchs characteristically have spiral heads (Metten, 1939, Mellinger, 1965). Metten was able to detect the spiral of the heads only in live material, but the sections figured here, from a different species and stained differently, clearly show the spiral at 325 X magnification under the microscope. Metten found that sperm were more numerous in those portions of the tubules proximal to the shell gland lumen; I found the same. The sections figured here came from a shark which had complete, fertile eggs in the caudal oviducts.
2. Egg-case Structure
Before considering the remaining sections, we must develop a clear picture of the structural details of the egg-case. -17-
Figure 2. Oblique section of a keratin secretory tubule.
Sperm are indicated by the arrow. Magnification, about 2000X.
-18-
Figure 3. Section at base of lamellae of keratin secretory area of a shell gland. Sperm (arrow) shown between two lam• ellae. Magnification, about 2000X.
- 19 -
Edwards (1920), Daniel (1934 and earlier editions), and
Cox (1963) have all figured single egg-cases of Cenhalosc.yllium ventriosum (= _C. uter; = Catulus uter). Edwards gave the dimensions of her single specimen as 116 by 49 mm. Cox gave the range of lengths as 90 to 125 mm., and the widths as 28 to 55 mm., but included no sample data.
Appendix 3 contains the measurements of some of the eggs laid by sharks from two different locations in two different years. The configuration of the eggs is such that length measurements cannot be taken with any consistent accuracy. However, the widths and weights can be reproduced accurately.
Figures 4-7 show various egg-cases; each will be used to illustrate specific points. The upper ends of the eggs figured are posterior in the oviduct; the lower ends are anterior. These designations wi 11 be retained in subsequent discussion. The egg-cases vary considerably ih configuration and color, as well as size. The circle enclosed by the horns at the post• erior end is usually slightly lees than a cm. (fig. 5)» but can vary from completely closed (fig. A), to nearly 2 cm. The shape of the anterior end varies from a deeply lunate curve to an almost straight edge between the horne. The size and sharpness of the lateral bulges just posterior to the anterior end varies, also. I could detect no apparent pattern in these variations. -20-
Figure 4. Swell shark egg-case, showing tendrils. The arrows indicate the area where the incision was made on another egg- case for the insertion of a newly-hatched shark, to simulate hatching.
-21-
Figure 5. A typical pair of egg-cases. Both, show a b^nd of shell material of a different shade across their width.
-22-
Figure 6. A typical quartet of egg-cases.
-23-
Figure 7. Egg-case containing an embryo. The arrows indicate the positions of the four respiratory slits.
-24-
Figure 8. Diagramatic sections of egg-cases. A. Longitudinal. B. Cross. The dashed lines a-a and b-b indicate the planes of sections A and B. The dotted line indicates the approximate plane of the anterior end of the egg-case (removed from section B), when viewed from the anterior end.
- 25 -
Notice that the eggs in figures 4 and 7 have long, coiled tendrils at the four corners of the cases, while those in figures 5 and 6 do not. The tendrils of these latter eggs have neither been deliberately or accidentally removed, nor are the eggs malformed. This is a consistent population dif• ference, and will be expanded upon in section 7.
The color and opacity of the egg-cases .vary both during the course of aging of the eggs, and from one egg to another. Shell material which has just been secreted is a milky white, becoming yellow and translucent in the posterior portion of the shell gland. Eggs in the caudal oviduct are slightly darker in color, and more transparent. Newly laid eggs are a light olive-brown and usually quite transparent. As eggs age, they darken and become less transparent. However, individual variation is such that some eggs are as transparent when several months old as others are when newly laid. A variable streaking of opaque yellow is seen on some eggs. The darken• ing of the shell is accompanied by a change in consistency; it becomes tougher and more resilient. Earlier writers sug• gested or assumed that this was the result of the egg-case ex• posure to sea water. This may well be so, but Threadgold
(1957) has presented histochemical evidence which suggests that some type of quinone tanning is the operative process, at least in the shell gland itself.
Figure 8 shows longitudinal and cross sections of the egg-case. Kotice the thickened portions at either edge of - 26 - the case in the cross section. These are continuous with the filaments extending from the corners of the case. These thick• ened edges are displaced toward opposite sides of the egg-case at both ends of the egg-case (see next paragraph, figure 7). The egg-cases also show a slight torsion at their anterior ends. If the cross section is taken as being viewed from the anterior end, the dotted line indicates the approximate plane of the anterior end. Close examination of the anterior ends of the egg-cases in figure 6 will show the same thing; the left corner of each was slightly closer to the camera.
The arrows in figure 7 indicate the position of four openings which are formed into the egg-case, known as resijir- atory slits. The positions of these slits conform to the dis• placement of the edges of the case, which was mentioned above. Only the two on the right are visible because of this; those on the left are on the other side of the case. This displace• ment is in the same direction in all eggs, whether they come from the right or left oviduct. These slits are plugged with a dense albumen when the egg is laid. Their opening,and function is discussed in section 4.
3. Mechanics and Sequence of Egg-case Formation.
The process and timing of egg-case formation in elasmo• branchs have been subjects of speculation for numerous authors - 27 - for about the last hundred years. Hobson (1930), Metten (1939), Nalini (194C-), Setna and Sarangadhar (1948) , and Prasad (l95l) have reviewed this earlier literature, and all but the last author cited have added the results of direct observations.
It was variously held by earlier authors that: 1 . The egg-case was formed in the caudal oviduct from :the secretions of the shell gland.
2. The albumen and shell were simultaneously laid down around the ovum in the shell gland as the ovum passed through. 3. Secretion started in the shell gland before the ovum reached it, but the whole of the egg-case is formed around the ovum in the shell gland. 4. The caudal portion of the egg-case is formed before the ovum reaches the gland, and the anterior end of the case is closed after the ovum has entered the case. Hobson supported this last view. After examining some 150 egg-carrying females of the ray, Raja radiata. he found one in which the shell glands contained partially formed egg- cases. I quote his description: "The half-formed egg case is quite empty [^italics mine] . Its dorsal end ventral walls are well separated from one-another so that at the anterior end there is an opening which will admit the egg [italics mine] when it is ready to pass into the case ... At least half, and possibly considerably more, of the egg case is already formed before the egg comes in contact with the nidamental organ at - 28 -
all."
Referring to Hobson's work, Metten described a number of incomplete egg-cases in the shell glands of £>. caniculus. He found that none which were less than three-fourths completed contained ova, but gave no further description of their con• dition.
Nalini discounted Hobson's hypothesis that part of the egg—case is formed first, then receives the egg into it. To her, this implied a discontinuous process which might be ex• pected to result in a suture in the egg-case, which is not seen.
Prom what she described as "... the egg case in fully formed condition inside the nidamental organ of ' [jthe sharlTj Chilos- cyllium griseum.", she adhered to the premise that "... the secretions are poured over the egg after it reaches the nidament• al."
But, Setna and Sarangadhar found egg-cases about two- thirds complete in C. griseum, and described them thus: "The fertilized eggs, surrounded by dense masses of albumen, had already entered [italics mine] the egg-cases, and more albumen was seen to be still entering through the open ends."
Prasad suggested that "... when the egg reaches the nida• mental gland there will be a partly formed egg-case to receive
Qltalics mine] the egg.", but that subsequent secretions are
continuous with those which have formed the first portion, thus explaining the lack of a suture. - 29 -
Despite the apparent conflict between the accounts quoted and described above, I believe that they are all partially cor• rect. First, consider those portions of the quotations which I have italicized. Hobson described his skate egg-cases as "quite empty", yet the walls were separated. I can but assume that he meant only that the ovum was not present, for if the open egg-case end contained no albumen, it would not only be contrary to the other author's and my own findings, but would mean that the case, to be open, must contain some gas which would have to be displaced by subsequent albumen secretions; this seems to me to be a structural impossibility.
The other italicized portions point out that the various authors state or imply that the ovum and/or albumen move into the previously formed posterior portion of the egg-case. In contrast, remember that Nalini held that the secretions took place as the ovum passed through the gland. On three occasions I examined partially formed egg-cases of C_. ventriosum. In the first instance, dissection of a shark revealed partially formed tendrils extending posteriorly from the shell gland into the caudal oviducts. The posterior end of the egg cases had not yet formed, so that the two tendrils in each oviduct were not connected to one another. The second instance was the laying of two incomplete eggs. These are shown'. in figure 9 with a normal egg for - 30 -
comparison. The eggs are slightly more than half completed. They both contained albumen, which also protruded from the in• complete ends. Both contained fragments of broken ova. Notice the thin edges of the incomplete ends. The third involved the dissection of a female killed while in the process of forming eggs. Figure 10 shows one of the distended shell glands containing a partially formed egg, the incomplete egg from the other side, and an inactive shell gland from another shark for comparison. Both of these eggs contained complete ova, with albumen both posteri^and anterior to the ova.
From the descriptions of the authors quoted, examination of a variety of other elasmobranch eggs, and my own observa• tions just described, I have deduced a somewhat different sequence of egg-case formation. I will describe it here for C. ventriosum. but I suspect it to be generally valid for a wide variety of elasmobranchs. The initial keratin secretions move posteriorly in the grooves at either side of the lumen of the shell gland, which form them into the posterior egg-case tendrils. As these near completion, the albumen portion of the gland begins secreting. As this initial albumen moves posteriorly, it is covered with successive layers of keratin produced from between the rows of lamellae of the keratin secreting area (see fig. X). In the dissections mentioned earlier, of the shell glands which -31-
Figure 9. Two incomplete eggs laid by a swell shark. The com• pleted egg is included for comparison.
-32-
Pigure 10. Active and inactive shell glands. The gland to the left contains a partially completed egg. The egg re• moved from the opposite gland of the same shark is at lower right. An inactive gland from another shark is at upper right. The anterior ends of the glands and the posterior end of the egg are uppermost.
- 33 - contained partially formed eggs, I found that the first thin layer of milky white shell material was issuing from between the first two lamellae. This initial mass of keratin-covered albumen is formed into the posterior end of the shell gland lumen, \i?hich functions as a female mold, exactly matching the shape of the finished egg, with its respiratory slits and their albumen plugs. As this molding takes place, keratin and albumen continue to flow. The shell gland extends longitudin• ally somewhat, allowing the posterior portion of the egg to "sef'in the "mold", despite the continuing secretion of material and increasing egg size. Thus, there would be no hal• ting of the secretions, and resulting suture in the egg-case. The ovum arrives, is coated with the continuing secretions of albumen and keratin as it moves along, and is added onto - does not "enter"-the enlarging egg. Albumen secretion slows and ceases. The sheets of keratin come together as the last of the albumen passes the lamellae. These keratin sheets seal together to form the anterior end of the egg-case, and the formation of the sheets ceases. The keratin which forms the anterior tendrils continues to flow into the lateral grooves of the lumen, then ceases as the egg continues down the oviduct, and the tendrils are finished. The egg is complete. - 34 -
I see the shell gland as functioning both as a mold and as an extensible extrusion die for its own secretions. The posterior end first functions as a mold, shaping the/first secretions reaching it into the characteristic shape of the posterior end of the egg. This portion:does not move in the gland until the shape is "set". The cross sectional shape of the lumen, with its lateral grooves, acts as extrusion die, forming the continuing secretions into the characteristic cross section of the egg. The characteristic longitudinal shape of the egg, tapering from the ends to a maximum thick• ness in the middle, may well be controlled simply by the amount of albumen and ovum arriving at the shell secreting lamellae to be coated. This is suggested by the fact that "wind eggs" - eggs normal in shape, but containing only albumen and no ova - are smaller than normal eggs. These have been described for other species by several of the authors cited, and are also formed by the swell shark. Another phenomenon described by those authors mentioned who have studied egg-case formation is the synchrony of devel• opment of the eggs in both oviducts. I observed the same thing. In the three instances described earlier, in which I examined partially completed egg-' cases, both sides of the system were in the same stage of devel• opment. The same thing is seen in that they are usually laid in pairs, or quartets. Figure 6 shows a typical quartet - 35 -
of eggs. The two eggs at the top form a pair, those at the bottom another. Notice that the members of a pair are nearly- identical in size and configuration, but differ noticeably from the other pair. Notice also that the pattern of longitudinal color striations is from the top to the bottom eggs on'each side of the figure, showing that the eggs on the left came from, one "extrusion die" - shell gland - and those on the right from the other.
Figure 5 shows another pair of eggs. The transverse, light-colored line is in the same place on both eggs, indicat• ing that some physiological event, changing the character of the keratin secretions momentarily, afi"ected both eggs at the same place.
4. Development of the Embryo and Egg-case Changes.
The embryology of several elasmobranchs has been described in great detail, as in Balfour (1878) and Smith (1940, 1942). Harris (1952), describing the development of another scylio- rhinid, has stated that its development to the gill filament stage is identical to that of Squalus acanthias. a live-bearing shark of a different taxonomic order. From these published accounts it appears that the course of development of a wide variety of selachians is much the same, particularly in the early stages. C_. ventriosum seems to fit this picture. Its embryology 36 - is not the subject of this thesis, and only a brief sketch will be given. While the development of the embryo itself seems to correspond to the pattern seen in other sharks, there are other structures present to which I have been able to find no reference in the literature. I didn't realize their pos• sible significance at the time the work was being carried on, and paid them no special attention.
Nelsen (1953, P. 165), drawing from the work of Balfour, describes the egg membranes of Scyliorhinus caniculus (= Scyllium canicula). He states that an outer "homogenous vitelline membrane", and an inner zona radiata, combine in the mature egg to form a thin, composite vitelline membrane. He states that, at about the time of fertilization, this membrane separates from the egg's surface, enclosing the perivitelline space thus formed. However, Balfour (1881, p. 35)» states that the elasmo- branch ovum is without a vitelline membrane at the time of im• pregnation. It has, by his account, disappeared by the time the egg enters the oviduct (Balfour, 1880, p.50). Smith (1957) states that during the early cleavages, when the embryo is a relatively small mass of dividing cells, the ovum is covered by a thin, non-cellular membrane, which is replaced by organized embryonic tissue after gastrulation. - 37 -
Read (1968) in hie study of osmoregulation in Ra.ia hi&oculata. mentions an "extremely delicate membrane" func• tioning in urea retention in undeveloped eggs.
Figure 11 is a back-lit photograph of an embryo in the case. The embryo is dead, and not typical in form. But the membrane extending up from the yolk and surrounding the embryo is typical of embryos of this age, with the exception of the distortion of the embryo's tail touching it.
I saw this membrane on numerous occasions. It was read• ily visible when the more transparent egg-cases of younger embryos were "candled". It increased in size as the embryos grew. I did not notice at what stage it disappeared, but it was certainly gone by the time the respiratory slits in the egg-case opened.
The other undiscribed structure cannot be seen dir• ectly, but can only be inferred from its effect on the mem• brane just described. If an egg is viewed as in figure 11, and tilted back and forth, the embryo and yolk rotate slightly, as if the embryo were more buoyant than the yolk mass. But, the movement is limited. This might be attribut• ed solely to the confined space, were it not for a distortion which appears in the apex of the membrane over the embryo.
Imagine an invisibly thin thread attached to the sur• face of a partially inflated balloon, and pulled just taut.
If the balloon is then rotated slightly, the distortion of its surface by the pull of the thread can be seen, even though the -38-
Figure 11. Backlit photograph of an embryo in its case, showing the membrane enclosing it and the yolk sac.
- 39 -
thread cannot. The distortion of the membrane in the egg fol• lows this analogy, and changes its orientation appropriately as the egg is tilted from one side to the other. From this, I deduce the presence of a structure in the albumen of the eggs of this shark which is analogous to the chalaza of a hen' egg, and which is likely homologous with it. Clark (1922) noticed that the embryos of rays in newly- laid eggs were in different stages of development. The same thing occurs in the swell shark; the embryos may be in any stage from blastodisc to an embryo up to 4mm., on a yolk stalk and showing muscular contractions. These early contractions are myogenic (Harris,1962).
Development of the shark in the aquarium to hatching takes 7T-10 months; I assume that the rate of development is, as has been found in other sharks (Harris, 1962), temperature- dependant. The egg-case is closed for only about a third of this time, until the respiratory slits begin to open. It may take as long as a month for the albumen plugs to disappear from all four slits. The dense albumen forming the plugs is apparently metabolized by the shark; little if any erosion of the surfaces exposed to the surrounding water occurs.
The time of disappearance of the external gill filaments roughly corresponds to the complete opening of the slits. At this point, the embryos can be removed from the egg-case and - 40 -
survive, if placed in a smooth-walled container through which the aquarium water may circulate. At hatching, the sharks are about 15 cm. total length. The external yolk sac may have been completely absorbed, or there may remain a stalk of up to a cm. long, attached to a sac of 3 or 4 mm. diameter.
In addition to an even covering of small, pointed dent• icles over the entire body, the young have two longitudinal, dorso-lateral rows of larger denticles of different form. Ford (l92l) described these denticles as primary, and listed eight oviparous species of sharks - seven scyliorhinids and an orectolobid - the young of which have such denticles. The young of some chimaeroids also have two similar rows of denticles, though the adults have naked skin (Norman and Greenwood, 1963). A newly-hatched swell shark, showing the primary denticles, is shown in figure 12.
5. The Emergence of the Shark from the Egg-case.
The details of egg-case structure have been described. Remember that the anterior end of the case, through which the shark emerges, is different in structure from the posterior end. Look at the longitudinal section in figure 8. Imagine the two sides of the case, where they join at the anterior end, -41-
Figure 12. A newly-hatched swell shark, showing the larger primary denticles on its back. Life size.
- 42 -
as two pages of a book which have been lightly glued together at their edges. A finger inserted between these pages and moved outward will separate them. The first step in this sequence is the opening of the case by the shark. The shark's snout is the "fingertip" between the "pages". The shark's body is longer than the interior of the egg-case, and the necessary force is supplied by the bent tail thrusting against the posterior end of the case.
When this first step is completed, the head and gill region of the shark - the widest portion of its body - are protruding from the opened anterior end of the egg-case, which is just large enough to allow passage. The tail is now straightened out, and can no longer thrust against the closed posterior end. In the second step, the shark completes its exit from the case by a series of exaggerated lateral flexions. Recall the two dorso-lateral rows of primary denticles described earlier. It appears that these larger denticles interact with the edge of the egg-case opening in the manner of a ratchet with a pawl, providing a purchase which renders the lateral movements of the shark effective in moving it out through the opening, after the tail pan no longer thrust against the other end. Each successive lateral movement brings more posteriorly placed denticles in contact with the edge of the opening, which is close against the shark's body. This - 43 -
"anchoring" allows the lateral movement of the forward portion of the body to be translated into forward thrust against the edge of the opening, which moves the shark out through it. The distribution of these primary denticles on the shark's body is optimum for this function. The rows are lateral to the midline, effecting the maximum forward movement for a given lateral movement. They begin posterior to that portion of the body which is thrust through the opening by the initial thrust of the tail, and are continued posteriorly to the area where the body cross section is sufficiently smaller to allow easy passage of the rest of the body through the opening. The reduction in comparative size and disap• pearance of these denticles in the ensuing months of growth suggests that, whatever their function, it has been served.
6. Predation on Egg-cases.
I have found no mention in the literature of predation on elasmobranch eggs, let alone in the manner to be described. In 1969, a "nesting area" of the swell shark was dis• covered at Ship Hock, which is about a mile offshore from Santa Catalina Island. In the crevices of the submarine talus slopes at the base of this rock, in 25-35 m. depths, literally hundreds of egg-cases are to be seen. This is in contrast to nearby Bird Rock, where the sharks can also be found, but 4 44 - where I have found only two eggs, despite having spent much more time diving in the latter area.
Forty-two eggs were collected in one dive at Ship Rock.
Of these, only two contained embryos. Of the remainder, 29 had one or more holes in them - 11 had one hole, 11 had two holes,
6 had 3 holes, and one had 5. The holes were circular, with an irregularly serrated edge which tapered inward toward the center of the hole through the thickness of the egg-case. Most were 2-5 mm. across, but one was 15 nim. Several egg-cases showed partially completed holes.
The only candidate I consider likely to perforate the eggs in this manner would be a gastropod of the suborder Steno- glossa; "Marine snails with...radula narrow, often adapted for a-carnivorous diet." (Light, et al., 1957)• I eliminat• ed Octopus bimaculatus. a common carnivore in the area, after comparison of its beak shape with the holes in the egg cases, and their positions. While the octopus also posesses a radula, it seems likely that the egg-cases would show some beak, marks, at least along the edges, if the octopus were the predator.
I have seen one other type of predation upon shark eggs.
In the 2,000,000 liter marine community tank at Marineland of the Pacific, the large labrid teleost, Pimelometopon pulerum, will, bite and puncture the eggs of C.ventriosum and Hetero- dontus francisci which are laid in the tank. This has not - 45 -
been reported from nature, but would be a possibility, consider• ing the opportunistic feeding habits of this fish, which is sympatric with the sharks mentioned.
7. Population Differences.
As mentioned earlier, swell sharks from some localities produce eggs with long tendrils at the four corners; eggs from other places have short tendrils. Aiyar and Nalini (1938) have described a similar difference in tendrils on the eggs of Ghiloscyllium griseum at Madras, India, and those of Malabar.
Figure 13 shows sample source areas, sizes of egg samples, and the known distribution of the presence or absence of tendrils. My first samples came from mainland sharks, and had tendrils from 80-200 cm. long. The next sample came -from the
inshore side of Santa Catalina Island; the eggs had no tendrils over two cm. long.
Numerous descriptions of various tendril-bearing elas- mobranch egg-cases state or assume that the tendrils are for attachment, to prevent the egg's being tossed about by wave action or currents. Indeed, those eggs with tendrils are often found entangled in marine algae strands and detritus. The mainland coast where tendril-bearing swell shark eggs are found receives almost continuous moderate to heavy surf. The inshore side of Santa Catalina Island, from whence came my second sample, which produced tendril-less eggs, rarely has any surf. To check this correlation between surf and tendrils, I obtained sharks from the offshore side of the island, which is, like the mainland, surfswept. Their eggs had no tendrils. (With the exception of one pair which had 15 cm. tendrils at one end.)
So, the correlation was between the presence of tendrils and being found on the mainland, not between tendrils and surf. As shown in figure 13, two egg-cases (in the S.I.O. Museum collection) from Isle G-uadaloupe, Mexico, which is some 260 km. from the mainland, and 420 km. from Santa Catalina Is. also lack tendrils. The difference between the eggs from sharks from Santa Catalina Is. and those from the mainland is consistent, whether the eggs are found in nature, or are laid in the aquarium. This suggests genetic rather than environmental control. The absence of exceptions to or intergrades between the two tendril types in the two locations studied (with the exception of the one instance mentioned) further suggests reproductive iso• lation. Santa Catalina Island is only some 30 km. from the main• land, but the intervening basin ie 3000 m. deep. When it is recalled that this is a reef-dwelling shark not given to swimming in midwater, it is conceivable that this basin is an - 47 -
effective geographical barrier which prevents, or at least severely restricts, migration between these two populations.
This reasoning led me to look for other differences.
Table 1 shows the results of two-tailed t-test comparisons of the lengths, widths, and weights of the two samples of eggs in appendix 3; one sample laid in one year by sharks captur• ed on the mainland, the other laid the next year by sharks captured at Catalina. The comparisons of all three parameters show that there is a:significant difference between the sizes of the eggs in the two samples. It is conceivable that there was a treatment difference - feeding, handling, temperature, etc. - between the two samples of sharks in the two different years, which might possibly have caused the egg size differences.
Nevertheless, I consider the data to be supportive, when considered together with the other differences.
In search of other differences, I turned to the sharks themselves. Simple visual comparison of sharks from all of the various locations described revealed no consistent dif• ferences inthe color pattern variations, or apparent differ• ences in gross morphology. But, statistical comparisons showed that both the first dorsal fin and the caudal fin were relatively larger in one population than in the other.
The ratios obtained by dividing the total length by, respectively, the height of the first dorsal (T,L./H.D.I.), and the width of the caudal (T.L./c.W.), for the samples of -48-
Pigure 13. Source locations of egg-cases. The triangles mark the locations. Underlined numbers are the approximate numbers of egg-cases laid in the laboratory by sharks from that location. Plain numbers are numbers of egg-cases seen which were found in nature at that location. - The + and - signs indicate the pres• ence or absence of tendrils on that sample of egg-cases. L.A. = Los Angeles; S.D. = San Diego; S.C. Is. = Santa Cata- lina Island. IH Jsla Guadaloupe - 49 -
Table 1. Comparisons by two-tailed t-test of measurements of egg samples from sharks captured at Isthmus Cove, Santa Catalina Island, and the mainland near Los Angeles.*
Source n x var. p_
Length: M 2Q 114.700 78.01? , <0.01 C 28 1 21.186 2 5.769
Width: M 20 41.850 19.819 05 C 28 44.714 4.138
Weight: M 20 26.593. 34.503 <0. 01 C 28 32.546 11.865
Weight**: M 14 29.593 11.874 01 C 26 32.996 9.581 4-
C .= Santa Catalina Is.; M =? mainland; p = probability that the samples are from the same population. The calculations were done by computer. The results were rounded to three decimal places for this table.
This comparisonbetween weights was done between only those eggs containing an ovum, because of the difference in weight of eggs without an ovum, and the difference in the propor•
tion of such eggs in:the two samples. (See appendix 3.) - 50 - the populations of the mainland and Santa Catalina Is. were compared by two-tailed t-test. The results of comparisons between various groupings are given in Table 2.
Note that both samples had sex ratios which were strongly biased, but in opposite directions. (See appendix 1). Because of this bias, the difference in fin proportions.between both entire samples could be attributed either to a population dif• ference, or to sexual dimorphism, which is found for some para• meters in some scyliorhinids (Brough, 1937). However, as shown in Table 2, the differences in those parameters tested are significant between populations, and not between sexes within the same population.
My contention that the sharks of Santa Catalina Island are a separate population from those of the mainland is based primarily on the difference in egg-case tendrils. The statis• tical data given on the other differences found - egg-case size and fin proportions - are too few to stand alone, but are supportive. When the local submarine topography and the shark's habits are also considered, the most logical conclusion is that the sharks form separate populations. - 51 -
Table 2. Comparisons by two-tailed t-test of morphometric ratios of different samples of sharks from Isthmus Cove, * Santa Catalina Island, and Point Dume, California.
Source Sex n ac T.L./H.D.I. x T.L./C.W. var.
D both 11 15.509 0.613 / 6).01 C . both 12 13.767 0.781 X
D both 16 11.638 0.061 / <0.01 C both 13 11.031 0.116
D P 10 15.600 0.580 / <0.05 C P 2 13.950 0.605 N
D ' M 5 11.760 0.027 / D M 5 11.760 0.027 1.00 C P 11 11.580 0.070 C M 11 11.000 0.114 1 .00 c P 2 11.200 0.180 c M 10 13.730 0.878 , 1 .00 C ' P 2 13.950 0.605 * C = Santa Catalina Is.; D = Pt. Dume; T.L. = total length; CW. = width of the caudal fin; H.D.I. • height of the first dorsal fin; p = probability that samples are from the same population. The calculations were done by computer. The results were rounded to three decimal places for this table. - 52 - DISCUSSION This discussion will generally follow the same sequence of topics as the foregoing text, with certain exceptions. Some of the descriptive portions are just that, and require no further attention. Other topics which were dealt with sep• arately in the text are structurally or functionally related, and are discussed together. Simultaneous formation of egg-cases in both oviducts is the general pattern seen in the swell shark, as it is in those other elasmobranchs for which the process has been described. It has been amply demonstrated that the synchronisation of the secretory processes in the two shell glands is quite close. But, in C_. ventriosum, and other species such as S. caniculus which have but a single ostium, the means by which this syn• chronization is effected presents a problem. The ostium is of a size which allows the passage of but a single ovum at a time. The ova must pass serially through the single ostium into the two oviducts, and the encapsulation of ova in each oviduct is simultaneous. Considering these two facts, it becomes apparent that synchronization of the two ova - one "waiting" for the other to "catch up" - must occur in the cranial oviducts, before the ova reach the shell glands. I have found no clue as to how this might occur in the swell - 53 - shark. Initiation and control of shell gland secretion is another unsolved problem. While I have suggested that al• bumen secretion might exert some influence on keratin secre• tion, the formation of "wind eggs", by the swell shark and other elasmobranchs, shows that the presence of ova in the oviducts or at the shell .gland is not the stimulus initiating secret• ory activity. Te Winkel (1950) has suggested that ovarian hormones may control shell gland secretory activity. I have described the torsion shown by the anterior ends of the swell shark's egg-cases (fig. 8), and the displacement of the respiratory slits; those on one edge of the egg-case are one side of the case, those on the other edge are on the op• posite side (fig. 7). The interesting thing about these two features of egg-case morphology is that, despite the fact that these eggs come from right and left paired shell glands, all eggs have the same "handedness"; the anterior end torsion and the slit displacement are the same, irrespective of the side of the shark from which the egg came. Here is, then, another example of departure from bila• teral symmetry in the elasmobranch reproductive system. Others mentioned earlier are the development of a single ovary in many species, and the asymmetry of the oviduct ostium in S. caniculus. Another which I have noted is the direction of spiral of the flanges on the egg-cases of heterodontid sharks; - 54 - it ie homologous to the much smaller thickening of the egg-case edges of swell sharks. The phenomenon may well be general in oviparous sharks. Despite Balfour's statements to the contrary, it seems most probable that the membrane surrounding the embryo and yolk, described in section four, is the vitelline membrane. It is unlikely that such a structure is unique to this species, considering the conservative nature of ontogeny. It is under• standable how it might be missed in studies of other species; it is extremely thin and transparent, and can only be seen by its refraction of a fairly strong light shining through the more transparent of the cases of the swell shark, which are generally more transparent than those of other elasmobranchs I have seen. Upon dissection to obtain the embryo and yolk, it is easily lost in the viscous albumen. From this, it follows that the chalaza-like structure I have described, which cannot itself be seen, but only inferred from its action on the membrane, could not have been previously described. Dissections of all sorts are normally carried out under strong incident illumination. If the eggs of the swell shark nnd other species were carefully dissected using strong trans• mitted illumination, the membrane in question could possibly be detected and separated from the albumen. - 55 - The main points of the pattern of reproduction in the swell shark are: internal fertilisation, an extended breeding season, and the production of a relative small number of large eggs, which have an extended development period resulting in fairly large young. This pattern is broadly typical of a wide variety of elasmobranchs, and suggests certain conclusions. The extended breeding season has at least two possible advantages. The production of large eggs, even though few in number - the known maximum in a year for any elasmobranch is 114 (Springer, 1967) - still entails a considerable energy ex• penditure, which is thus spread out over a long period. Also, the loss of any "year class", due to an unfavorable seasonal change in the environment, is reduced. Sperm storage in the female renders this extended egg production independent of repeated courtship and copulation. Sperm storage has now been directly demonstrated in the swell shark and two other species, and inferentially ehowm.in a few species of rays, but may well prove to be of more general oc• currence. Another possible advantage of sperm storage is implied by the fact that many elasmobranchs segregate by sex during part of the year (Springer, 1967). The data in appendix 1 suggest that this may occur in the swell shark. Also, copu• lation often results in/physical damage to the female in some species (Springer, 1967). - 56 - The extended development period and the large yolk supply allow the production of relatively large, mature young. The major portion of this development period is after the res• piratory slits in the egg-case have opened, after which the egg-case serves only as a protective barrier lessening the chances of physical damage to the embryo or the fragile yolk sac. Price and Daiber (1967) have suggested that the advantage of more efficient osmoregulation for the embryo has been the selection pressure favoring the evolution of intra-uterine development, from the oviparous mode, in elasmobranchs. Read (iy68) discounts thisj and suggests that protection against predation and mechanical injury are more likely selection pressures, but he describes no specific instances of such damage to egg-cases. I have described two types of predation upon shark egg- cases; one actually occurring in nature at present, and the other conceivably so. If such damage as this to elasmobranch egg-cases has occurred on any sizeable scale during their evolutionary history, it could well have provided the selec• tion pressure that Read has postulated. While describing my investigations of tendril-bearing vs. tendril-less eggs, I mentioned that many authors have stated or assumed that these tendrils are for attachment. This may - 57 - well be - or have been,in the course of evolutionary history - true. I have no data on survival rates of the tendril-less egg-cases on the calm and the surf-swept sides of Santa Catalina Island. Another function ascribed to these tendrils by Nalini and others is assisting in oviposition, by becoming entangled in seaweeds, etc., as they protrude from the cloaca, and al• lowing a shark to lay an egg by swimming away from it, so to speak. But, those elasmobranchs bearing tendril-less eggs seem to do so successfully - for example, I've never seen an "egg-bound" swell shark - and those bearing tendriled eggs can void them in a tank having a completely smooth bottom and walls. Until such time as some direct proof of one or more of these supposed functions for tendrils on egg-cases is set forth, I believe that the following quote must stand as an ac• curate description of the state of knowledge of egg-case tendrils. Kyle (1926), in a discussion of various elasmobranch egg forms, says, "In dogfishes ^includes scyliorhinids in England^ ...the long tendrils at the corners curl round the seaweed... "These are sometimes called adaptations, in their various ways, and it is significant that the egg-capsules of the Chimaeridae laid in deep water, and of most of the rays, do not have the attaching tendrils. But the purpose usually as• cribed to the special adaptations become mutually contradict- - 58 - ory when we take all cases into consideration, so that the sup• posed advantages are purely hypothetical." While I have indicated that the large, primary denticles on the young shark's back appear to serve a function in the shark's emergence from the egg-case, I do not wish to suggest that the shark's success in this maneuver is entirely depend• ant upon these primary denticles. The other denticles cover- t ing the young shark are smaller, but also have posterior- directed points, and could well function in the same manner as that postulated for the larger primary denticles, if some• what less efficiently because of their smaller size. However, the opening in the egg-case is a tight fit, and these young sharks have little stamina. If irritated in an aquarium, they will; swim actively for a very short time, then sink to the bottom, apparently exhausted. It seems to me' that any adap• tation that reduced the energy requirements of escaping from the egg-case would have definite survival value. In that section of this discussion ending with the quote from Kyle, it should have been apparent that I have had no success in developing a factually-supported explanation of the difference in tendril morphology between mainland and island sharks. But, the difference exists, nevertheless. In addition, I have described the difference I found in egg sizes, and in the body proportions of the sharks themselves. Some doubt will perhaps be cast on the later two; the egg size difference may have been the result of some treatment - 59 - difference, and the reliability of the morphometries would be greater if they were based upon larger sample sizes, in which there were no sex biases, despite the statistically significant differences between the samples obtained. Thus, the main burden of proof that there are separate populations at Santa Catalina Island and the mainland falls upon the difference detected first, that between the tendril forms. This difference, the presence or absence of a struc• ture, has proved consistent through time, and in both laborat• ory and field situations. It cannot, I believe, be consider• ed an artifact. This difference in egg-case morphology is as valid as any other consistent morphological difference between the in• dividuals of two populations. It has been suggested that I should separate the Santa Catalina Island and mainland popu• lations into species, on the basis of this difference alone (pers. comm., C. Hubbs). I have not, however, because of the lack of knowledge of the distribution of the.difference (no data at all from the other coastal islands), and the un• certain state of the relationships within the genus as a whole along its eastern Pacific distribution (pers. comm., S. Springer). In reflecting back over this thesis, and the checkered progress of the research leading to it, it eeemsto me that I have raised more questions.than I have answered, and I see - 60 - a good marry things left undone, as well as things which could have been more profitably done differently. But these are, after all, some of the reasons which justify the effort. - 61 - SUMMARY The swell shark, Cephaloscvllium ventriosum Garman, is a small, reef-dwelling, nocturnal scyliorhinid. Aspects of its oviparous reproduction, related structures of the sharks and eggs, and population differences in these, are the subjects of this thesis. 1. Descriptive anatomy of the female reproductive system. a. Ovary, ova, and peritoneal ciliation functioning in ovum transport. These are generally as described for relat• ed species, with a difference in the pattern of ciliation. b. Ostium and oviducts. The single ostium admits ova to the two oviducts, and is dissimilar in structure to relat• ed species previously described. c. Circulatory system. The structure and volume of the vessels draining the oviducts and shell glands is unique, and is not described in detail elsewhere. Each oviduct, including the shell gland, is surrounded by a large sinus. These drain to the postcardinals, which are confluent in this area. d. Shell gland structure, secretions, and sperm storage. The keratin-secretory tubules of the shell gland function as a sperm storage site. While sperm storage has been assumed or indirectly demonstrated for several species of elasmobranchs, the site has been histologically demonstrated for only two other species. Photomicrographs are included which show sperm - 62 - in the keratin secretory tubules. 2. Egg-case structure. No detailed description of the egg- case and the variations which occur has been previously published. This forms a necessary background to following sections. 3. Mechanics and sequence of egg-case formation. Prior ob• servations and speculations are discussed in detail, and re• interpreted in the light of my own observations, 4. Development of the embryo and egg-case changes. The sequence of development of the embryo itself is generally in accord with prior descriptions published for other species, and it was not an objective of this study to record them in detail. However, I have observed two structures which, to my knowledge, have not been previously described; a membrane surrounding the embryo and yolk during the earlier stages of development, and a chalaza-like structure attached to this membrane. 5. Emergence of the shark from the egg-case. The young of several species of sharks and chimaerids have two longitudinal, dorsolateral rows of large denticles, which disappear some time after the young emerge from their egg-cases. No function has previously been attributed to these denticles. Prom direct observation and analysis of motion pictures, it appears that these specialized denticles function in the emergence of the young from their egg-cases. a 63 - 6. Predation on egg-cases. I have found no mention in the literature of predation upon elasmobranch egg-cases. Evidence is presented showing predation, probably by a stenoglossid gastropod, on a high proportion of a number of egg-cases found at one location. 7. Population differences. The egg-case from sharks from two locations show consistent differences in morphology. This and other evidence from the ecology of the species, the local submarine topography, and morphometries of the eggs and of the sharks themselves, is presented in support of the hypothesis that there exist at least two reproductively isolated popula- 30 tions, separated by as little as 33f km. - 64 - REFERENCES Aiyar,R. G. , and K. P.Nalini, 1938. Observations on the repro• ductive system, egg-cases, and breeding habits of Chilio- sc.yllium griseum Mull, and Henle. Proc. Ind. Acad. Sci. B7:252-269. Balfour, F.M. 1878. A monograph on the development of elasmo- branch fishes. Macmillan , London. , 1880. Comparative embryology. Vol. I. Macmillan, London. , 1881. Comparative embryology. Vol. II. Macmillan, London. Brough, J. 1937. On certain secondary sexual characteristics of the common dogfish, Scyliorhinus caniculus. Proc. Zool. Soc. London. 107B:217-223. Chieffi, G. 1962. Endocrine aspects of reproduction in elas- mobranch fishes. Gen. Comp. Endocr. Suppl. 1;275-285. Clark, E. 1947. Note on the inflating power of the swell shark, Cephaloscyllium uter. Copeia 1947:279-280. Clark, R.S.1922. Rays and skates (Raiae) No. I; Egg capsules and young. J. Mar. Biol. Assoc. U.K. 12:577-643. Collenot, G. 1966. Observations relatives au developpement au laboratoire d' embrypns et d'individus juve'niles de Scyliorhinus canicula L. Cahier de Biologie Marine 7:319-330. - 65 - Cox, K. W. 1963. Egg-cases of some elasmobranchs and a cyclo- stome from Californian waters. Calif. Pish and Game 49:271-289. Daniel, J.P. 1934. The elasmobranch fishes. Berkeley, Calif. Edwards, H.M. 1920. The growth of the swell shark within the case. Calif. Pish and Game. 6:153-157. Pord, E.1921. A contribution to our knowledge of the life histories of the dogfishes landed at Plymouth. J. Mar. Biol. Assoc. U.K. 12:469-505. Harris, J. E. 1952. A note on the breeding season, sex ratio, and embryonic development of the dogfish Sc.vliorhinus canicula. J. Mar. Biol. Assoc. U.K. 31:269-275. , 1962. Early embryonic movements. J. Obstet. Gynaec. Brit. Cwlth. 69:818-821. Herald, E. 1962. Living fishes of the world. Doubleday, N.Y. Hobson, A. D. 1930. A note on the formation of the egg case of the skate. J. Mar. Biol. Assoc. U.K. 16:577-581. Kato, 3., S. Springer, and M. H. Wagner, 1957. Field guide to eastern Pacific and. Hawaiian sharks. U.S. Bureau Comm. Fisheries Circular 271. Kyle, H. M. 1926. The biology of fishes. Sidgwick and Jackson, London. Libby, E. L. 1959. Miracle of the mermaid's purse. Nat. Geog. Mag. 116:413-420. Light, S.P., et al. 1957. Intertidal invertebrates of the central California coast. Univ. of Calif. Press, Berkeley. - 66 - Mellinger, J. 1965. Stades de la spermatogenese chez So.vlio- rhinus caniculus (L.): description, donates histochemi- ques, variations normales et experimentales. Z. Zellforsch. 67:653-673. Metten, H. 1939. Studies on the reproduction of the dogfish. Phil. Trans. Roy. Soc. London 230:217-238. , 1944. The fate of spermatozoa in the female dogfish. (Scyliorhinus canicula). Quart. J. Micr. Sci. 84:283- 294. Nalini, K.P. 1940. Structure and function of the nidamental gland of Chilosc.vllium griseum (Mull, and Henle). Proc. Indian Acad. Sci. Sect. B 12:189-214. Norman, J. R.,and P. H. Greenwood. 1963. A history of fishes. Hill and Wang, N.Y. Prasad, R.R. 1945. Further observations on the structure and function of the nidamental glands of a few elasmobranchs of the Madras coast. Proc. Indieja Acad. Sci. Sect. B 22:368-373. , 1951. Observations on the egg-cases of some oviparous and vivaparous elasmobranchs, with a note on the for• mation of the elasmobranchs egg-case, J. Bombay Nat. Hist. Soc. 49:755-762. Price, K.S., and F. C. Daiber. 1967. Osmotic environments during fetal development of dogfish, Mustelus canis (Mitchell) and Squalus acanthius Linnaeus, and some 67 - comparisons with skates and rays. Physiol. Zool. 40: 248-260. Read, L.J. 1968. Urea and trimethylamine oxide levels in elasmobranch embryos. Biol. Bull. 135:537-547. Setna, S.B., and P.N. Sarangadhar. 1948. Observations on the development of Chiloscyllium griseum M. & H., Pristis cuspidatus Lath, and Rhynchobatus d.iiddensis (Porsk.). Rec. Indian Mus. 46:1-24. Smith, B.G. 1940. The breeding habits, reproductive organs, and external embryonic d evelopment of Chlamydoselachus. based on notes and drawings by Bashford Dean. Am. Mus. Nat. Hist. The Bashford Dean Memorial Volume. Archaic Pishes. Article 7:525-633. (E.W. Gudger, ed.) , 1942. The heterodontid sharks: their natural history, and the external development of Heterodontis .iaponicus based on notes and drawings by Bashford Dean. Am. Mus. Nat. Hist. The Bashford Dean Memorial Volume. Archaic Pishes. Article 8. 651-770. (E.W. Gudger, ed.). Smith, S. 1957. Early development and hatching in Physiol• ogy of fishes. Vol. 1:323-359. (M. Brown, ed.) Academic Press, N.Y. Springer, S.I967. Social organization of shark populations, in Sharks, skates, and rays. (P. Gilbert, R.P. Mathew- son, and D.P. Rail, eds.) Johns Hopkins Press, Baltimore. - 68 - Te Winkel, L. 1950. Notes on ovulation, ova, and early development in the smooth dogfish, Mustelus canis. Biol. Bull. 99:474-486. Threadgold, L.T. 1957. A histochemical study of the shell gland of Scyliorhinus caniculus. J. Histochem. Cytochem. 5:159-166. Appendix 1. Body measurements (in cm. used for morphometric comparisons. T,L. = total length; H.D. I. = height of the first dorsal fin; CW. = width of the caudal fin; F = fin frayed, not meas• urable . Part A. Sharks captured in or near Isthmus Cove, Santa Catalina Island. Sex T.L. H.D.I. CW. Female 796 55 69 805 60 74 Male 906 68 82 . 875 67 80 771 55 70 837 — 78 700 59 61 869 64 81 895 65 79 « 662 49 62 750 52 65 « 794 55 71 787 51 74 Appendix 2. Pre-caudal vertebral counts of sharks from various locations. Santa Catalina Island. 69, 72, 70, 70. California mainland - Los Angeles area. 70, 72, 70, 70, 68, 72, 70, 71 . Isla G-uadaloupe, Mexico. 67, 67, 68, 68. Appendix 3. Measurements of shark eggs laid in the laboratory. (Dimensions in mm., weight in grams.) An asterisk by the weight indicates those eggs not containing an ovum. Part A. Eggs laid in 1963 by sharks captured near Los Angeles. Date Length Width Weight 22 July 115 48 29.0 n 114 49 29.2 28 July 134 47 34.5 it .111 46 32.0 it .134 47. 35.0 ii : 115 45 32.0 1 1 1 46 31.5 II 115 45 *23.2 26 August 105 37 *18.1 II 111 37 24.3 3 September 128 44 33.1 II 118 42 *23.7 18 September 115 39 28.3 II 117 39 26.8 25 October 103 36 *16.4 it 103 37 *16.4 19 November . 105 37 *17.9 II 110 38 25.4 26 December 118 39 26.5 II 112 39 26.7 Appendix 1. Part B. Sharks captured near Point Dume, Sex T.L. H.D.I. CW. Female 824 50 71 II 831 52 71 ti 820 52 70 n 802 53 69 II 738 P 62 it 809 51 71 II 764 53 67 it 812 53 72 it 731 48 63 II 759 51 68 II 845 50 70 Male 803 55 68 II 718 P 62 it 659 P 57 it 792 P 66 ti 734 62 P = Fin frayed Appendix 3. Part B. Eggs laid in the laboratory in 1964 by sharks captured in or near Isthmus Cove, Santa Catalina Island. Date Length Width Weight 26 June 119 47 30.5 it 119 46 30.6 II 117 45 29.7 ti 116 46 30.0 27 June 127 45 34.9 ii 125 41 * 24.8 II 124 46 35.6 II 124 47 34.6 17 July 134 47 38.1 II 134 47 38.3 II 126 45 36.1 II 125 45 37.0 20 July 125 47 36.9 ti 125 47 36.0 II 122 4'6 35.6 ti 121 47 35.2 31 July 120 44 30.7 it 120 44 30.1 II 118 45 29.6 II 118 44 29.6 21 August 115 43 28.3 ti 117 43 30.4 ii 118 43 30.5 ii 118 44 30.7 8 October 117 4:2 32.4 II 115 43 * 28.6 14 December ** 116 39 34.1 II •* 121 44 32.4 ** These two eggs were taken while diving at Isthmus Cove.