Reproduction and Development in Chondrichthyan Fishes

Transcription

1 AMER. ZOOL., 17: (1977). Reproduction and Development in Chondrichthyan Fishes JOHN P. WOURMS Department of Zoology, Clemson University, Clemson, South Carolina SYNOPSIS Patterns of chondrichthyan reproduction and development are diverse. Species either are reproductively active throughout the year, or have a poorly defined annual cycle with one or two peaks of activity, or have a well defined annual or biennial cycle. Based on embryological origin and adult morphology, their reproductive system is more similar to tetrapods than to teleosts. Primordial germ cells are of endodermal origin. The Wolffian ducts in males and Mullerian ducts in females become the functional urogenital ducts. Differentiation is under hormonal control. Unusual features of the reproductive system include an epigonal organ in males and females. It contains lymphoid and hemopoietic tissue. Leydig's gland, a modified region of the kidney, produces seminal fluid. In some species, sperm passing through the vas deferens, is enclosed in spermatophores. Rotating about their long axis, helical spermatozoa can move forward or reverse direction. Spermatogenesis often occurs in bicellular units, spermatocysts. These consist of a spermatogonium enclosed in a Sertoh cell. Fertilization is internal. Claspers, modified portions of the pelvic fins act as intromittent organs. In many viviparous sharks and rays, the female reproductive system is asymmetrical. Eggs of some sharks are the largest known cells. Yolk platelets contain lipovitellin. Oocytes have lampbrush chromosomes. Eggs released from the ovary into the body cavity are transported by ciliary action to the ostium of the oviduct. There they are fertilized. Physiological polyspermy is normal. The shell gland, a specialized region of the anterior oviduct, functions both in long term sperm storage and in egg case production. Egg cases of sharks and skates consist of unique collagenous protein with a 400 A period, organized as a cholesteric liquid crystal. Chimaeroid egg cases contain 550 A pseudotubules in orthogonal lattices. In small sharks, males copulate by coiling around the female. A parallel position is assumed by large sharks. Skates and rays copulate with ventral surfaces apposed or by a dorsal approach. Biting is a pre-copulatory release mechanism. Parental care, except for selective oviposition, is lacking. Heavily yolked eggs undergo meroblastic, discoidal cleavage. Development is lengthy, shortest (2-4 months) in rays, longer in skates (3-8 months) and longest (9-22 months) in sharks and chimaeras. Most sharks and all rays are viviparous. Chimaeras, skates, and some sharks are oviparous. Viviparity either involves a yolk sac placenta or is aplacental. If aplacental, the embryo derives nutrients either from yolk reserves, or by intra-uterine embryonic cannibalism, or from placental analogues which secrete "uterine milk." Phylogenetic position, geographical distribution, benthic vs. pelagic habitat, adult size, egg-embryo size, feeding ecology, and embryonic osmoregulation are factors in the retention of oviparity or the evolution of viviparity. INTRODUCTION the two groups have in common than by the ways in which they differ. This is The chondrichthyan or cartilaginous especially true with respect to patterns of fishes include the sharks, skates, rays, and reproduction and development in which chimeras. They are one of the oldest living structural and functional similarity that is groups of jawed vertebrates. They are manifest at the anatomical level becomes often considered to be primitive or ar- even more striking at the level of tissue, chaic. Yet, when one considers that the cell, and molecule. These fishes almost chondrichthyan fishes and the amniotes seem to constitute an experimental system represent two extremes of vertebrate in which many of the novel structures and evolution, one is more impressed by what processes characteristic of the vertebrates were first developed. I am grateful to Dr. James W. Atz of the American. Chondrichtyan fishes are of particular Museum of Natural History for reading portions of interest to reproductive and developmenthe manuscript and for many helpful suggestions. tal biologists since, for the first time in the 379

2 380 JOHN P. WOURMS vertebrate line, the following processes either make their appearance or else become well established: 1) internal fertilization; 2) viviparity; 3) placental mechanisms for fetal maintenance; 4) patterns of genital tract development and sex differentiation, closely resembling the ones in amniotes; 5) vertebrate type of reproductive endocrinology. The first four topics will be reviewed as well as related areas of interest such as: patterns of reproduction; gametogenesis; fertilization and early development; egg case ultrastructure; the role of the shell gland in egg case formation; and the significance of viviparity. Reproduction and development of holocephalans (chimaeras) will be discussed in terms of available information. The endocrinology of reproduction will not be treated in detail since it has been extensively reviewed in recent years (Chieffi, 1967; Dodd, 1972, 1975). HISTORICAL REVIEW The earliest recorded observations on the reproductive biology of chondrichthyan fishes are those of Aristotle. He distinguished between oviparous and viviparous species and described the egg cases of the former. He discovered the yolk sac placenta in Mustelus laevis Risso [= M. canis (Mitchill)] and was aware that it differed from the mammalian placenta (Aristotle, Peck translation 1965, 1970). Although Aristotle's observations were neglected for many centuries, it is unlikely that knowledge of elasmobranch reproduction was ever lost entirely. Since skates and dogfish have continued to be staple items of diet since ancient time, both fishermen and cooks would undoubtedly have distinguished between oviparous and viviparous species and would have been familiar with their anatomy. A revival of formal interest in elasmobranch reproduction coincided with a revival of interest in natural history during the Renaissance. Rondelet (1554), one of the early zoological encyclopedists illustrated part of the ovary and the egg case of a skate. He also depicts a female shark (probably M. laevis) which is connected to a fetus by an elongated yolk stalk which passes from the fetus through the cloaca of the female. A placenta is not illustrated, however. By 1673, the anatomist Steno had rediscovered and published an illustrated account of the placenta in - Mustelus laevis. What happens next constitutes a curious chapter in the history of biology. It has been generally claimed that the works of Aristotle, Rondelet, and Steno were overlooked until the time of Johannes Muller (1842). This does not seem to have been the case. Review works such as that of Bohadsch (1776, also 1761) cite all three of these authorities. Moreover, Muller (1842) discusses in detail the work of his immediate and distant predecessors. It is more accurate to say that Muller, an outstanding anatomist and one of the founders of physiology, was better able to appreciate the significance of his findings and to communicate them to a receptive audience. His classic paper marks the beginning of modern studies of chondrichthyan reproduction and development. Not only did he review all previous studies, but he also greatly expanded on them with his own original observations. The structure and probable function of the placenta in the dogfish M. laevis and the blue shark Prionace glauca (Linnaeus) were treated in detail. A comparative approach was used to study reproduction in non-placental viviparous species, ovoviviparous species, and oviparous species. In the latter, not only were shark and skate egg cases studied but also those of the chimaera, Callorhynchus. Following Muller, research has tended to progress through areas of current interest to the developing science of biology. From Miiller's time to the present, certain papers can be recognized as landmarks. A few are listed here. Embryological studies were firmly established by Balfour (1885). Ruckert (1899) and Ziegler and Ziegler (1892) worked out the early stages of development. Dean and his associates produced monographs on: the reproduction and development of chimaeras (Dean, 1906); the frilled shark Chlamydoselachus (Smith, 1937; Gudger, 1940); and the heterodontid sharks (Smith 1942). Van-

3 CHONDRICHTHYAN REPRODUCTION 381 ^ debroek (1936) carried out the principal study of morphogenetic movements associated with gastrulation. Needham (1942) reviewed the physiology of de- velopment. A number of studies have dealt with the anatomy of the reproductive system (c/., Dean for early references). In this respect, Borcea's (1905) study is outstanding. He paid particular attention to the role of the shell gland in egg case production. Chemical studies of the egg case and its cellular origin originate with Faure-Fremiet and Baudoy (1938) and Filhol and Garrault (1938). Beginning in 1922, Leigh-Sharpe reported on a series of investigations of secondary sex characteristics. Viviparity has been an area of special interest (Gudger 1912, 1951; Te Winkel, 1943, 1950). In a unique series of papers Ranzi (1932, 1934) reported on comparative studies of adaptations for viviparity. In addition to placental species, he also considered non-placental species that displayed special embryonic and maternal adaptations, viz., "placental analogues." Intra-uterine oophagy was reported by Shann (1923) and Springer (1948). The physiology of gestation has attracted the attention of Daiber and his associates (Price and Daiber, 1967; Graham, 1967). Gilbert and his co-workers have contributed not only to the study of placentation (Gilbert and Schlernitzauer, 1966; Schlernitzauer and Gilbert, 1966) but also to an understanding of clasper-siphon sac function (Gilbert and Heath, 1972). Reproduction and reproductive seasons have been treated by Matthews (1950) in the basking shark Cetorhinus, the blue shark Prionace glauca (Tucker and Newnham, 1957), the spiny dogfish Squalus acanthias Linnaeus (Hishaw and Albert, 1947; Jensen, 1966), and the skate Raja erinacea Mitchill (Fitz and Daiber, 1963; Richards et al., 1963). REPRODUCTIVE CYCLES On the basis of Breder and Rosen's (1966) massive review offish reproductive patterns and cycles, it is apparent that chondrichthyan fishes are either oviparous or viviparous. Information on reproductive cycles tends to be incomplete or fragmentary. The present state of knowledge is unsatisfactory especially when compared to rigorous studies of invertebrate reproductive cycles (Giese, 1959; Giese and Pearse, 1974). Sampling is a major problem. Large samples need to be taken at appropriate intervals over a period of several years. This has been done only for a few small, inshore species or for larger species which have been the subject of commercial fisheries. Based on a limited survey of three inshore forms, Squalus, Mustelus, and Scyliorhinus, Dodd (1972) postulated the existence of sophisticated annual reproductive cycles. The situation is more complex. Three basic types of cycles are encountered: 1) reproduction throughout the year; 2) a partially denned annual cycle with one or two peaks; and 3) a well defined annual or biennial cycle. The first category consists of those species which are either reproductively active throughout the year or for the major part of the year, e.g., Scyliorhinus, Chlamydoselachus, and Heterodontus (in part). Ford (1921) and Metten (1939) report that a population of Scyliorhinus canicula (L.) from the English Channel in the vicinity of Plymouth breeds throughout the year. Although somewhat more prolific in spring and summer, they have no definite breeding season. Based on 2000 specimens collected in the IIfracombe region, Harris (1952) concluded that the spawning season lasts about 8-9 months starting in November and continuing until July. Dodd's (1972) observations that ovaries of female dogfish from areas of the Irish Sea become quiescent during the summer support Harris' view. Are these differences real or due to sampling bias? Harris (1952) raised the question of sampling error since Ilfracombe probably represents a spawning ground into which females migrate at the spawning season. Gudger (1940) reported that the frilled shark, Chlamydoselachus anguineus Garman, a viviparous species, breeds throughout the year. He attributes its continuous reproductive activity to the relative constancy of a deep sea habitat. Finally, a variety of cycles are encountered within

4 382 JOHN P. WOURMS the genus Heterodontus. Barnhart (1932) states that the California species H. francisci (Girard) spawns throughout the year. Although the population of H. japonicus Macleay in the vicinity of Misaki, Japan, does deposit eggs throughout the year, there is an apparent peak in activity in March-April (Smith, 1942). Recent field studies of McLaughlin and O'Gower (1971) reveal a well defined August-September spawning season for Australian populations of//, portusjacksoni (Meyer). The second category includes those species with a partially defined annual cycle. Although reproductively active throughout the year, they tend to exhibit one or two peaks in activity. Raja erinacea Mitchill and Hydrolagus colliei (Lay and Bennett) exemplify this pattern. Richards et al. (1963) reported that R. erinacea is reproductively active throughout the year. (Their study lasted almost 10 years and included detailed examination of 15,000 specimens per year). Once having attained maturity, adult males produce sperm continuously. Mating, fertilization, and the production of egg cases take place throughout the year. The study compared two distinct, but geographically close populations, one in Block Island Sound and the other in Long Island Sound. The presence of egg cases within a female was considered evidence of its reproductive activity. The Block Island Sound populations displayed two peaks. During November-January, 30-60% of the adult females were "gravid" and during June and July, 15-30% of the females were "gravid." During the remainder of the year, the per cent of "gravid" females was under 25% and tended to average about 10%. The Long Island Sound population had a similar pattern with peaks of 5-15% during the November-January and June- July periods as well as a low level of "gravid" females during the rest of the year. Similar patterns were found in a Delaware Bay population although the summer peak was greater than the fall (Fitz and Daiber, 1963). In a related species, R. eglanteria Bosc they found that egg maturation and spawning occurred only during the spring. Differences in reproductive cycles between two populations of the same species as well as differences between species, suggest a need for careful study and cautious generalization. Among the chimaeroids, only Hydrolagus colliei (Lay and Bennett) of the Pacific Coast of North America is readily accessible for study. Dean (1906) reported that although it is reproductively active throughout the year, a peak in activity probably occurs during late summer and early fall. More recently, Stanley (1961, cited in Johnson and Horton, 1972) confirmed that the summer was a period of peak reproductive activity although 33% of the females and all the males showed evidence of activity throughout the year. The third category includes those species with a well defined annual or biennial cycle, viz.,squalus acanthias L.,Mustelus canis (Mitchell) and Urolophus halleri Cooper. It may also include other migratory forms such as Eulamia milberti (Miiller and Henle) and Cetorhinus maximus (Gunner). The spiny dogfish, Squalus acanthias, widely distributed throughout the northern regions of the Atlantic and Pacific oceans, shows a striking periodicity. Males display an annual cycle. Females display a biennial cycle which can be attributed to a gestation period of 22 months. Parturition usually occurs in the autumn. Shortly thereafter copulation ensues (Hisaw and Albert, 1947; Holden and Meadows, 1964; Jensen, 1966). Simpson and Wardle (1967) discovered an annual cycle of activity in the male testes. Maximum sperm accumulation coincided with the January- February breeding period of the biennial female cycle. The smooth dogfish, Mustelus canis, has also been reported to have an annual cycle (Hisaw and Abramowitz, 1939). The details of the cycle were established by Graham (1967) for a migratory population in Delaware Bay. This species is also viviparous. The gestation period, however, is 11 months long (Te Winkel, 1950). M. canis winters off North Carolina. Northward migration begins in early spring. In Delaware Bay, males first appear during the last week of April and the first week of May. Females first appear during the next two weeks. Parturition

5 CHONDRICHTHYAN REPRODUCTION 383 occurs at this time. Ovulation occurs during the first three weeks of June. Mating is restricted to the time after parturition and before ovulation. Ovulation is said to be dependent upon copulation (Hisaw and Abramowitz, 1939). The population seems remarkably well synchronized since all gravid females carry embryos at the same stage of development. In the round sting ray of California, Urolophus halleri Cooper, the major reproductive season occurs in late May, June, and early July (Babel, 1967). At this time, most males are in mating condition, and the majority of females ovulate. Shortly thereafter small embryos are to be found in their uteri. Quantitative studies demonstrated an annual cycle both of spermatogenesis and oogenesis. A small number of females are out of synchrony with the rest of the population and ovulate in December. They are successfully fertilized with sperm which has been stored for several months. Annual cycles have also been reported in some of the larger migratory species of sharks, both inshore and pelagic. Available information although fragmentary is of interest. The primary winter range of the sandbar shark, Eulamia (=Carcharhinus) milberti is off the southeastern coast of the United States. It is extended as far north as Long Island and Cape Cod to form a primary nursery range where from late March to early August the young are born. Although the period of gestation is nine months, females only seem to reproduce every other year. Sexes tend to be segregated, except during courtship and mating. In southeastern Florida, June is the time of maximum mating activity (Springer, 1960). The basking shark, Cetorhinus maximus (Gunner), illustrates the complications which seasonal migrations, both vertical and horizontal, introduce into the study of reproductive cycles. In his definitive study, Matthews (1950) presents circumstantial evidence that this shark is viviparous. Direct evidence in the form of gravid females is lacking. This is remarkable considering that this species, the second largest shark, is common and has been the subject of commercial fisheries. Matthews (1950) is of the opinion that the reproductive cycle of the basking shark is correlated with its seasonal migration. Off the west coast of Scotland where his studies were conducted, these sharks begin to appear in April and become most numerous in May and June. Similar spring inshore movements have been reported in Norway and British Columbia. All of the adult fish which were examined were in breeding condition and showed signs of recent copulation. Based on these observations, there appears to be a single yearly reproductive cycle which is at its peak during the second half of May. There remains, however, the problem of the nature and duration of the female cycle. Since gravid females have not been reported in modern times, Matthews (1950) suggests that they desist from basking at the surface sometime before the embryo reaches a recognizable size. One assumption is that they migrate to deep, off shore water during pregnancy. The period of gestation obviously is not known, hence the duration of the female cycle is not known. Older reports, cited by Matthews, suggest that males may be sexually active during the entire year. DEVELOPMENT OF THE CONADS AND GENITAL DUCTS On the basis of its embryological origin and morphology, the reproductive system of chondrichthyan fishes is more similar to amphibia and amniotes than to teleosts. In most vertebrates, including chondrichthyan fishes, the somatic portion of the gonad has a dual origin, the cortex and medulla. These two tissues, although in close proximity, are distinct and have different developmental histories. This pattern is in contrast to cyclostomes and teleosts where the somatic tissue of the gonad has a single origin, the peritoneal epithelium. Hence the somatic portion of the gonad is comprised entirely of the cortex (Chieffi, 1967; Hoar, 1969). Chieffi (1967) has provided a modern account of gonad development and differentiation in Torpedo ocellata and Scyliorhinus caniculus. Gonads develop in the dorsolateral lining of the peritoneal cavity in the posterior

6 384 JOHN P. WOURMS half of the body. Usually there is one gonad on each side of the dorsal mesentery. The undifferentiated gonad is derived from two sources, the cortex and the medulla. The cortex which is more laterally located develops first. It appears as an elongated strip of the mesodermal epithelium which forms the peritoneal wall. This thickening becomes a multilayered convex mass of cells which protrudes into the coelomic cavity to form the germinal ridge. On its dorsal side, the ridge develops a hollow which is filled by migrating mesoderm cells. These cells comprise the medulla. In Scyliorhinus, the medulla is derived from the nephrogenic cord (=interrenal blastema) while in Torpedo, it is derived from the same center of proliferation which forms the nephrogenic cord. Under normal conditions, genetic factors determine whether the embryonic gonad will differentiate into an ovary or testes. The first step involves the migration of primordial germ cells to the gonad. Hoar (1969) states that there is considerable evidence for a widespread origin of the primordial germ cells. In the elasmobranchs, however, it is well established that the primordial germ cells segregate from the primitive entoderm quite early in development (prior to embryo formation?) and migrate via the mesoderm into the site of the developing gonad (Beard, ; Woods, 1902; Hardisty, 1967). There they settle in the cortical region of the gonad. At this stage, the gonad is considered indifferent. In genetic females, the primordial germ cells once having settled in the cortex retain their cortical location. In Torpedo, a few germ cells do migrate from the cortex to the medulla in mm female embryos. Transient connections are established with the mesonephric tubules, but these soon degenerate. Formation of the primary ovarian follicles occurs by the 75 mm stage. In genetic males, primordial germ cells migrate from the cortex to the medulla (at 22 mm in Torpedo; at mm in Scyliorhinus). Formation of the sex cords and the seminiferous ampullae occur during the mm stages of Torpedo (Chieffi, 1967). Once germ cell migration has been completed, that region, either cortex or medulla, which will form the definitive gonad grows rapidly while the remaining region fails to develop. Thus, the sex of the individual is determined 4 quite early. Secondary sex characters also make an early appearance. According to Chieffi (1967), their appearance coincides with sexual differentiation in the genital ridges. The reproductive ducts provide the means for conveying gametes from the gonads to the exterior. In common with most vertebrate embryos, elasmobranchs develop two sets of urogenital ducts, only one of which will function as a reproductive duct. In males, the functional duct is the mesonephric or Wolffian duct and in females, it is the Miillerian duct. The mesonephric duct is derived, by direct transition, from the pronephric duct. In males, the excretory and genital functions of the Wolffian (mesonephric) duct become segregated. In Squalus acanthias, mesonephric tubules establish connections between the seminiferous tubules of the testes and the Wolffian duct. These tubules become the vasa efferentia or efferent ducts. The remainder of the duct differentiates into an epididymis, vas deferens, and seminal sac. The urinary or opisthonephric duct is independent of the mesonephric duct. In female embryos, however, the upper part of the Wolffian duct atrophies while the lower part serves as urinary duct (Balfour, 1885; Kerr, 1919; Goodrich, 1930; Nelsen, 1953). In female elasmobranchs a second set of ducts, the Miillerian ducts, function in reproduction. They become the oviducts. Although the embryonic development of the Miillerian ducts in elasmobranchs differs from that of other vertebrates, their function and anatomical relationships in the adults are remarkably similar. The Miillerian ducts of elasmobranchs are well developed and appear early during the course of ontogeny. The anterior end of each duct opens into the coelom by a funnel, the ostium tubae. Not infrequently, the funnels of the right and left ducts combine to form a single median ostium. The duct, proper, is of large size, regionally specialized, and opens inde-

7 CHONDRICHTHYAN REPRODUCTION 385 pendently into the cloaca. Miillerian ducts develop from the pronephros and the pronephric duct. The funnel region is derived directly from one or more prok nephric nephrostomes. The main portion of the Miillerian duct develops from the pronephric duct. The pronephric duct undergoes a gradual longitudinal splitting into an anterior-posterior direction to produce a dorsal and ventral tube. The ventral tube is continuous with the pronephric funnel and becomes the Miillerian duct. The dorsal tube receives the kidney tubules. It is a true Wolffian (mesonephric) duct which persists as the functional urinary duct of the opisthonephros. Both ducts open separately into the cloaca (Kerr, 1919; Goodrich, 1930). Miillerian ducts also develop in males. In immature males of some species, e.g., Notorynchus maculatus Ayers, they may persist as rudimentary right and left oviducts (Daniel, 1928). Normally, they atrophy or survive only as vestigal funnels (Goodrich, 1930). Chieffi (1967) reviewing the reaction of the duct systems to steriod hormone treatment presents evidence for hormonal control of differentiation. Other important secondary sex characters are external and are found in males, viz., the pelvic claspers or copulatory organ, found in almost all Chondrichthyes; the frontal (cephalic) clasper of the chimaeras; and alar spines on the pectoral fins of skates. Of these, only the pelvic claspers have received any significant attention. The claspers are derived from the medial margin of the pelvic fins and develop simultaneously with the sexual differentiation of the testes. In Torpedo, growth of the clasper is not affected by treatment with steriod hormones. Chieffi (1967) suggests that they are a somato-sexual character. MALE REPRODUCTIVE SYSTEM Functional Organization The male reproductive system consists of the testes, accessory glands, genital ducts, and secondary sex organs. Its organization has been the subject of numerous studies {cf., Borcea, 1905; Dean, 1906; Daniel, 1928; Matthews, 1950; Stanley, 1963) so it will not be treated in detail here. The testes are paired organs attached to the body wall along either side of the vertebral column by a mesorchium. Testes vary in size and shape and are often enlarged during the breeding season. Closely associated or even sometimes combined with the elasmobranch testes are the epigonal organs. These consist of lymphoid or hemopoietic tissue (Matthews, 1950). According to Stanley (1963), the epigonal organ and hemopoietic tissue are not pressent in the reproductive system of holocephalans. Spermatogenesis occurs within the testis, in units termed ampullae. Hoar (1969) has pointed out inter-specific variation in the basic organization of the testis. The testis of the basking shark consists of many lobules, separated by connective tissue trabeculae (Matthews, 1950). Each lobule is equivalent to the entire testis of the dogfish, Scyliorhinus (Mellinger, 1965). In the dogfish testis, the spermatogenic ampullae are arranged in six zones which correspond to stages in ampullae formation and spermatogenesis (Mellinger, 1965). Mature sperm are discharged from the testis through the efferent ducts (vasa efferentia). In sharks, the number of efferent ducts range from two to six. In skates and rays, there is a single efferent duct (Daniel, 1928; Babel, 1967). The efferent duct(s) joins the epididymis. The epididymis usually assumes the form of a coiled tubule but in some species, e.g., Cetorhinus, it may form a compact mass of highly convoluted tubules (Matthews, 1950). The epididymis passes into the vas deferens. There is usually a well defined region of demarcation between the two structures. The anterior portion of the vas deferens tends to be coiled while the posterior portion extends as a straight tube to the urogenital sinus. In many elasmobranchs, "sperm sacs" arise as diverticula from the posterior region of the vas deferens. Their size varies according to species. Sperm passes from the vas deferens into the urogenital sinus and from these via the urogenital papilla into the cloaca. Leydig's gland empties into the vas deferens. Leydig's gland is the anterior part

8 386 JOHN P. WOURMS of the kidney which has lost its excretory spermatozoa has been reviewed by function in males and has acquired a secretory function. It consists of a mass of exceeding 100 /A in total length. The head Ginzburg (1972). Sperm are very large, convoluted tubules embedded in connective tissue. According to Matthews (1950), times the length of the sperm head * portion is also long, \x, which is it is responsible for secreting the greater in most teleosts. The sperm is characterized by a long, pointed, spirally twisted part of the seminal fluid. The size of the vas deferens varies considerably among the elasmobranchs. It is ance (Retzius, 1902). The helical twisting head which gives it a corkscrew appear- often expanded to form an ampulla which also includes the midpiece and tail. The is used for sperm storage, e.g., Urolophus helical shape of the sperm is correlated (Babel, 1967). This tendency becomes extreme in the basking shark where the ex- primarily by rotation about their long axis with its locomotion. Squalus sperm move panded ampulla may contain liters rather than by a lateral lashing motion. of spermatophores. In addition to size, the Rotation of the gyres of the helix apparently can be reversed, allowing the sperm ampulla of the vas deferens in the basking shark has been structurally modified. The to reverse direction without turning (Stanley, 19716). Both at the microscopic and interior consists of a series of transverse folds each of which forms a circular diaphragm with an eccentric aperture. The sperm are conservative in structure and do ultrastructural levels, chondrichthyan ampulla is lined with an epithelium that is not differ significantly from other vertebrate sperm (Boisson, et al., 1968; Stanley, made up of two cell types, viz. tall ciliated cells and short cells. The latter occupy a 1971<z,i). Attempts to determine the ultrastructural basis of spiralization have not basal position between the ciliated cells. The short cells secrete the material which been successful (Stanley, 19716). forms the cortex of the spermatophores A number of investigations have dealt (Matthews, 1950). The vas deferens serves with spermatogenesis in elasmobranchs one of two functions. In most species, (cf., Mellinger, 1965; Stanley, 1966, sperm diluted with seminal fluid is stored I97la,b; Boisson et al., 1968 for earlier in it. In other species, e.g., the basking references). Stanley's 1966 study of shark, sperm is packaged into spermatophores. Spermatophores of Cetorhinus elasmobranch testis, spermatogenesis takes Scyliorhinus will be reviewed here. In the may attain a size of 30 mm. They consist of place in spherical seminiferous follicles or a cortex of hyaline material which surrounds a central mass of sperm. The pack- branched system of collecting ductules. ampullae located at the termini of a highly aging of sperm into spermatophores varies Follicles originate at fixed sites on the in different species from simple sperm dorsal or dorsal-lateral margins of the testis. New seminiferous follicles are formed aggregation to the complex structures of Cetorhinus (Hoar, 1969). Spermatophores when one or two spermatogonia are surrounded by several epithelial cells. The are not present in the chimaera, Hydrolagus colliei but may occur in Chimaera monstrosa epithelial or follicle cells are considered to (Stanley, 1963). The basic organization of be homologous with mammialian Sertoli the male reproductive system in the cells (Stanley, 1966). Both the spermatogonia and follicle cells undergo an chimaera, H. colliei closely resembles other chondrichthyans. Important differences inital period of mitotic activity. At the end do exist, e.g., the complex chambered ampulla of the vas deferens whose epithelial cells in a Scyliorhinus follicle and about 250 of this period there are about 500 Sertoli lining is regionally differentiated into several secretory regions (Stanley, 1963). present in equal number. Within a follicle, in a Torpedo follicle. Spermatogonia are the two cell types segregate into two concentric single layers. Sertoli cells surround Spermatogenesis the central lumen, while spermatogonia The organization of chondrichthyan are adjacent to the limiting membrane.

9 Each spermatogonium is engulfed by a single follicle cell. The result is a bicellular unit, the spermatocyst. After this, the Sertoli cell undergoes no further division. Within a seminiferous follicle, there are many spermatocysts all of which differentiate synchronously. Each spermatogonium undergoes four successive divisions to produce 16 spermatogonia. These transform into 16 primary spermatocytes which undergo meiosis to produce 32 secondary spermatocytes and then 64 spermatids. Spermatids differentiate into mature sperm. Differentiating sperm cells are connected by intercellular cytoplasmic bridges. Spermiogenesis has been described at the ultrastructural level by Boisson et al. (1968) and Stanley (\91\a,b). Except for details previously noted, it differs little from spermiogenesis in other vertebrates. Spermiation takes place in mature follicles, i.e., those with fully differentiated spermatozoa, when an opening forms in the follicle wall and continuity is established with the attached terminal branch of the collecting ductule system. The bundles of mature sperm pull away from each of the Sertoli cells and flow into the ductules. Following this, the follicle contracts until the Sertoli cells form a solid mass in the interior. Then they degenerate and are resorbed. In the chimaera, H. colliei, the general features of spermatogenesis are similar to those in Scyliorhinus (Stanley, 1963). Secondary sex characters CHONDRICHTHYAN REPRODUCTION 387 Highly developed male secondary sex characters are found in the Chondrichthyes. These include the claspers or copulatory organs and the siphon sac. In skates and rays, the siphon sac is replaced by a clasper gland (La Marca, 1964; Babel, 1967). Fertilization is internal in the Chondrichthyes. The claspers function as intromittent organs during copulation (Gilbert and Heath, 1972; Hoar, 1969). They represent modifications of the male pelvic fin. Their structure varies in different species (Leigh-Sharpe, , cited in Hoar, 1969). The clasper is formed in part by cartilaginous elements which support the medial margin of the pelvic fin and extend beyond the posterior margin as a rod. The clasper can be looked upon as a part of the fin rolled up to form a tube whose edges overlap. The proximal opening of the clasper tube is the apopyle and its distal opening is the hypopyle (Leigh- Sharpe, 1920). The mechanism of the clasper-siphon sac or clasper gland function has been subject to experimental study in rays, Urolophus (La Marca, 1964; Babel, 1967) and in the sharks, Squalus and Mustelus (Gilbert and Heath, 1972). During erection, the clasper in Urolophus, bends forward to lie along the ventral surface of the animal (Babel, 1967). La Marca (1964) reports a medial flexure of 85. At the same time, the clasper rotates so the dorsally located apopyle widens and contacts the cloaca. Sperm then passes from the urogenital papilla through the cloaca and into the clasper tube. Contraction of the striated muscles which sheath the clasper gland expels a secretion into the clasper tube. The secretion moves the sperm through the tube and out of its distal end (Babel, 1967; La Marca, 1964). Although some details differ, clasper function is similar during copulation in Squalus and Mustelus (Gilbert and Heath, 1972). One clasper is flexed medially about 90 and inserted into the oviduct where it is anchored to the wall by a cartilaginous complex at its tip. During copulation, sperm is passed from the urogenital papilla into the clasper groove (=tube) where it is then washed into the oviduct by sea water expelled from the siphon sac. The siphon sac had previously been filled with water by repeated flexing of the clasper (Gilbert and Heath, 1972). In sharks, the siphon sacs are paired, subdermal structures located in the pelvic region on either side of the mid line between the skin and the abdominal wall. The sacs are blind pockets, closed at their anterior end and opening into the clasper groove at their posterior end. Sacs are sheathed with muscle and lined with a secretory epithelium. Upon electrical stimulation of the muscular wall, the sacs contract to 85 per cent of their original length. The relative sizes of the sacs vary. In

10 388 JOHN P. WOURMS S. acanthias they measure 12 per cent of the total body length and 30 per cent of the body length inm. canis (Gilbert and Heath, 1972). Secretory cells occur throughout the entire epithelium of the siphon sac of sharks and in localized regions of the clasper gland in skates and rays (La Marca, 1964; Babel, 1967; Gilbert and Heath, 1972). Mann (1960) has shown that the undiluted siphon sac secretion of sexually mature spiny dogfish, S. acanthias, contains a high concentration of 5-hydroxytryptamine (5- HT), % or about 16 mg/animal. The siphons of immature males contain 200 times less 5-HT. Mann and Prosser (1963) demonstrated that application of either 5-HT or siphon sac fluid to spiny dogfish uterus in vitro initiated a brief but powerful contraction which was succeeded by periodic contractions. They suggested that 5-HT, introduced during copulation, caused uterine contractions which would aid in sperm transport. This generalization may be premature since 5-HT could not be found in the clasper glands of Torpedo and Raja and was present only in traces in Mustelus canis. The apparent discrepancy could be explained by a possible diversity of function. Clasper glands of skates and rays produce a white, viscous, slightly acid fluid which coagulates on contact with sea water. La Marca (1964) reported that the secretion of the stingray, Urolophus, contained a muco or glycoprotein and a phospholipid. He proposed that some of the secretion coagulates, sealing the margins of the clasper groove and thus converting it into a closed tube. The remainder serves as a vehicle for sperm suspension and transport. One also wonders whether these secretions participate in the formation of "sperm plugs" found in the uteri of Raja erinacea (Richards, et al., 1963). FEMALE REPRODUCTIVE SYSTEM Functional organization The female reproductive system consists of the ovaries and oviducts. These are in close association but are not morphologically continuous. Oviducts display a considerable degree of regional differentiation. The morphology of the reproductive system has been well described (Borcea, 1905; Dean, 1906; Daniels, 1928; Gudger, 1940; Metten, 1939; Matthews, 1950; Stan- A ley, 1963; Hoar, 1969). Organization of the ovary and oviduct is highly variable due to species diversity and a wide range of reproductive patterns. Adaptation for viviparity have a profound effect on the organization of the oviduct. Although the ovaries and oviducts begin development as paired structures, they often become asymmetrical in adults. In the sharks, Scyliorhinus, Pristiophorus, Carcharhinus, Galeus, Mustelus, and Sphyrna, the right ovary is functional and the left ovary atrophies. Both oviducts are present (Daniel, 1928). With the exception of Scyliorhinus, these sharks are viviparous. Among the viviparous rays, the right ovary and oviduct undergo varying degrees of reduction or loss. In Urolophus, the right ovary is non-functional but both oviducts are functional. In contrast, both the right ovary and oviduct are absent in Dasyatis bleekeri (Babel, 1967). In skates which are oviparous, both ovaries and oviducts are present and functional. The oviducts often function in synchrony (Wourms, unpublished). This is also true for the chimera, Hydrolagus colliei (Dean, 1906; Stanley, 1963). The ovaries are paired structures, except as noted above. They are attached on either side of the vertebral column to the anterior-dorsal wall of the body cavity by a mesentery, the mesovarium. Structure and shape are variable. The ovary is usually elongate in sharks and some rays (Metten, 1939; Babel, 1967) but may be nearly spherical in skates (Wourms, unpublished). In most Chondrichthyes, the ovaries are naked (gymnovarium condition). The germinal epithelium covers the outer surface of the ovary. Ovarian follicles develop from the germinal epithelium. Ripe follicles burst through the surface to discharge ripe ova into the abdominal cavity. In most instances the ovary is solid. When the ovary is hollow, lymph spaces develop within the ovarian stroma. Development of the ovarian follicle has been reviewed

11 CHONDRICHTHYAN REPRODUCTION 389 (Hoar, 1969; Dodd, 1972). Except for several points to be considered elsewhere, it is not noticably different from other vertebrates. Although the ovary of the basking shark, Cetorhinus, differs considerably from that of other elasmobranchs (Matthew, 1950), it is often erroneously used to illustrate the viviparous condition. In Cetorhinus, the surface of the ovary is invested by a fibrous coat. The germinal epithelium invaginates from the surface to form a network of tubules. The ovary is, in effect, hollow. The tubules open to the surface in a pocket on the right side. Ova are discharged into the pocket and pass from there, via the peritoneum, to the oviduct. In Cetorhinus and some other elasmobranchs, epigonal organs are associated with the ovary (Matthews, 1950). Corpora atretica and corpora lutea have been described for a number of species. Their probable role in hormone production has been considered (Hoar, 1969; Dodd, 1972). The organization of the oviduct varies according to function and reproductive pattern. Hoar (1969) summarizes the functions as: 1) egg collection; 2) transport of eggs to exterior; 3) egg case formation; 4) site for development of young in viviparous forms; 5) site of sperm reception and storage; and 6) dissolution of the spermatophore cortex. The oviduct ( = Mullerian duct) originates as a simple tube which then undergoes regional differentiation. Four regions can be distinguished: an anterior ostium tubae or funnel; the shell gland or nidamental gland; a connecting isthmus; and an expanded posterior uterus. The ostium is a funnel at the anterior end of the oviduct which serves to collect ovulated eggs. It is formed either by the fusion of the anterior end of the oviducts, e.g., Cetorhinus, or by asymmetrical development of one primitive funnel, e.g., that of the right side in Scyliorhinus (Metten, 1939). From the ostium, a tubular portion of the oviduct leads to the shell or nidamental gland. This gland is best developed in oviparous species. In viviparous species, it tends to be reduced or vestigial. In its fully developed state, the shell gland is a compound tubular gland. It synthesizes and secretes albumin and mucus in all species (Threadgold, 1957). In oviparous species and those viviparous species which produce egg cases, it also secretes egg case proteins (Borcea, 1905; Filhol and Garrault, 1938). In some species, e.g., Scyliorhinus, it is involved in sperm storage (Metten, 1939). An isthmus leads from the shell gland to the posterior region of the oviduct. The latter region can be expanded, especially in viviparous species, to form a uterus. In oviparous species, the uterus normally serves only as a passageway for the eggs. An apparent exception is the chimaera, H. colliei where the uterine epithelium participates in the morphogenesis of the egg case (Dean, 1906). The uterus of viviparous species is highly developed and displays various modifications for viviparity. These will be discussed in the section on viviparity. Oviducts sometimes merge at their extreme posterior end to form a common vagina (Daniels, 1928; Matthews, 1950). In most species, the oviducts either singly or as a common vagina open into the cloaca usually dorsal to the rectal opening. A hymen or tissue membrane may be present near the posterior end of the oviduct (Daniels, 1928). In the chimaera, H. colliei, both oviducts open directly to the exterior (Dean, 1906). Oocytes, oogenesis, egg transport Chondrichthyan fishes produce small quantities of large eggs. Mature eggs range in size from one mm in Scoliodon sorrakowah (Prasad, 1951, cited in Ginzburg, 1972) to 100 mm or more in Ginglymostoma and Chlamydoselachus (Gudger, 1940). The eggs of the latter two sharks are probably the largest known cells of any living animal. Egg size generally reflects the reproductive strategy of the species. In Scoliodon and Gymnura (= Pteroplatea) where the egg is much reduced in size, the developing embryo receives almost all of its nutrients from the mother via a placenta or trophonemata (Ranzi, 1934). Massive accumulation of yolk occurs in oviparous species and in viviparous species such as Ginglymostoma and Chlamydoselachus in which the developing embryo is solely de-

12 390 JOHN P. WOURMS pendent on its yolk reserves (Gudger, 1940). Mature ovarian and ovulated eggs have a regular spherical shape. The egg may assume an ellipsoidal shape when enclosed in an egg case. Yolk reserves dominate the structure of the mature egg. They consist of granules and platelets associated with small amounts of cytoplasm. In section, the eggs of some fishes, e.g., Torpedo, display concentric layers of bright and dark yolk similar to what is seen in the avian egg (Ruckert, 1899). Yolk color tends to be characteristic of a species. It is usually yellow or orange, but may be pink or light green. A lens shaped blastodisc is located at one pole. The blastodisc also may have a distinctive color which differs from that of the yolk. The egg nucleus is located within and near the surface of the blastodisc. According to Ginzburg (1972) the nucleus is arrested at metaphase of the second maturation division. The egg is surrounded by an extracellular egg envelope, closely apposed to the egg surface. Some confusion exists as to the number of egg envelopes and their origin (Ginzburg, 1972). Most of the material is produced by the oocyte, hence is a primary egg envelope (Balfour, 1885). Follicle cells may contribute additional material. The older literature makes mention of a zona radiata. In teleosts, ultrastructural studies have shown that it consists of the egg envelope matrix and closely spaced microvilli of the oocyte surface. The latter may interdigitate with follicle cell microvilli (Wourms, 1976). The chondrichthyan zona radiata is probably of similar structure. Mature eggs contain a relatively small amount of cytoplasm located in the blastodisc and at the peripheral. The cytoplasm contains the standard cell organelles (Jollie and Jollie, 1967a). Most of the egg, however, is made up of yolk inclusions. Yolk platelets predominate together with a diverse spectrum of inclusion bodies (Jollie and Jollie, 1967a). The yolk platelets were described by Riickert (1899) who pointed out that their size and shape differed according to species. An important chemical and morphological study of yolk platelets in Raja batis was done by Faure-Fremiet (1933). Re-examined in terms of current knowledge, his results indicate that the platelets contain livetin, lipovitellin and probably phosvitin. Fujii (1960) used modern analytical techniques to confirm the presence of lipovitellin. He also demonstrated that the physical and chemical properties of the lipoproteins of the dogfish Scyliorhinus stellaris were very much similar to those of a frog and the hen. With regard to structure, platelets occur in the form of strongly birefringent, rectangular or pyramidal polygons enclosed in a fluid-filled vacuole. Faure-Fremiet's experiments (1933) on the platelets indicate that yolk proteins of skates are assembled in a paracrystalline lattice which is probably similar to that of amphibian yolk (Wallace, 1963; Karasaki, 1963). The yolk platelets of Mustelus canis closely resemble those of R. batis (Grodzinski, 1958). Electron micrographs of Squalus acanthias eggs while confirming the general structure of yolk inclusions were not of sufficiently high resolution to demonstrate the presence or absence of periodicities in yolk platelets (Jollie and Jollie, 1967a). Of the early work on the chemical composition of eggs, reviewed by Needham (1942), that part dealing with total composition and lipid fractions is still valid. Eggs contain a significant amount (5.9% wet weight) of urea as do the tissues of adult fishes. Protein and fat content varies according to species. Raja eggs contain 28% protein and 7% fat whereas eggs of the shark Heptranchus contain 25% protein and 23% fat. The fat fraction contained 88% neutral fats and 12% unsaponifiable material. Oleic, linolenic, and iwaskic acids accounted for 79% of the neutral fats. The remaining 21% was made up of isopalmitic, palmitic, and stearic acids. The unsaponifiable fraction contained octadecyl, cetyl, selachyl alcohols, and cholesterol. Subsequent studies have confirmed these figures, shown squalene to be present in quantity, and added considerably to the list of fatty acids. Studies on the eggs of deep sea sharks have shown an increase in the unsaponifiable fat fraction (21-44%) (Higashi etal., 1953; Zama et al., 1955; Shimma and Shimma, 1968).

13 CHONDRICHTHYAN REPRODUCTION 391 ^ Although a challenging and perhaps ideal system for the study of oogenesis, there have been few modern investigations of chondrichthyan eggs. Certainly nothing approaches the combined chemical, structural, and endocrinological studies of amphibian eggs (Wallace, 1972). A major exception to this are those studies which have been concerned with the endocrine function of the ovary. The general pattern of oogenesis does not differ appreciably from that of other lower vertebrates with heavily yolked eggs. Details and further references can be found in Balfour (1885), Wallace (1903), Marechal (1906), Matthews (1950), Bertin's review (1958), and Babel (1967). Oogenesis in chimaeras was described by Dean (1906) and Stanley (1963). Only two topics will be considered here, lampbrush chromosomes and specialized structures associated with yolk formation. Riickert (1892, cited in Callan, 1957) described lampbrush chromosomes in the oocytes of the shark Pristiurus only ten years after they had been discovered by Flemming. He appears to have been the first to use the term, lampbrush. In his definitive study of lampbrush chromosomes in Pristiurus and Scyllium, Marechal (1906) described them as filaments, mostly in the form of loops projecting laterally from the chromosome axis. In 1957 Callan re-examined the lampbrush chromosomes in the oocytes of Scyllium. He was able to demonstrate, for the first time, the presence of axial chromomeres and to show that they closely resembled the classical amphibian lampbrush chromosome. Due to yolk accumulation during oogenesis, there is a massive increase in egg size. Sometimes yolk accumulation has involved the modification of pre-existing structures. Babel (1967) reported an unusual proliferation and infolding of the follicular epithelium in the oocytes of the ray Urolophus halleri. Extensive folding carries the follicular epithelium far into the egg's center. This infolding amplifies the surface area of the follicular epithelium. It also alters its spatial distribution so that the transport of metabolites or yolk precursors would occur not only at the surface but also in the interior of the oocyte. A similar follicular infolding has been observed in the heavily yolked eggs of cephalopods (Cowden, 1968). In addition to the infolding, cells of the follicular epithelium appear to function as nutrient cells (Wallace, 1903). Babel (1967) has convincingly demonstrated that enlarged follicle cells contain inclusions which are structurally identical to the yolk granules of the oocyte. It would appear that these follicle cells synthesize and transport yolk as do follicle and nurse cells of some invertebrates. Since a direct connection between the ovary and the oviduct is lacking in most chondrichthyans, mature eggs are discharged from the ovary directly into the body cavity. They are then transported to and enter the oviduct via the ostium or funnel at its anterior end. In some species, e.g., Cetorhinus, the ostium is in close proximity to a specialized ovarian pocket from which the ova are discharged. Ova pass directly into the oviduct (Matthew, 1950). In most species, ovulation occurs at any point along the surface of the ovary. Ova released into the body cavity are then transported in the oviduct. In Scyliorhinus Metten (1939) has experimentally demonstrated that this is effected by continuously beating cilia arranged in tracts within the abdominal cavity. Selective orientation of the cilia with respect to the direction of their power strokes enables these tracts to act as unidirectional pathways. Cilia occur on: the peritoneal wall; outer surface of the oviducts; and portions of the liver, bile duct, and hepatic portal vein. The absence of cilia in males and immature females (Metten, 1939) suggests that ciliation is under hormonal control. Sperm storage Sperm storage occurs in females of several species of sharks and skates. Information on chimaeroids is insufficient to draw conclusions (Stanley, 1963). In addition to demonstrating sperm storage in sharks and skates, Metten (1939) and Richards et al. (1963) reviewed the previous evidence. A series of observations have established that female elasmobranchs maintained in

14 392 JOHN P. WOURMS isolation are able to repeatedly deposit fertilized eggs after an initial mating. For example, Clark (1922) reported that a female skate, in the absence of males, deposited fertilized eggs during a period of five-six weeks. Metten (1939) concluded that the shell gland is the site of sperm storage since he found sperm there but not in other regions of the reproductive tract. In freshly excised glands, clusters of active sperm were found within the lumen of those tubules which secrete the egg shell. Sperm were not found in the albumin or mucous secreting regions of the gland. Egg cases and egg case formation The eggs of all oviparous species and many viviparous species are enclosed in leathery egg cases. Chondrichthyans produce two types of egg cases. Oviparous species produce permanent egg cases which are usually deposited on or near the bottom. Embryonic development is completed within the egg case. Both egg case and embryo are subject to physical and biological hazards. In viviparous species, diverse reproductive strategies govern the fate of egg cases. In some instances, e.g., the stingray Urolophus, an egg case is not formed (Babel, 1967). In many species, a temporary egg case is formed from which the embryo emerges to complete development in utero. Finally the egg case may be retained during the entire period of intra-uterine development and even incorporated into the placenta, e.g., M. cams (Te Winkel, 1963). Size and shape of egg cases vary according to species. The egg case of the whale shark Rhincodon is 150 x 300 mm (Baughman, 1955) whereas that of Scoliodon sorrakowah is 3 x 5 mm (Prasad, 1951, cited in Ginzburg, 1972). Four basic shapes are encountered (cf. Cox, 1963, for illustrations). Egg cases of skates and some sharks, e.g., Rhincodon, are quandrangular with horn-like processes at each corner. The dorsal surface is usually arched while the ventral surface is flattened. Species differences which may be associated with environmental adaptations have been described in skates (Ishiyama, 1958). Egg cases of sharks tend to be more rounded and resemble ellipsoids. There are a number of exceptions. Eggs of the heterodontid sharks resemble a wood auger (Smith, 1942). The ellipsoidal body of the egg case is surmounted by two flanges which spiral around the case. The egg cases of chimaeras are intricate and morphologically complex {cf., Dean, 1906 for detailed descriptions). They are spindle or tadpole-shaped and are exceptionally large ( mm) both in absolute and relative terms. Their shape conforms not only to the shape of the egg but to the shape of the fully developed embryo (Dean, 1906). They possess a series of respiratory pores and elaborately sculptured lateral flanges. Newly formed egg cases are light colored, often white. They rapidly darken after oviposition (Dean, 1906). Modern work on the structure and composition of egg cases begins with Faure-Fremiet. Working with Scyliorhinus and two species of Raja, he showed that the egg case was made up of many layers. When examined with polarizing optics, alternating layers were strongly birefringent. Protein was found to be the principal structural component. On the basis of its relative insolubility and sulfur content, the structural protein was considered to be a type of keratin, ovokeratin (Faure- Fremiet, 1938; Faure-Fremiet and Baudouy, 1938). Subsequent chemical and physical studies, however, indicated that the structural protein(s) was not keratin but collagen (Gross et al., 1958). Early electron microscope studies (Gross et al., 1958) failed to find axial periodicities. Wourms and Sheldon (1971, 1972) reported that shark and skate egg cases contain an imperfectly ordered orthogonal array of structural components. One component displays a 400 A periodicity whose banding pattern remains in lateral register over long distances. The repeat units consists of: 1) a 90 A dense band; 2) a 125 A light band traversed by fine filaments in ladder-like fashion; 3) a second 90 A dense band; and 4) a 90 A light band not traversed by filaments. Each dense band can be resolved into two dense sub-bands separated by a light band. Equidistant A

15 CHONDRICHTHYAN REPRODUCTION 393 fe A point densities observed in transverse sections probably are sectioned filaments. The periodicity and banding pattern differs from that of most vertebrate collagens. It closely resembles the elastoidin form of collagen found in sharks by McGavin and Pyper (1964). The organization of egg case collagen does not appear to be fibrillar. A cholesteric liquid crystal model of egg case collagen best explains both the completed structure and its mode of assembly (Wourms, unpublished). The same periodicities and banding patterns have now been found in Heterodontus, Apristurus, Ginglymostoma, Galeorhinus, Halaelurus, CephaloscyIlium, and Raja (Wourms and Sheldon, unpublished). Recently, Knight and Hunt (1974, 1976) have confirmed and extended these observations in the egg cases of Scyliorhinus caniculus using biochemical, X-ray diffraction, and ultrastructural methods. Their results were corroborated by Rusaoven et al. (1976). While collagen is the major structural component of shark and skate egg cases, the question remains whether there are other components present. Egg case collagen may have unique physical and chemical properties. Chemical studies have shown that the tyrosine content is unusually high for collagen. Tyrosine residues may provide the basis for phenolic crosslinking. This is consistent with the demonstration of a quinone-tanned protein in the egg case (Brown, 1955) and phenol oxidases in the shell gland (Threadgold, 1957; Krishnan, 1959). The presence of sulfur in egg cases, originally reported by Faure-Fremiet, is not inconsistent with collagen since Blanquet and Lenhoff (1966) have found disulfidelinked collagenous proteins in nematocysts. Egg cases of chimeras display a considerably different structural organization which probably reflects a basic chemical difference. Egg cases of Hydrolagus, Callorhynchus, and Harriotta, representing members of the three families of living chimeras, have an identical structural organization. The egg case is made up of three structural components arranged in discrete, birefringent layers. The number of layers and their relative positions vary. Ultrastructural examination revealed: 1) a granular layer smilar to the outer layer of the dogfish egg case; 2) layers of fibrils without banding patterns or periodicity; and 3) a population of tubular components. Granules are of different sizes and they seem to be aggregates of smaller units. The tubular component forms the egg case surface, associated sculpturing, and the filamentous projections. The tubular component is a lattice of 550 A pseudotubules. Lattices are arranged in layers in which all of the pseudotubules are oriented in one direction. Adjacent layers are aligned at 90 angles to form an imperfect orthogonal array. The 550 A unit is a pseudotubule since the walls of adjacent tubules are shared in common. The walls contain subunits about 90 A in diameter. The subunits appear to be hollow cylinders (Wourms and Sheldon, 1971; unpublished). Illustrations of fossil chimaeroid egg cases (Dean, 1906) differ little from modern egg cases. The remarkable difference in structure and the probable difference in chemical composition of elasmobranch and holocephalan egg case proteins is consistent with Patterson's (1965) view that a direct relationship between the two groups cannot be demonstrated. The chondrichthyan egg case is a tertiary egg envelope (Wilson, 1925, following Ludwig, 1875) since its structural proteins are synthesized and secreted by the oviduct. At the time of spawning, an egg enters the oviduct, is fertilized and is enclosed in an egg case. The shell gland, a specialized region of the anterior oviduct synthesizes and secretes egg case structural proteins (Borcea, 1905; Dean, 1906; Filhol and Garrault, 1938). It also appears to control the deposition and morphogenesis of the egg case in sharks and skates (Borcea, 1905; Fitz and Daiber, 1963). In the chimaera, H. colliei, the posterior region of the oviduct appears to participate in the moulding of the lateral flanges and respiratory pores (Dean, 1906). The sequence of egg case formation in the skate R. eglanteria proceeds according to the following sequence: 1) formation of the

16 394 JOHN P. WOURMS anterior horns; 2) formation of the anterior two-thirds of the egg case; 3) fertilized egg flows into case; 4) posterior third forms; and 5) posterior horns are completed (Fitz and Daiber, 1963). In the chimaera H. colliei the sequence is similar. The egg containing capsule is formed first and then the spindle shaped tail portion (Dean, 1906). The shell gland is a compound tubular gland (Borcea, 1905; Filhol and Garrault, 1938). The epithelial cells forming the tubules are secretory cells. Zones of specific secretory activity can be distinguished. Although there is variation, especially in viviparous species, three regions have been distinguished using simple staining techniques, viz., an anterior zone of ovalbumin synthesis, a zone of mucous synthesis, and a zone of shell protein synthesis (Metten, 1939). Histochemical studies of the same species of Scyliorhinus (Threadgold, 1957) showed five distinct regions. "These zones secrete respectively a carbohydrate, a carbohydrate substance which is metachromatic, a polyphenol oxidase, a protein, a phenol, and perhaps a phenolic protein and a basic protein." Similar results were obtained by Krishnan (1959). Combined histochemical and incorporation studies with radioactive proline suggest that two classes of cells are present within the same shell secreting tubules and occupy alternate positions (Rusaouen et al., 1976). One cell secretes collagen and the other secretes a protein containing large amounts of tyrosine and sulfhydryl groups. These findings would be consistent with a collagen cross linked by phenolic tanning and disulfide linkages. Wourms and Sheldon (1972; unpublished) reported on the ultrastructure of the shell gland in Raja inornata Jordan and Gilbert. They found that within the tubule of the shell secreting region all of the cells in a given region were identical. Shell secreting cells have an abundant and well developed rough endoplasmic reticulum. They also contain many granules which consist of an outer ring of light material and an inner core of dark material. Granules are formed within the Golgi complex from. material derived from the rough endoplasmic reticulum. Granules are secreted into the lumen where they coalesce into fibrils. Light material within the fibrils has a 400 A periodicity and the banding pattern of egg case collagen. It is possible that both egg case collagen and the phenolic tanning enzymes are packaged in the same granule. The terminal stages of secretion agree with Borcea's (1905) scheme. Shell secreting tubules empty into common ducts at the base of ciliated lamellae. Parallel rows of lamellae extend across the inner surface of the gland. It is suggested that nascent fibrils leaving the tubules fuse into sheets which are sequentially extruded from the structurally polarized lamellae. Each lamella would control the deposition of a single layer in the egg case. At this point the advantages of a liquid crystal model of collagen organization are apparent. It provides the plasticity necessary to make rearrangements at the molecular and supramolecular level. Mating MATING AND PARENTAL CARE In chondrichthyan fishes, fertilization is internal and obviously requires close contact between the sexes. Copulation, however, has been observed only on infrequent occasion. Most accounts deal with small, inshore fishes, e.g., Scyliorhinus, Heterodontus, and Raja. In spite of the paucity of information, mating appears to follow one of several patterns. These seem to be determined by the size and shape of the fishes. In small sharks and probably also chimaeras, the male coils himself around the female. This was first observed in Scyliorhinus and probably also occurs in Squalus and Mustelus (Gilbert and Heath, 1972). A more complete description of mating behavior is available for Heterodontusfrancisci (Dempster and Herald, 1961). Courtship commenced with the male biting the female on almost any part of the body. Seizing the left pectoral fin of the female in his mouth, the male manoeuvred his tail over the back of the female immediately in front of the second dorsal fin. m

17 With the purchase thus afforded, he inserted the right clasper into the cloaca. Copulation lasted about a half hour during which the female was passive. Although copulation has not been observed in chimaeras, there is circumstantial evidence that the series of actions involved is similar to that of small sharks. Male holocephalans possess a second pair of claspers, the antero-pelvic claspers. They also possess a cephalic clasper. Neither organ is found in elasmobranchs (Dean, 1906). The clasper is a cartilagenous rod equipped with denticles. Movement is effected through attachments to the musculature of the lower jaw and labial cartilage (Raikow and Swierczewski, 1975). Dean (1906) suggests that the cephalic clasper is used to grasp the female since the pattern of scars found near the dorsal fin of egg-laying females matches the arrangement of denticles on the clasper. Dean (1906) concludes that in Hydrolagus colliei, the male wraps his body around the female and gains purchase by attaching the cephalic clasper near the female's dorsal fin. The antero-pelvic claspers which are erectile probably serve as additional means of attachment. Relatively little is known about copulatory behavior in large sharks. Clark (1963) reports observations made by Brown on the lemon shark Negaprion brevirostris Poey. Copulation took place at night. The pair assumed a parallel position with the heads slightly apart and the posterior half of the bodies in very close contact. The pair moved in tandem with closely synchronized swimming motions. It is reasonable to assume that this parallel position is typical of copulation in large sharks. Their less flexible bodies preclude them from assuming the coiled position of smaller species. There are two main patterns of copulation in skates and rays. Smaller species of skates mate with the ventral surfaces apposed while males of larger species make either a dorsal or ventral approach (Richards et al., 1963). Libby and Gilbert (I960) observed mating in Raja eglantaria. It lasted for more than two hours. The male bit the caudal margin of the female's pectoral fin. Copulation took place with CHONDRICHTHYAN REPRODUCTION 395 the pair resting ventral side down on the bottom. The male bent his tail 75 beneath the female's, flexed one clasper medially 90 and inserted it into the cloaca and oviduct. Spines (=alar spines?) on the upper anterior surface of the male's pectoral fin assisted in holding the female. Small species of skates mate with the ventral surfaces apposed. Richards et al. (1963) state that "after the male mounts the female, she rolls her pectorals inside his allowing him to maintain a firm hold on her back with his alar spines." Alar spines are retractile, claw-like spines found in adult male skates where they occupy one to five rows at the outer margin of the dorsal surface of each pectoral fin (Bigelow and Schroeder, 1953). Although definitive evidence is lacking, rays seem to copulate as do the small skates. La Marca (1964) states that the length and manner of flexion of the claspers in the stingray Urolophus rules out any method of copulation except ventral apposition. Female sharks of a variety of species have been reported to bear the scars of tooth cuts on their bodies (Springer, 1960, 1967; Stevens, 1974). In the blue shark, P. glauca, tooth cuts which are found only on female sharks longer than 180 cm are considered to be courtship scars (Stevens, 1974). Somewhat earlier Springer (1967) had reached a similar conclusion based on the examination of a number of species of large sharks. Drawing on Clark's account of copulation in the lemon shark, Stevens (1974) concluded that the bites were not made to aid the male in hanging on during copulation. Instead, biting serves as a necessary pre-copulatory release mechanism in females. Biting behavior observed during the courtship of'heterodontns (Dempster and Herald, 1961) and Raja eglantaria (Libby and Gilbert, 1960) supports this view. Tooth sexual dimorphism reported in some sharks (Springer, 1967) seems to be related to courtship behavior. Parental care In the strict sense of the term, parental care of the young is unknown. Early reports of parental care based on the obser-

18 396 JOHN P. WOURMS vation of family groups composed of mother and young are misinterpretations (Breder and Rosen, 1966). It was not parental care but rather the tendency of small sharks to follow any large moving object. The closest approach to parental care appears to involve the selection of incubation sites and the specific orientation of egg capsules within these sites. McLaughlin and O'Gower (1971) present circumstantial evidence for this in Port Jackson sharks,//, portusjacksoni. Spawning probably occurs in open areas. They suggest that the female takes the newly extruded egg in her mouth, swims to a protected site, and places the egg in a crevice. After hardening, the egg due to its corkscrew shape is effectively anchored in place. If confirmed, this would account for the "nest" of H. japonicus eggs described by Smith (1942). not seem to occur. Riickert (1899) reports that all of the spermatozoa entering the blastodisc are transformed into pronuclei of similar size and structure. The female pronucleus fuses with the closest male pronucleus. At first, supernumerary male pronuclei are evenly distributed in the blastodisc, but are subsequently displaced by derivatives of the zygote nucleus. The supernumerary pronuclei undergo mitosis, display typical metaphase figures, and remain viable throughout the blastula stage. Although they do not participate in embryogeneses, their exact fate is not known. According to Riickert (1899), they become the periblast nuclei, a theory which is the basis of an historical controversy (Nelsen, 1953). Although they may persist for long periods, it is likely that the supernumerary pronuclei degenerate. Periblast cells probably arise from peripheral blastoderm cells. EARLY DEVELOPMENT Fertilization and polyspermy Fertilization is internal in all chondrichthyan fishes. Gametic encounter takes place in the upper part of the oviduct, anterior to the shell gland when this gland is present. Riickert (1899) demonstrated that eggs which were at the entrance of the shell gland had been fertilized and contained spermatozoa which were transforming into pronuclei. In some species, e. g., Scyliorhinus where sperm storage occurs, fertilization is believed to occur in the shell gland (Metten, 1939). Further study is needed. In either case, it follows that fertilization occurs before the egg case is fully formed. Physiological polyspermy, as a normal aspect of fertilization has been recognized since the latter part of the 19th century (Ginzburg, 1972). Chondrichthyan eggs are heavily yolked and their development superficially resembles that of avian and reptilian eggs. Fertilization and early development occur within the blastodisc. Riickert (1899), Dean (1906) and others have shown that the blastodisc is penetrated by a number of spermatozoa (over 100 in some sharks). Monospermy does Development An overview of chondrichthyan development is given by Balfour (1885); Ziegler (1902); Nelsen (1953); and Pasteels (1957). The chondrichthyan egg is markedly telolecithal. Cleavage is meroblastic and is usually confined to a small cap of cytoplasm, at one pole, the blastodisc. Eggs of the horned shark, Heterodontus, and the chimera, Hydrolagus, also undergo a series of cleavages within the yolk mass (Smith, 1942; Dean, 1906). Prior to the the first cleavage, the zygote nucleus divides several times producing four or more nuclei and establishing a temporary syncytium. The first four cleavages are meridional or vertical. The resulting blastoderm consists of a central group of three or four cells surrounded by marginal cells. The fifth cleavage plane is horizontal and produces an upper and lower layer of blastomeres in the central region. A cavity subsequently appears between the two layers. Successive cleavages establish three major cell populations: surface blastomeres, deep blastomeres, and periblast. The surface blastomeres, consisting of a single layer of cells, encloses the segmentation cavity. Deep blastomeres are numerous and lie

19 CHONDRICHTHYAN REPRODUCTION 397 within the segmentation cavity. Only these two populations participate in embryo formation. The third population, the syncytial periblast, occupies a peripheral posim. tion and also extends centrally as the floor of the segmentation cavity. The blastoderm acquires a flattened disc shape as cleavage continues. Its upper surface is made up of a single layer of flattened cells beneath which is a tightly packed mass of deep blastomeres. The segmentation cavity is present only in the caudal portion of the blastoderm. Vandebroek (1936) has provided a fate map of the presumptive organ forming areas in the blastoderm of the shark, Scyllium. Most of the future endoderm lies on the surface in a small sector at the caudal margin of the blastoderm. Some endodermal cells also lie deep within the blastoderm. The epidermal ectoderm occupies almost the entire cephalic portion of the blastoderm. Immediately behind it is a large semi-circular region of presumptive neuroectoderm. The prechordal plate forms a small median region on the surface immediately cephalic to the endoderm. A thin crescent of notochord lies between the pre-chordal plate and the neural ectoderm. The presumptive mesoderm is found in two crescent shaped regions along the lateralcaudal margins of the blastoderm (Vandebroek, 1936; Nelsen, 1953). In addition to the classical accounts, Vandebroek (1936) has provided an analysis of the complex morphogenetic movements which culminate in embryogenesis. During gastrulation, the presumptive epidermal and neural ectoderm retain their surface location and expand greatly. Notochordal, pre-chordal plate, mesodermal and endodermal cells migrate from the surface, over the caudal edge of the blastodisc, into the segmentation cavity. The cells of the notochord and prechordal plate move into a median position beneath the neural plate. As the embryo begins to take shape, it rises above the surface of the blastoderm. A head fold is present at the anterior end while a notochord has formed in the median plane. The mesoderm and endoderm lie on either side of it. Subsequently, the endoderm establishes a complete floor by growing medially beneath the mesodermal plates and fusing below the notochord (Vanderbroek, 1936; Nelsen, 1953). Vandebroek's (1936) fate map and account of morphogenetic movements should be approached with considerable caution. The recent drastic revision of the classical account of morphogenetic movements and the fate map of the trout (Ballard, I966a,b, 1968) calls into question other studies of the same vintage and employing similar methods. In point of fact, preliminary studies by Ballard (personal communication) have shown that Vanderbroek's account is in error and must be redone. In this respect, it is worth noting that there are very few experimental studies of the early development of elasmobranchs. Vivien (1955) reported that the pregastrula blastoderm of Scyliorhinus could be divided into two or four segments without affecting development. After formation of the subgerminal cavity, sagittal section of the blastoderm caused twin formation. Isolated marginal regions did not form embryos. Two embryos with normal orientation were obtained by transverse section of the caudal region of the blastoderm, i.e., over the subgerminal cavity. Loss of regulatory ability coincided with the onset of active morphogenetic movement. The later phases of development have been dealt with on numerous occasions and except for the following topics will not be considered here (cf., Balfour, 1885; Ziegler, 1903; Dean, 1906; Nelsen, 1953; Pasteels, 1958). Dean (1906) in his study of the chimaeroid fish, H. colliei, states that their development is shark-like. Several points of difference exist and should be re-examined. The behaviour of the yolk during development is remarkable. By day 32, and early embryo, the yolk mass has been partitioned into fragments of varying size. Prior to that, it underwent a modified total cleavage. Only a small portion of the yolk is enclosed within the yolk sac. The rest, an estimated 80-90%, forms a creamy mass. Dean (1906) suggests that this yolk either is absorbed via the external gills or is swallowed. Dean (1906) also describes and

20 398 JOHN P. WOURMS figures an open blastopore which communicates with an archenteron. The archenteron is lined with several layers of cells and is distinct from the segmentation cavity. Since Dean worked with a limited amount of preserved material, the need for critical re-examination is obvious. Embryonic development is a lengthy process. It is longest in sharks and chimaeras, of intermediate length in skates, and shortest in the rays. Developmental rate is temperature dependent (Harris, 1952) and also species specific. Development is relatively rapid in the viviparous rays. The gestation period is 4 months in Myliobatis bovina, 3 months in Urolophus halleri, and 2 months for Dasyatis (=Trygon) violacea (Ranzi, 1934; Babel 1967). Development in the oviparous skates is of intermediate duration. In six species of skates within the genus Raja, Clark (1922) observed incubation times which ranged from months in R. clavata Linnaeus to 8 months in R. naevus Miiller and Henle. An apparent exception is R. marginata Lacepede with a duration of almost 15 months. In R. eglantaria, Libby and Gilbert (1960) found an incubation time of 9 weeks in a Florida population. Whereas Fitz and Daiber (1963) reported 12 weeks for northern (Delaware-New York) populations. Among sharks and chimaeras, especially those from temperate waters, the longest incubation or gestation periods occur. Dean (1906) reports incubation times of 9-12 months for the chimaera, H. colliei. Similar times have been reported by McLaughlin and O'Gower (1971) for three species within the oviparous shark genus Heterodontus. In Scyliorhinus the incubation time is 6-8 months (Ranzi, 1934; Harris, 1952). Gestation periods of 11 months and 8-12 months respectively have been reported for the carcharhinid sharks, Mustelus canis (Te Winkel, 1950) and C. milberti (Springer, 1960). The month gestation period of the spiny dogfish, S. acanthias is the longest period thus far documented. At the other extreme, an Indian shark, Chiloscylium grispem Miiller and Henle lays eggs that take months to complete development (Aiyar and Nalini, 1938 cited in Breder and Rosen, 1966). One would like to know whether the rate of development is intrinsically more rapid in warm water species or is brevity due to a higher temperature. REPRODUCTIVE PATTERNS VIVIPARITY Reproductive patterns in chondrichthyan fishes have been reviewed by Breder and Rosen (1966) and others (Ranzi, 1932, 1934; Needham, 1942; Budker 1958; Amoroso, 1960; Hoar, 1969). An overall picture emerges. All recent chondrichthyan fishes employ internal fertilization. With few exceptions, e.g., Cetorhinus (Matthews, 1960), they produce a relatively small number of large, heavily yolked eggs. These fishes are either oviparous or viviparous (Tables 1, 2). If they are oviparous (chimaeras, skates, some sharks) their eggs are enclosed in an egg case and deposited in the external environment. Development is completed outside of the body of the mother. Eggs are retained in viviparous species and embryonic development is completed in utero. Traditionally, these fishes have been categorized as oviparous, ovoviviparous, or viviparous species. Budker (1958) and Hoar (1969) pointed out that the distinction between ovoviviparity and viviparity is artificial. Ranzi (1934) has shown that in live bearing species fetal/maternal nutritional dependency ranges from nil to almost complete. Placental species only occupy an intermediate position in this series. Moreover, the placenta develops only after a period of yolk reserve dependency. Budker (1958) subdivided the viviparous fishes into placental and apla- TABLE 1. Reproductive patterns in chondnchthyan fishes. I. Oviparity II. Viviparity A. Aplacental viviparity 1. Dependent solely on yolk reserves 2. Oophagy: Intrauterine embryonic cannibalism 3. Placental analogues uterine milk secretion and/or uterine trophonemata B. Placental viviparity 1. Yolk sac placenta: Ontogenetic transition from yolk reserve dependency

21 Class Chondrichthyes Subclass Elasmobranchii Order Squaliformes Suborder Hexanchoidei 1. Family Hexanchidae (Cow Sharks) Viviparous 2. Family Chlamydoselachidae (Frill Sharks) Viviparous Suborder Heterodontoidei 3. Family Heterodontidae (Bullhead or Horn Sharks) Oviparous Suborder Lamnoidei 4. Family Odontaspidae (Sand Sharks) Viviparous 5. Family Scapanorhynchidae (Goblin Sharks) Viviparous 6. Family Lamnidae (Mackerel Sharks) Viviparous 7. Family Cetorhinidae (Basking Sharks) Presumed viviparous 8. Family Alopiidae (Thresher Sharks) Viviparous 9. Family Orectolobidae (Carpet or Nurse Sharks) Oviparous and viviparous 10. Family Rhincodontidae (Whale Sharks) Oviparous 11. Family Scyliorhinidae (Cat Sharks) Oviparous, one viviparous species 12. Family Carcharhinidae (Requiem Sharks) Viviparous 13. Family Sphyrnidae (Hammerhead Sharks) Viviparous Suborder Squaloide 14. Family Squalidae (Dogfish Sharks) Viviparous 15. Family Pristiophoridae (Saw Sharks) Vivipa- Modified from Breder and Rosen, 1966and Budker, CHONDRICHTHYAN REPRODUCTION 399 TABLE 2. Modes of reproduction in chondrichthyan fishes. cental species. Here, the aplacental species have been categorized as: 1) dependent solely on yolk reserves; 2) oophagous; or 3) possessing placental analogues (Table 1). Table 2 summarizes the occurrence of oviparity and viviparity within the Chondrichthyes (cf., Breder and Rosen, 1966; Bigelow and Schroeder, 1948, 1953 for additional detail). Viviparity, in varying degrees, is widespread. It almost seems characteristic of the elasmobranchs. Oviparity, in contrast, is confined to the three extant families of chimaeras, the three or more families of skates in the suborder Rajoidei, and four families of sharks. Knowledge of the developmental physiology of oviparous species is derived chiefly from studies on the sharks, Scyliorhinus and skates, Raja. Following oviposition, the embryo enclosed in the 16. Family Squatinidae (Angel Sharks) Viviparous Order Rajiformes Suborder Pristoidei 17. Family Pristidae (Sawfishes) Viviparous Suborder Rhinobatoidei 18. Family Rhinobatidae (Guitarfishes) Viviparous 19. Family Rhynchobatidae (Guitarfishes) Viviparous Suborder Torpedinoidei 20. Family Torpedinidae (Electric Rays) Viviparous 21. Family Narkidae (Electric Rays) Viviparous 22. Family Temeridae (Electric Rays) Viviparous Suborder Rajoidei 23. Family Rajidae (Skates) Viviparous 24. Family Arhynchobatidae (Skates) Oviparous 25. Family Anacanthobatidae (Skates) Oviparous Suborder Myliobatoidei 26. Family Dasyatidae (Sting Rays) Viviparous 27. Family Myliobatidae (Eagle Rays) Viviparous 28. Family Mobulidae (Manta Rays) Viviparous Subclass Holocephali Order Chimaeriformes 29. Family Callorhynchidae (Chimaera) Oviparous 30. Family Chimaeridae (Chimaera) Oviparous 31. Family Rhinochimeridae (Chimaera) Oviparous egg case completes its development while exposed to the vagaries of the surrounding environment. Harris (1952) and Collenot (1966) have shown that the rate of development is temperature dependent. The egg case affords a considerable degree of mechanical protection. Phenols contained within it may serve as anti-microbial agents. During the early phases of development, the embryo is sealed within the egg case and is effectively isolated from the outside environment (Ouang, 1931; Smith, 1936). The egg and embryo retain urea, even though the egg case is structurally permeable to it (Needham and Needham, 1930). Sometime during early development, at 20 days in Raja eglanteria (Libby and Gilbert, 1960) and at the mm stage in Scyliorhinus, (Collenot, 1966), sea water is admitted into the egg. This is brought about by the dissolution of mu-

22 400 JOHN P. WOURMS cous plugs in preformed passages (Collenot, 1966) or the opening of respiratory pores (Dean, 1906). Rhythmic movements of the embryo bring about a continuous flow of sea water through the egg case. The egg contains all of the organic material required for development. Although mostly used in forming embryonic structures, some serves as an energy source. This accounts for the 21% loss of organic material during the development of Scyliorhinus (Ranzi, 1932; Amoroso, 1960). Water and inorganic material are derived from the environment (Amoroso, 1960). Organic material, stored in the yolk, is transferred to the developing embryo by several different processes (Beard, 1896; Te Winkel, 1943; Jollie and Jollie, 1967a). In the earliest phase, yolk is incorporated into blastoderm cells, probably by phagocytosis, and digested intracellularly. In the next phase, yolk was once believed to be digested extracellularly by merocytes, i.e., extra-embryonic yolk cells. Jollie and Jollie (1967a) have shown that in Squalus the merocytes are part of the peripheral syncytial cytoplasm of the yolk sac. The products of digestion pass into the vitelline circulation. Extracellular digestion by the endodermal epithelium of the yolk sac and subsequent absorption of digested yolk into the vitelline circulation may also occur. In the Chondrichthyes, all three germ layers take part in the epiboly of the blastodisc to form a three-layered yolk sac (Ruckert, 1922). In the later phases of development, yolk is mainly digested within the intestine of the embryos (Beard, 1896; Te Winkel, 1943). Yolk platelets are moved directly from the external yolk sac into the gut by ciliated epithelia. In some instances, e.g., Scyliorhinus (Collenot, 1966), the yolk duct or umbilical canal is enlarged at its terminal end to form an internal yolk sac which empties directly into the intestine. The least specialized of the viviparous fishes are those aplacental species which depend solely on yolk reserves. This category corresponds in part to Types la and Ib of Ranzi (1934). Fishes exhibiting this pattern, e.g., Squalus acanthias, Scymnus, Centrophorus, display a considerable (15-55%) loss of organic material during development (Ranzi, 1934; Hisaw and Albert, 1947; Amoroso, 1960). S. acanthias is typical. One to four fertilized eggs are enclosed in an elongated egg case. The egg case remains intact for about six months, after which the embryos hatch and complete the remaining months gestation in the uterus. The processes of yolk absorption and utilization are similar to those of oviparous species, especially Scyliorhinus (Te Winkel, 1943). The syncytial yolk sac cytoplasm digests and absorbs yolk during early stages (Jollie and Jollie, 1967a). During later stages, yolk platelets are moved from the external sac up the yolk stalk into the internal sac and from there into the intestine. Movement is effected by ciliated epithelia. The intestine becomes functional when the embryo is approximately mm long. During development, the internal yolk sac increases in size while the external yolk sac is diminished. Absorption is almost complete at parturition (Te Winkel, 1943). Jollie and Jollie (19676) describe ultrastructural changes in the mucosa of the pregnant uterus. It is transformed into a respiratory membrane which may also regulate the transport of water and electrolytes. Changes involve extensive vascularization and a reduction in the quantity of tissue between the maternal blood and the uterine lumen. Oophagy, a curious adaptation for viviparity, has evolved in several families of shark. It is a form of intra-uterine embryonic cannibalism and can be said to represent a final solution to sibling rivalry. The phenomenon was first recognized and described by Lohberger (1910) and subsequently confirmed by Shann (1923) in embryos of porbeagle sharks, Lamna spp. Porbeagle embryos are quite large and massive, over 55 cm in length. They appear to have an enormous yolk sac. The yolk sac, however, is absorbed at an early (6 cm) stage (Shann, 1923). What appears to be a yolk sac is really the cardiac stomach, enormously distended by eggs which have been ingested by the developing embryo. In Lamna cornubica, each oviduct may contain two embryos. Some

23 authors (Gudger, 1940) have overlooked the phenomenon of oophagy. As a result they have concluded that the enormous size of the porbeagle embryos is due to eggs which are of enormous size. More recently, Springer (1948) has reported on oophagy in the sand shark Odontaspis taurus (Rafinesque) (=Carcharius taurus). Living embryos were exceedingly active in utero. They dashed about, open mouthed, inside the oviduct snapping at whatever they encountered, including the investigator's hand. In this case, there was only one embryo in each oviduct. Normally, several fertilized ova are enclosed within one egg capsule, but only a single active embryo hatches. This strategy is strikingly similar to that of some gastropods, e.g., the Buccinidae and Muricidae. Once hatched, the developing embryo feeds on successive crops of ovulated eggs. Springer (1948) suggests that oophagy offers two advantages. It produces very large embryos (up to 105 cm) which carry an extra reserve of nutrients. Also, as a result of its intrauterine behaviour, the fetal shark emerges at parturition as an experienced predator. The frequency with which oophagy occurs is not known. Springer (1948) and Budker (1971) suggest that additional species of mackerel sharks (Lamnidae) and some thresher sharks (Alopiidae) are oophagous. Even without chemical analysis, it is obvious that oophagous embryos undergo a great increase in organic content. Added material is of maternal origin. The final category of aplacental viviparity includes species which have placental analogues. It corresponds to Group III of Ranzi (1934). Placental analogues are modified regions of the uterine epithelium which secrete an embryotrophe or "uterine milk" which is absorbed or ingested by the embryo (Amoroso, 1960). Leucocytes are sometimes found in the secretion. Their origin is unknown. In the ray, Dasayatis violacea, the "milk" has an organic content of 13% and a total fat content of 8% (Ranzi, 1934). The secretion process in the epithelial cells of Dasayatis and Myliobatis commences with the entrance of a leucocyte into the cell. The leucocyte disintegrates. Shortly thereafter CHONDRICHTHYAN REPRODUCTION 401 lipid and protein containing granules appear, mature, and are secreted. The most highly developed placental analogues occur in the form of villous tufts of uterine epithelium, e.g., Dasyatis violacea and Myliobatis bovina or trophonemata, e.g., Gymnura micrura (Ranzi, 1934). Trophonemata are long, flattened glandular appendages. They enter the embryo through the spiracles and pass into the esophagus where they release secretory product into the gut. Wood-Mason and Alcock (1891) introduced the term, trophonemata, to distinguish them from intestinal villi. The efficiency of placental analogues surpasses that of the yolk sac placenta. They account for a % increase in organic material during development compared to the 840% and 1050% increase in two placental species, the blue shark, P. glauca, and the dogfish, M. laevis (Ranzi, 1934; Needham, 1942). The placental analogues found in these three species of rays represent the culmination of a series of adaptations which originate with yolk dependent species. Stages in the evolution of placental analogues become apparent when the electric rays, Torpedo ocellata and T. mormorata are considered. Both show a net loss of organic material during development, 22% and 34% respectively (Ranzi, 1934; Amoroso, 1960). While the uterine lining has elaborated prominent folds and lamellae, relatively few secretory cells are present (Ranzi, 1934). The uterine milk contains little (1.2%) organic material and less fat (0.1%). Weight loss occurs since the quality and quantity of secreted material is inadequate for the energy requirements of the embryo. The comparative study of Ranzi (1934) reveals a progressive shift in the energy budget of development from a negative to positive balance. This change apparently is accomplished by an increase in efficiency of the placental analogues. There is an increase in the total number of secretory cells in the uterine epithelium as well as an increase in the organic content (5-9%) of their secretory product, e.g., Mustelis vulgaris, M. antarcticus (Ranzi, 1934). Parallel evolution of efficient placental analogues in sharks and rays proba-

24 402 JOHN P. WOURMS bly reflects a basic selection for increased efficiency in maternal-fetal maintenance. In some instances, evolution of efficient placental analogues and placentae have resulted in reduction of egg size, e.g., the ray, Gymnura, and the shark, Scoliodon. The placental form of viviparity is confined to two families of sharks, the Carcharhinidae (requiem sharks) and the Sphyrnidae (hammerhead sharks). Within these groups, placentae are of frequent occurrence and appear to have evolved independently. In the Carcharhinidae, species within the genera Carcharhinus, Prionace, Scoliodon, Carcharias, Hemigaleus Paragaleus and Mustelus have placentae. Within a genus some species, e.g., Mustelus canis may have a placenta while others, e.g., M. antarcticus do not. In the Sphyrnidae, Sphyrna is the principal placental genus (Schlernitzauer and Gilbert, 1966). Placental structure has been described in detail for Mustelus canis (Miiller, 1840; Ranzi, 1934) Prionace glauca (Miiller, 1840; Calzoni, 1936), Carcharhinus falciformis (Gilbert and Schlernitzauer, 1966), Scoliodon surrakowah and S. palasorrah (Mahadevan, 1940), and Sphyrna tiburo (Schlernitzauer and Gilbert, 1966). The placenta in all instances is a yolk sac placenta. Variation exists with respect to the intimacy of fetal-maternal contact and the thickness and number of intervening tissue layers. In most species, e.g., M. canis, P. glauca, Sphyrna tiburo, there is an intimate interdigitation of the maternal and fetal tissues. In contrast, the placentae of Carcharhinus dussumerieri (Mahadevan, 1940) and C. falciformis (Gilbert and Schlernitzauer, 1966) rest on a thickened, vascularizied region of the uterine wall. Schlernitzauer and Gilbert (1966) state that all exchange between the fetal and maternal blood systems involves up to five tissue layers. These are: 1) maternal endothelium; 2) maternal epithelium; 3) egg case; 4) fetal epithelium; and 5) fetal endothelium. Reduction or loss of one or more of these layers may occur. Only a few sharks retain the egg envelope and incorporate it into the placenta, e.g., M. canis and S. tiburo. Schlernitzauer and Gilbert (1966) report that the fetal epithelium in C. falciformis is greatly reduced while the maternal epithelium remains relatively unchanged. Both maternal and fetal epithelia are greatly reduced in the placentae of M. canis and S. tiburo. For details of the histological organization of placentae, reference should be made to the studies listed above. Several peculiarities are associated with placentae in the sharp-nosed shark, Scoliodon. In S. palasorrah, the yolk sac established a specialized connection with a trophonematous cup (Mahadevan, 1940). In S. sorrakowah, due perhaps to small egg size and the consequent need for establishing a placental connection, the yolk sac is not filled with granules but with a network of cellular strands and blood vessels. In addition, a yolk stalk does not form. Instead of an umbilical stalk, a placental cord carrying two blood vessels connects the embryo to the placenta. In Scoliodon as well as Paragaleus and Sphyrna, the umbilical stalk or placental cord is festooned with fingerlike processes called appendiculae. An absorptive function is attributed to them (Budker, 1958). The details of placenta formation are best known in M. canis (Te Winkel, 1950, 1963; Graham, 1967). Information on Sphyrna and Scoliodon can be found in Schlernitzauer and Gilbert (1966) and Mahadevan (1940). In Mustelus canis, the gestation period is months. During the first three months, the developing embryo is yolk dependent. It undergoes an ontogenetic transition during the third and fourth months and establishes a placental connection which is retained until birth. This pattern is common to all placental sharks. The process begins with the fertilization of three to ten eggs. Each egg is enclosed in a thin membranous capsule and released into the uterus at hour intervals. Development proceeds for the next three months. During this period, transverse folds grow from the endometrium and form compartments around each embryo (Graham, 1967). Compartmentalization occurs in most placentai species. The endometrium undergoes other changes. At first, it is slippery due to secreted fluids. As placentation begins, the

25 endometrium becomes "sticky." Changes occur in the yolk sac and egg capsule. During the second month of development, the yolk sac begins to expand. Expansion continues until the embryo is completely surrounded by the sac. The yolk sac then contracts and becomes localized in a small area where the placenta is forming. Changes in adhesivity are associated with placentation. Prior to placentation, yolk sac, egg capsule, and endometrium are easily separated. As placentation commences, they adhere but can be separated. They can no longer be separated after the placenta forms. The egg capsule is incorporated into the placenta, and on the basis of histological evidence seems to undergo chemical changes. Placentation also involves folding of the yolk sac and uterine epithelia and the subsequent interdigitation of the two tissues. Structural differences between the distal and proximal portions of the yolk sac often occur (Gilbert and Schlernitzauer, 1966). In most carcharhinids, connection between the yolk sac, placenta and the embryo is by a modified yolk stalk or umbilical stalk. This contains the yolk duct (vitellointestinal duct) and the umbilical vein and artery (Budker, 1958). In the 40 years that have elapsed since Ranzi's classic work, a surfeit of research problems have been available yet have gone untouched. Regretably, there is still little to report on the physiology of viviparous species, especially placental ones. An exception to this is Graham's (1967) study of placentation in Mustelus canis. Using 3 H-glucose, he demonstrated that low molecular weight organic substances "filter" across the placental tissues into the yolk sac cavities and from there are passed up the yolk duct into the intestine. He concluded that nutrient transfer through the placenta of late term fetuses is nonhemotrophic. Obvious questions of embryonic nutrition and placental function are raised. The % increase in organic material in the embryos of placental species indicates an efficient mode of nutrient transfer (Ranzi, 1934). What is involved? The uterine milk of M. canis has a high (9%) organic content. What role CHONDRICHTHYAN REPRODUCTION 403 does it play? Alternate pathways of absorption do exist. After the establishment of the placenta in M. canis, there is a dramatic increase in the uptake of labelled tracers from the uterine cavity (Graham, 1967). The state of knowledge is even more unsatisfactory in other species. The formation of a trophonematal cup at the site of placental attachment in Scoliodon as well as the elaboration of "absorptive" appendiculae on the placental cords of Scoliodon, Sphyrna, and Paragaleus (Budker, 1958) suggest that intra-uterine secretions are being absorbed. One would like to know the balance between absorption and placental transfer. In addition the functional difference between the occluded, vascular placental cord of Scoliodon and the patent yolk stalk of the carcharhinid placenta should be pursued. Viviparity The reproductive strategies of chondrichthyan fishes are diverse and successful. Oviparity and placental vivparity are the two extremes in a continuum of reproductive adaptations. Current interest centers about the retention of oviparity and the evolution of viviparity. Both processes are subject to the forces of selection. Since selection operates primarily at the level of the individual and population, the advantages or disadvantates of each pattern must be considered in terms of the individual and the species (cf., Wourms and Evans, 1974a,b; Wourms and Cohen, 1975, for examples in teleosts). A study involving the 600 or more chondrichthyan species is premature. What follows is a prolegomena to a more detailed study of reproductive strategies. Basic trends in chondrichthyan reproduction and some factors which may effect the evolution of viviparity will be explored. Eight factors seem to be important in the evolution of viviparity and the retention of oviparity (Table 3). The first of these is the phylogenetic position of a species. Oviparity is the least specialized and primitive pattern of chondrichthyan reproduction. From it, viviparity has independently evolved in several different groups.

26 404 JOHN P. WOURMS TABLE 3. Factors in the evolution of viviparity. 1. Phylogenetic position 2. Geographical distribution 3. Habitat: Benthic vs. pelagic 4. Feeding ecology 5. Adult size 6. Egg/embryo size 7. Osmoregulation 8. General reproductive strategy of viviparity Oviparity as it occurs in these fishes is a specialized strategy since it is based on the advantages of producing small numbers of large eggs. As a result the fecundity of oviparous species is low. That of viviparous species does not differ appreciably. Holden et al. (1971) reported the maximum egg laying rate of the skate, R. clavata, to be one egg per day. At best, this could account for 360 eggs per year, probably far less. Harris (1950) estimated that the maximum production of eggs in Scyliorhinus is 120 eggs per year. Breder and Rosen (1966) give a figure of 108 embryos per brood for the viviparous shark Hexanchus. These are maximum values. Production of 2 to 50 young per year seems more likely. The initial low fecundity of oviparous species may aid in explaining the widespread occurrence of viviparity in this group. The advantages of viviparity could be acquired without substantial loss of fecundity. A necessary feature of viviparity is internal fertilization. This appears to have evolved early and is found in all extant oviparous and viviparous species (Matthews, 1955; Tortonese, 1950; Breder and Rosen, 1966). Subsequent steps in the evolution of viviparity involved retention of fertilized eggs, thinning and loss of the egg case, elaboration of mechanisms for fetal maintenance, and reduction in egg size. Simple egg retention occurs in several diverse taxa, e.g., Ginglymostoma and Chlamydoeslachus. The diversity of mechanisms for fetal maintenance in present day chondrichthyans suggests that viviparity is still evolving. In this respect, it would appear that once viviparity has evolved, it is retained. In terms of phylogenetic position (Table 2) several points are of interest. All living chimaeras and probably all fossil chimaeras are oviparous (Dean, 1906; Bigelow and Schroeder, 1953). Twelve of the sixteen families of elasmobranchs are viviparous. Placental viviparity is confined to two of these families. Oviparity occurs only in four families. Two of these, the Orectolobidae (carpet sharks) and the Scyliorhinidae (cat sharks) have viviparous species. The former includes Ginglymostoma which is considered to have recently made the transition from an oviparous to a viviparous condition (Gudger, 1940). Within the Scyliorhinidae, one member of genus Galeus, G. polli Cadenat is marginally viviparous while other species within the genus and family are oviparous (Breder and Rosen, 1966). Within the order Rajiformes, derived from sharks or sharklike ancestors, only the skates have retained oviparity whereas members of the other four suborders are all viviparous. The geographical distribution of the major groups of the Rajiformes tends to be correlated with their reproductive strategies. Skates (suborder Rajoidei) which are oviparous occur chiefly in temperate and polar regions. The other four suborders, sawfishes, guitarfishes, and rays, which are viviparous, are found in tropical and sub-tropical regions (Bigelow and Schroeder, 1953). A similar correlation is not found in sharks. Tortonese (1950) was the first to call attention to a possible relationship between habitat and reproductive strategies in sharks. He noted that oviparous species were benthic, littoral, and not of large size. Viviparous species were more diverse in habitat. Association of oviparity with a benthic habitat is still valid in sharks and can be extended to skates and chimeras. Skates and chimeras also tend to be of moderate size. Many skates and some chimeras tend to be littoral. Many large species of pelagic sharks, e.g., the blue shark, are viviparous. The advantages of viviparity to a pelagic species are readily apparent. Oviparity in benthic species may be opportunistic or may offer advantages. In the case of viviparous rays many of which are benthic, other factors may be operable. Feeding ecology is a function in part of d

27 CHONDRICHTHYAN REPRODUCTION 405 size and also habitat. Large sharks and sawfishes, active predators of fishes, tend to be viviparous. Smaller sharks e.g., Heterodontidae and Scyliorhinidae, and the skates, both of which feed on benthic invertebrates and small fishes, are oviparous. Torpedos, sting rays, and eagle rays which have a similar feeding ecology are viviparous. Why? One family and two species of chondrichthyan fishes are considered macro-plankton feeders: the whale shark, Rhincodon; the basking shark, Cetorhinns; and the devil rays, Mobulidae (Bigelow and Schroeder, 1948, 1953). They are large, pelagic fishes. The first is oviparous (Baughman, 1955). Cetorhinus is presumed viviparous (Matthews, 1950) and the Mobulidae are viviparous. If any significance can be attached to the difference, one is tempted to suggest that oviparity is less advantageous in view of the apparent rarity of the whale shark. Since gigantism in the marine environment is often associated with plankton feeding, size rather than feeding ecology may be more relevant to reproductive strategies. Tortonese (1950) stated that viviparity was a function of large adult size in sharks. This generalization can be extended to the skates, rays, and chimaeras. Viviparous species tend to produce larger offspring. The largest oviparous embryo is that of the whale shark, circa 35 cm (Baughman, 1955). Most oviparous embryos tend to be considerably smaller, under 15 cm. In contrast, viviparous embryos of cm are not uncommon (P. glauca, Wourms, unpublished). Embryos of about 100 cm have been reported in Odontaspis (Springer, 1948). Absolute size is an advantage to the large offspring of viviparous species. Embryos of cm length are considerably above the median size of cm for adult fishes (Marshall, 1971). In terms of Hutchison and MacArthur's (1959) model of size distribution, several advantages accrue from increased size: 1) reduction in number of potential predators; 2) reduction in number of competitors; and 3) a greater number of potential food organisms. Increased embryo size also results in greater efficiency, since swimming speed is a function of absolute size (Marshall, 1971). Growth phenomena may be another reason why large species tend to be viviparous. As a generalization, early phases of postembryonic growth tend to almost be exponential. Embryos whose initial size is large tend to grow to a large adult size (Marshall, 1953). The factor of egg/embryo size is related in part to adult size. As a rule, large adults develop from large embryos. Post-partum embryos of viviparous species are larger than newly hatched oviparous embryos. For this reason large species tend to be viviparous. Size differences can be accounted for in terms of embryonic and maternal energetics and upper limits to egg size. Oviparous embryos consume 25% or more of their organic content during development (Ranzi, 1934). Viviparous species suffer the same loss but in many instances have evolved efficient means of continuous fetal nutrition. Where mechanisms for continuous fetal nutrition have evolved, the fetus attains a large size. The maternal energy budget is more efficient since nutrients are delivered on demand rather than sequestered in the egg prior to demand (Wourms and Cohen, 1975). In oviparous species, the size of the embryo is limited by the size of the egg. The eggs of some sharks are gigantic cells. The egg of the whale shark may represent an upper limit in absolute size. In spite of this, the embryo is only half the size of the average viviparous shark embryo. Price and Daiber (1967) suggested that the inability of elasmobranch embryos to regulate urea content and osmotic pressure during early development constituted a selective disadvantage which led to the evolution of viviparity. Read's (1968) demonstration of ornithine-urea cycle enzymes in all stages of development appears to contradict this. Pang, et al. (1967) reviewed the subject but did not resolve the issue. It would seem that even if early stage oviparous embryos can control their urea content, it can be done more efficiently and with less expenditure of embryonic energy in the uterine environment. Finally, the evolution of viviparity in the chondrichthyans cannot be divorced from the general strategy of viviparity. A bal-

28 406 JOHN P. WOURMS ance sheet of evolutionary advantages and disadvantages shows the following. The maternal environment offers the advantages of protection from predators and other hazards. It also provides physiological regulation of the environment. In some instances, supplemental mechanisms of fetal nutrition have evolved. Viviparity leads to increased size of full term embryos. Size increase is advantageous. An apparent reduction in fecundity, considered a negative aspect of viviparity, does not seem important in the Chondrichthyes. In an overview of chondrichthyan reproductive strategies, several themes emerge. The primitive reproductive pattern is oviparity. In the Chondrichthyes, it is a specialized strategy since the production of a small number of large eggs has been selected for. Reproductive success (Richards et al, 1963) and diversity of species in the skates attest to the advantages of this strategy. Viviparity seems to have evolved independently, almost on a group-specific or species-specific basis. The repeated evolution of viviparity in different taxa, its prevalence in the elasmobranchs, and the reproductive success of viviparous species attest to its advantages. Eight factors are considered to affect the retention of oviparity or the evolution of viviparity. They probably act in concert. The relative influence of each factor probably varies according to species. REFERENCES Aristotle. Historia animalium. A. L. Peck (trans.) Vol. 1 (1965), pp ; Vol. 2 (1970), pp Harvard University Press, Cambridge. Amoroso, E. C Viviparity in fishes. Symp. Zool. Soc. (London), 1: Babel, T. S Reproduction, life history, and ecology of the round stringray, Urolophus halleri Cooper. Calif. Fish and Game Bull. 137: Balfour, F. M The development of elasmobranch fishes. In M. Foster and A. Sedgwick (eds.), The works of Francis Maitland Balfour, Vol. 1, pp ; Vol. 4, pp MacMillan, London. Ballard, W. W. 1966a. Origin of the hypoblast in Salmo. Does the blastodisc edge turn inward? J. Exp. Zool. 161: Ballard, W. W Origin of the hypoblast in Salmo. II. Outward movement of deep central cells. J. Exp. Zool. 161: Ballard, W. W. and L. M. Dodes The morphogenetic movements of the lower surface of the blastodisc in salmonid embryos. J. Exp. Zool. 168: Barnhart, P. S Notes on the habits, eggs and young of some fishes of Southern California. Bull. Scripps Inst. Oceanogr. Tech. Ser. 3: Baughman, J. L The oviparity of the whale shark Rhineodon lypus with records of this and other fishes in Texas waters. Copeia 1955: Beard, J The yolk-sac, yolk and merocytes in Scyllium and Lepidosteus. Anat. Anz. 12: Beard, J The germ cells. J. Anat. Physiol. (London) 38:82-102; ; Benin, L Sexualite et fecondation. In P. P. Grasse (ed.), Traite de Zoologie, Vol. 13, Part 2, pp Masson, Paris. Bigelow, H. B. and W. C. Schroeder Fishes of the western North Atlantic. Part 1. Lancelets, cyclostomes, and sharks. Sears Foundation for Marine Research, New Haven. Bigelow, H. B. and W. C. Schroeder, Fishes of the western North Atlantic. Part 2. Sawfishes, guitarfishes, skates and rays. Sears Foundation for Marine Research, New Haven. Blanquet, R. and H. M. Lenhoff A disulfidelinked collagenous protein of nematocyst capsules. Science 154: Bohadsch, J. B Beschretbung einiger minderbekannten Seethiere und ihren Eigenschaften. N. G. Leske (trans.). Waltherischen Hofbuchhandlung, Dresden. Boisson, C, X. Mattel, and C. Mattei La spermiogenese de Rhinobatus cemiculus Geof. St.- Hilaire (Selacien Rhinobatidae). Etude au microscope electronique. (A): Bull. Inst. Fond. Afrique Noire 30: pis Borcea, I Recherches sur le systeme urogenital des elasmobranches. Arch. Zool. Exptl. Gen. 4: Breder, C. M. and D. E. Rosen Modes of reproduction in fishes. The Natural History Press, Garden City, New York. Brown, C. H Egg capsule proteins of selachians and trout. Quart. J. Micr. Sci. 96: Budker, P La viviparite chez les selaciens. In P. P. Grasse (ed.), Traite de Zoologte, Vol. 13, Part 2, pp Masson, Paris. Budker, P The life of sharks. Columbia Univ. Press, New York. Callan, H. G The lampbrush chromosomes of Sepia officinalis L., Anilocra physodes L. and Scyilium catulus Cuv. and their structural relationship to the lampbrush chromosomes of amphibia. Publ. Staz. Zool. Napoli. 29: Calzoni, M Ricerche sulla placenta del Carchanasglaucus. Publ. Staz. Zool. Napoli 15: Chieffi, G The reproductive system of elasmobranchs: Development and endocrinological aspects. In P. W. Gilbert, R. F. Mathewson, and D. P. Rail (eds.), Sharks, rays and skates, pp Johns Hopkins Press, Baltimore. Clark, E The maintenance of sharks in captivity, with a report on their instrumental conditioning. In P. W. Gilbert (ed.), Sharks and survival, pp D. C. Heath & Co., Boston.

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