Urogenital Morphology of Dipnoans, with Comparisons to Other Fishes and to Amphibians MARVALEE H

Home , Ceratodontidae, Cloaca, Lepidosirenidae, Neoceratodus, Protopterus

JOURNAL OF MORPHOLOGY SUPPLEMENT 1:199-216 (1986)

Urogenital Morphology of Dipnoans, With Comparisons to Other Fishes and to Amphibians MARVALEE H. WAKE Department of Zoology and Museum of Vertebrate Zoology, University of California, Berkeley, California 94720

ABSTRACT The morphology of the urogenital system of extant dipnoans is compared among the three genera, and to that of other fishes and amphibians. Analysis is based on dissections, sectioned material, and the literature. Urogenital system morphology provides no support for the hypothesis of a sister-group relationship between dipnoans and amphibians, for virtually all shared characters are primitive, and most characters shared with other fishes are also primitive. Urogenital morphology is useful at the familial level of analysis, however, and synapomorphies support the inclusion of Lepidosiren and Protopterus in the family Lepidosirenidae separate from Neoceratodus of the family Ceratodontidae.

Analysis of relationships of the Dipnoi to lized muscle characters, for example) and to other fishes and to tetrapods have largely generic and familial relationships. been based on the morphology of hard tis- This survey and review of urogenital mor- sues-the skeleton, tooth plates, and scales. phology of the three genera of extant dip- Several studies of other systems are avail- noans and of the literature is undertaken able in the literature, but most are descrip- with the following objectives: 1) to provide tive and few are comparative, at least in information from a direct comparison of rep- considering all three genera of living lung- resentatives of all three extant genera; 2) to fish. Soft-tissue systems have had limited see if the urogenital system offers support for evaluation in terms of assessing evolution any of the current hypotheses of dipnoan re- and relationships. lationships; and 3) to determine whether or Several current hypotheses of the relation- not the urogenital system offers additional ships of the Dipnoi exist. These include the characters for the familial allocation of the proposal by Romer (’66) that dipnoans are genera. Urogenital morphology and physiol- the sister group to actinistians and rhipidis- ogy have received little consideration in these tians; that of Miles (‘77) classifying dipnoans contexts (see, for example, Thomson, ’69; Ro- as the sister group of rhipidistians only; and sen et al. ’81). In this paper I review the that of Rosen et al. (’81) in which dipnoans morphology of the components of the urogen- are the sister group of tetrapods, with actin- ital system in dipnoans, adding new infor- istians the outgroup and rhipidistians (poro- mation where possible, and I compare the lepiforms) in an unresolved cladistic position. morphology of these components among dip- Within the Recent Dipnoi, Neoceratodus (one noan species as well as with the urogenital species: forsteri) is placed in the family Cera- structures of other fishes and amphibians. todontidae; Lepidosiren (one species: para- For each component I discuss the implica- doxa) and Protopterus (four species: amphi- tions of the morphology for assessment of bius, aethiopicus, annectens, and dolloi) are relationships and suggest directions for fur- placed in the Lepidosirenidae. Soft-tissue ther research. morphology, because of the extreme paucity of extinct forms, can offer virtually nothing to our understanding of the relationships of MATERIALS AND METHODS dipnoans to rhipidistians, and little to the Urogenital organs from five specimens of association of dipnoans with actinistians (La- Lepidosiren paradoxa (220-300 mm standard timeria not necessarily being “representa- length) from Laguna Oca, Rio Paraguay, For- tive”). However, soft-tissue morphology can mosa, Argentina, six of Protopterus aethiopi- contribute to information about dipnoan-te- cus (230-320 mm standard length) from Kusa trapod relationships (Rosen et al., ’81, uti- Beach, Lake Victoria, near Kisumu, Kenya,

0 1987 ALAN R. LISS, INC. 200 WAKE and four of Neoceratodus forsteri (520-560 paired, somewhat lobed, elongate, highly pig- mm standard length) from the Brisbane mented retroperitoneal structures (Fig. 1).As River system, Queensland, Australia, were illustrated by Kerr (’01) and confirmed in my dissected. Specimens had been fixed in 10% material (Fig. lB,E), the kidneys of Lepide formalin and stored in 70% ethanol. Males siren are nearly as long as the testes and are and females including immature specimens closely bound to the testes, some testicular of each species were examined; several ma- tissue invading the kidney posteriorly. The ture males and females were in breeding con- kidneys of Protopterus (annectens; Kerr, ’01; dition, with active spermatogenesis in the aethiopicus, this work) are shorter but stou- former and well-yolked eggs in the latter. ter, lying more posteriorly in the body cavity Parts of each urogenital structure were ex- (Fig. lA,D). Neoceratodus (Fig. 1C,F)also has cised and prepared for histological examina- short, stout, posteriorly situated kidneys, tion. Twenty histological preparations of the more lobular than those of other genera. Kerr kidneys, gonads, ducts, and cloacas of each (‘01) distinguished the genital or vesicular species were made by embedding in paraffin, posterior portion of the kidney from the an- cutting at 10 pm, and staining with hematox- terior urinary part, the latter recognizable in ylin-eosin. part by reduced pigment, especially in Pre The nomenclatorial format for the morpho- topterus. Jesperson (‘69) was not able to dis- logical comparisons is as follows: The generic tinguish two such regions in Neoceratodus epithet only is used in situations in which grossly or in section, nor can I in my material. morphological descriptions are 1) appropri- Histology. Several workers have examined ate to all species of a genus, 2) obviously my nephron structure in both the anterior and work on the species listed above, 3) relate to “vesicular” parts of the kidney. Hickman and the single species in the genera Neoceratodus Trump (‘69) summarize research on Protop and Lepidosiren, or, 4) if an author did not terus and Lepidosiren and describe the re- identify species. If the morphology is species- gions of the nephron-a typical corpuscle and specific, or if inter-specific variation is ques- glomerulus, a ciliated neck segment, a prox- tionable or unknown, the Protopterus species imal tubule of two components, a ciliated examined is identified. intermediate segment, a distal segment, and a collecting duct system. Jesperson (‘69) ad- DESCRIPTION AND ANALYSIS ded information on Neoceratodus and com- The kidney and the gonads pared its nephron cytology to that of Lepido Kidney siren and Protopterus dolloi and €! aethiop Considerable attention has been paid to icus. My observations agree with these re- kidney structure, especially its development, ports (Fig. 2). The structure of the urinary in considering vertebrate relationships. In nephrons approaches uniformity among the fact, few vertebrate organs have so extensive three genera. Hickman and Trump (’69)con- a developmental and morphological data clude that the cytological features of the ne- base. Those data have been treated in terms phron of lungfkhes are similar in all aspects of physiological and ecological correlates, and to those of a number of “typical” freshwater in terms of evolutionary relationships, in- teleosts. cluding the origins of major vertebrate The nephrons in the vesicular portion of groups. Several decades of pertinent litera- the kidney have all of the components de- ture on vertebrates in general are summa- scribed above, including a well-developed rized by Goodrich (’30) and Fraser (’50), and glomerulus (as in Kerr’s, ’01, Figs. 50 and 51 by Gerard (’54) and Hickman and Trump of Lepidosiren). Kerr (’01) did not find cap- (‘69) for fishes specifically. Fox (’60, ’61, ’62, sules and glomeruli in r! annectens, but I ’63) made significant contributions to under- find them in the posterior kidney in the spec- standing both dipnoan and amphibian kid- imens of I? aethiopicus I examined (Fig. 1A). ney development. (He emphasized the They differ little from anterior nephrons but similarities between those two groups, but have greater tubular diameters, as Jesperson seems not to have considered teleosts; see (‘69) noted. Anterior nephric tubules have below.) Torrey (‘65) reviewed the morphogen- outer diameters of approximately 0.1 mm esis of the vertebrate kidney. Jesperson (‘69) (Fig. 2B), while posterior tubules have outer examined kidney structure of Neoceratodus diameters of 0.15-0.3 mm. Capsules are ap- with reference to the testicular network and proximately 0.13 mm wide and 0.15-0.2 mm summarized much of the literature produced high in both regions of the kidney. The tu- during the 1930s on lungfish nephron bule dilation may be due to the recent pres- structure. ence of sperm, for capsule and tubule may be Gross morphology. Lungfish kidneys are packed with sperm at breeding season (Fig. DIPNOAN UROGENITAL SYSTEM 201

ado 0 D E F

Fig. 1. Urogenital morphology of dipnoans. A) Male Female Neoceratodus forsteri (dissection). Ventral view. Protopterus aethiopicw (dissection, and after Kerr, '01). Abbreviations: ad, archinephric duct; ado, archinephric B) Male Lepidosiren paradoxu (dissection, and after Kerr, duct orifice; b, bladder; c, cloaca; k, kidney; Id, lateral '01).C) Male Neocerutodus forsteri (dissection, and after duct; md, Miillerian duct; mdi, Miillerian duct infundi- Jesperson, '69). D) Female Protopterus aethiopicus (dis- bulum; 0,ovary; od, oviduct; t, testis. Not drawn to scale. section). E) Female Lepidosiren paradoxa (dissection). F) 202 WAKE 6D). The morphology suggests that the pos- dipnoans and amphibians in the body seg- terior nephrons are capable of urinary func- ment from which the functional pronephric tion. It is not known specifically whether they units are derived (Fox, ’63).Very few amphib- are functional at any time, nor is it known ians of each of the orders have actually been whether lungfish urine would inhibit sperm examined, and therefore there is inadequate in any way. Smith (‘30)found that l? aethiop information to permit generalizations on icus urine is very dilute (16.5-18.8 mOsm/l) variation or patterns of change. and contains 5 mEqA of Naf and 3.5 mEqD Pronephric development of few osteichth- of NH4+. yans has been examined, and the available The modification of the kidney for sperm data are crucial to consideration of relation- transport is distinctly different in dipnoans ships. Fraser (‘27) rigorously analyzed kid- when compared with amphibians, which ney development in the sturgeon Acipenser seems not to have been noted previously. In and Maschowzeff (‘34-’35) examined Salmo, dipnoans the posterior kidney is involved in as well as the sturgeon, a frog, and a sala- sperm transport, and the nephrons are es- mander. Fraser (‘27) also carefully compared sentially unmodified from typical urinary development among fishes and amphibians. structures. In amphibians the anterior meso- She found that Acipenser has six nephros- nephros is utilized for sperm transport if the tomic units in the pronephros, derived from kidney is used reproductively. In gymno- somites four through nine. This pattern also phiones the nephrons are unmodified; in an- occurs in Salmo; it has three functional pro- urans and urodeles the nephrons are altered nephric units which must be assumed to be for sperm transport, usually lacking a cap- the primitive actinopterygian condition. It sule and glomerulus (see Wake, ’70). seems logical that the pronephric units arise Development. Several workers have drawn from the anterior-most body segments as a attention to the similarity of dipnoan and property of cephalized development. Fraser amphibian pronephric development. In par- (’27) summarized her own work and the lit- ticular, Semon (’011, working on Neocerate erature, with the following conclusions: dus, Kerr (’19)examining Lepidosiren and l? 1. The pronephric chamber is segmented annectens, Kindahl (‘37) and especially Fox, early on in cyclostomes, Polypterus, and gym- from work on Neoceratodus (‘60, ’61) and nu- nophione amphibians, but slightly later in merous urodeles (summarized in ’63), indi- chondrosteans (her “ganoids”) and dipnoans. cated marked similarities of pattern. These The pronephric chamber is recognizable as similarities include: 1) A reduced number of segmental only in terms of numbers of tu- pronephric units is present in larval dip- bules in anurans and urodeles and is a sin- noans, urodeles, and anurans; 2) the prone- gle, never metameric chamber in teleosts. phroi in these groups are related positionally 2. In gymnophiones the pronephric ne- to the anteriormost spinal nerves; and 3) the phrostomal units remain serial; in cyclo- pronephric (archinephric) duct is similarly stomes, chrondrosteans, Polypterus, and dip- induced. noans the walls of the units break down, a The pattern of development is indeed very limited occurrence in anurans and urodeles. similar, but the conclusion that ontogenetic 3. In cyclostomes, Polypterus, amphibians, similarity suggests an evolutionary relation- and dipnoans the units open into the coe- ship between dipnoans and amphibians is lomic space; in chondrosteans only anterior faulty. First, the similarity in number of units are open (the units of dipnoans close functional nephrostomal units (two in larvae developmentally like those of chondroste- of all three dipnoan genera, two in a number ans); and the pronephric units of teleosts are of urodeles, and three in anurans) is a prop- closed off from the coelom. Goodrich (’30)also erty of derived amphibian (and fish) taxa. presented a useful summary of pronephric In fact, the more primitive urodeles have up development. Polypterus (Kerr, ’02) has two to five nephrostomial units (Fox, ’63). The functional tubules, Lepisosteus three, Amia only gymnophione amphibian examined, Hy- (Jungersten, 1900) three or four. The number pogeophis rostratus, has nine functional units varies among teleosts and includes retention (Brauer, ’O2), and the greater number is of a functional pronephros in adults of some thought to represent the primitive condition. species. Elasmobranchs have three to seven Many authors note that greater numbers of functional units (Goodrich, ’30). pronephric units develop in all of these taxa, It therefore seems apparent that chondros- but that many are eliminated or are non- teans, holostean neopterygians, amphibians, functional. There is slight variation among dipnoans, elasmobranchs, and even cyclo- DIPNOAN UROGENITAL SYSTEM 203 stomes share a primitive pattern of proneph- containing mature sperm (Figs. 3A,B; 4A). ric development and structure. Reduction in The tubules are enlarged and have nests of number of pronephric units is a general de- primary spermatogonia peripherally, second- rived state in each lineage. Teleosts show a ary spermatogonia somewhat more medially, similar pattern but with a greater diversity and spermatids in the lumen (Figs. 3A,B; of derived states. In addition, mesonephric 4A). Tubules open into a longitudinal testis development shows considerable diversity duct that extends posteriorly to a series of among cyclostomes, elasmobranchs, primi- lateral ducts, the “vasa efferentia,” which tive osteichthyans, dipnoans, teleosts, and carry sperm to the nephrons as described amphibians. Therefore there are no shared below and illustrated by Jesperson (‘69), Kerr derived states that support the hypothesis of (‘Ol), and Goodrich (‘30). a sister-group relationship between amphib- The regressed testis has much stromatous ians and dipnoans, unless one were to as- tissue. Tubules are reduced and few sperma- sume that gymnophiones are ancestral to togonia are stained, although a few mature sharks, osteichthyans, and other amphibi- sperm are still present (Fig. 3C). ans, and are a sister group to cyclostomes. Grier et al. (‘80) reevaluated testicular The pattern is one of convergence in reduc- morphology in several orders of teleosts and tion of numbers of pronephric units in all found two different tubular types. Salmoni- vertebrate lineages. It seems that workers form, perciform, and cypriniform fishes have who saw the similarity in the pattern: 1) spermatogonia distributed the length of the selected among amphibians to find data to tubule; atheriniform fishes have spermato- support the hypothesis; 2) ignored the work gonia only in the distal end of the tubule. My on osteichthyans or chose not to compare observations indicate that all three genera of them to dipnoans and amphibians; and 3)did dipnoans have a spermatogonial distribution not recognize that shared primitive charac- identical to the salmoniform, perciform, and ter states and patterns of convergence do not cypriniform type. Lungfish therefore share provide evidence of relationship except at the the primitive osteichthyan pattern with most general level. many teleosts and are unlike amphibians (reviewed in Wake, ’79). Gonads My material did not allow spermatogenesis The testis. The testes of dipnoans also have to be traced. However, spermatogenesis and been described extensively. Gunther (18711, the fine structure of sperm have been exam- Ballantyne (‘281, and Jesperson (’69)have ex- ined by several workers. Agar (’11)described amined Neoceratodus, and Kerr (’01) has de- spermatogenesis in Lepidosiren, as did Bois- scribed Lepidosiren and I? annectens. I have son (‘61, ’63), and Boisson et al. (’67) in F! examined material representing all three annectens. Jesperson (‘71) described the fine genera (one of the surprisingly few such ex- structure of the sperm of Neoceratodus, and aminations) and can largely corroborate ear- Boisson and Mattei (‘65) and Purkerson et al. lier descriptions, while putting the patterns (‘74) described spermatozoa of I? aethiopicus. of development and of spermatogenesis in a Sperm of Protopterus have a long tapering more current context. head, a short middle piece, and two flagella Dipnoan testes are elongate structures (Fig. each about twice the length of the head. The 1A-C), bound to kidney and dorsal body wall head piece of Neoceratodus is similar to that by mesenteries, and overlain by fat, espe- of Protopterus, but sperm of Neoceratodus cially before the dry season, as Kerr (‘01) have a single flagellum about three times noted. The testes are stout medially, taper- the length of the head. Agar (’11)did not ing laterally. In Protopterus and Lepidosiren describe mature sperm of Lepidosiren. In my the posterior part of the testis is not sperma- material the head pieces of Lepidosiren sperm togenic, but is “vesicular” (Kerr, ’01). The are similar to those of other dipnoans, but vesicular testis invades kidney tissue (Fig. the tail could not be distinguished. Purker- 30. Neoceratodus lacks a vesicular posterior son et al. (’74) note that biflagellate sperm region (Jesperson, ’69; confirmed by my dis- occur normally in diverse species of fishes. sections). The vesicular region is described This appears to be a convergence, assuming by Kerr (‘01) as spongy, trabecular, and lack- that the monoflagellate state is primitive, ing spermatogenic tubules; this too is con- and as such allows no evaluation of rela- firmed by the present study (Fig. 4B). tionship. During the breeding season the spermato- The ovary. Very little attention has been genic part of the testis is filled with tubules paid to the morphology of the female urogen- Fig. 2. Kidney structure in Protopterus aethiopicus. glomeruli and the tubules of the “urinary” part of the A) Posterior “vesicular” part of the kidney. Note the kidney. Abbreviations: Bc, Bowman’s capsule; bv, blood pigmented periphery, the orientation of tubules, and the vessel; g, glomerulus; nt, nephric tubule; p, pigmented large diameter of the nephric tubules. B) Anterior region region. Scale bar in A, 0.4 mm; in B, 50 pm. of the kidney, illustrating large Bowman’s capsules and

Fig. 3. Testis morphology of Protopterus aethiopicus. part of the testis. Note absence of spermatozoa and in- A) Spermatogenic tubules in transverse section. B) Tu- vasion of testicular tubules into kidney tissue. Abhrevi- bules in longitudinal section. Note primary spermato- ations: k, kidney; s, spermatids; t, spermatogenic tubule; gonia peripherally the length of the tubule, secondary 1, primary spermatogonia; 2, secondary spermatogonia. spermatogonia somewhat more medially, and sperma- Scale bar, 50 pm. tids projecting into the lumen of the tubule. C) Vesicular

206 WAKE

Fig. 4. Testis morphology of Neocerutodus forsteri and in diameter than those of Protopterus; both are large Lepidosiren purudoxa A) Spermatogenesis in Neocerute adult specimens. B) The vesicular region of the testis of dus. Tubules are elongate and lined with primary sper- Lepidosiren. It is “spongy” and virtually atubular. Ab- matogonia. Spermatids in the lumen are in small, breviations: s, spermatids; 1, primary spermatogonia; 2, sworled masses in contrast to the large amorphous mass secondary spermatogonia. Scale bar, 50 pm. of Protopterus. The tubules of Neocerutodus are smaller DIPNOAN UROGENITAL SYSTEM 207 ital systems, perhaps because it presents a gests the true vesicle, or enlarged, diplotene “typical vertebrate” pattern. Only the early nucleus but does not label it. My histological descriptive work of Owen (1841), Ayers (1885), examination of the ovaries of each of the and Parker (1892) on I! annectens, and three genera confirms Beddard‘s basic de- Gunther (1871) on Neoceratodus contain sub- scription, including staining characteristics stantive information, most of which has sim- of oocytes. In vitellogenic oocytes the yolk ply been restated by others. By dissecting stains differently medially and peripherally. adult females of each of the living genera The nucleoli at that stage are adjacent to the and examining the female urogenital struc- nuclear envelope, and are numerous. Bed- tures histologically, I corroborate their de- dard (1886a) did not comment on the pigmen- scriptions, 100 years later. tation of the ovary. Melanocytes are present The ovaries are large, elongate structures in ovaries of all three genera; they are few that extend from near the heart to the poste- and largely peripheral in Protopterus and rior end of the kidneys (Fig. 1D-F). They are Lepidosiren, whereas they are very numer- paired, and held to the dorsal body wall by a ous and distributed throughout the ovarian stout mesovarium. The ovaries are also stroma as well as densely in the periphery of tightly bound to the oviducts by a mesentery, the ovary in Neoceratodus (see Fig. 4). as illustrated by Goodrich (‘30). Beddard (1886a,b) paid much attention to Approximately 5,000 large mature oocytes what he called “multinuclear bodies” or C‘OV~”)may be present in each ovary (extrap- “multinuclear ova” in the ovaries of the three olated from my count of 46/cm3 of a mature genera. He described these as multicellular Neoceratodus ovary 350-mm long, 40-mm structures that coalesced, modified the follic- wide most of its length, and 8 mm deep; the ular layer, and acquired yolk. At maturity, female was 560 mm standard length). Green- they were fully yolked and the equivalent of wood (’58; this volume) found more than ova that had undergone the standard pattern 5,000 larvae in one F! aethiopicus nest and of vitellogenesis. He constructed a sequence more than 2,000 in another and noted that of development based on stages observed in the latter was an incomplete sample. Com- his sections but of course could not trace a munal nesting is not likely. Ovaries of the particular oocyte through development. I in- females of the three genera that I examined terpret his multicellular bodies rather differ- all contained oocytes of three sizes and mat- ently. I consider that they are simply atretic uration (state of vitellogenesis) classes (Fig. follicles and that Beddard (1886a,b) con- 5). None appeared recently spent of mature strued their place in the sequence of devel- ova. I have been unable to find data on size opment backward. I suggest that the bodies of mature ova. Ovarian oocytes of the largest observed to have some yolk, multicellular size class in a large Neoceratodus are 3.0 mm invasion of the center of the follicle, and a in diameter Pig. 5C). This probably repre- modified follicle wall are early in atresia; sents full development, for Kemp (‘81) illus- those that lack yolk and have a fully cellular trates a fertilized ovum beginning second center and virtually no follicle wall are late cleavage at 3 mm diameter, which should in atresia and are being resorbed. I note nu- reflect the size of eggs just laid and before merous red blood cells in the cellular mesh any embryonic growth would have occurred. of such bodies, which suggests capillary in- Ken- (1900) noted that the laid egg of Lept vasion during resorption (Fig. 5B), but Bed- dosiren is very large (6.5-7.0 mm diameter) dard made no mention of the presence of and surrounded by a capsule that is thick blood cells. and jelly-like before fertilization but “thin Ovarian structure and pattern of ovum de- and horny” after. The mature egg is unpig- velopment in dipnoans shed no light on dip- mented. Division is similar to that of large noan relationships. The pattern is a primitive yolky eggs in diverse fish and amphibians one, shared with most osteichthyans and (Kemp, ’81; ’82; this volume). Egg structure many amphibians, particularly frogs. Grod- and vitellogenesis were described 100 years zinski’s (‘72) work on yolk structure in Nee ago by Beddard (1886a,b). His description of ceratodus indicates closest similarity to that ovum maturation in all three genera is basi- of Latimeria. The yolk spheres are structur- cally accurate, though the terminology must ally the same, though much smaller in Nee be updated. His “germinal vesicle” is the ceratodus. There is also some resemblance to unfertilized nucleus; the “vesicular dots” are elasmobranch yolk granules, particularly in nucleoli; plate 29 in Beddard (1886a) sug- boundary organization, but less similarity to

DIPNOAN UROGENITAL SYSTEM 209 those of chondrosteans and holosteans which sonephrons, which lead to collecting ducts lack the peripheral organization of the yolk and then to the archinephric duct (Wake, ’68, spheres. ’70). The gymnophione testis is essentially unmodified for sperm transport. In urodeles The urogenital ducts and the cloaca the anterior part of the kidney is reduced, The urinary ducts and in anurans it is reduced or lost; in dip- In development and function, the archine- noans, as noted, the posterior part of the kid- phric (mesonephric, Wolffian) ducts of female ney is modified. The trend is for the dipnoans are typical of female vertebrates in archinephric duct to bear sperm from re- general, except teleosts. The archinephric duced anterior mesonephric tubules (re- duct transports urine from the adult meso- ceived by lateral ducts from the longitudinal nephric kidney to the cloaca. The ducts in all testis duct) and to bear a limited quantity of three genera are separate, rather than fused, urine from the medial part of the kidney. A as they enter the cloaca, according to Ayer’s further trend in urodeles, and especially in (1885) and Parker’s (1892) figures and my anurans, is for the development de nouo of a observations (Fig. 1D-F). variable (by taxa) number of accessory ducts Much more attention has been paid to the that evacuate urine to the cloaca from the ducts of males, for the archinephric duct posterior or urinary part of the kidney. Such transports both urine and sperm. The lengthy accessory ducts do not occw in dipnoans. Jes- literature is summarized by Jesperson C69), person (‘69) notes that in Polypterus the tes- and his own work on Neoceratodus sheds con- tis duct opens into the distal part of the ar- siderable light on the male system. In all chinephric duct. He commented that three genera of lungfish, the archinephric Lepisosteus and Acipenser resemble Neocera- duct drains the elongate kidney (Fig. 1A-C). todus, with several lateral ducts leading from In Neoceratodus 11-13 “vasa efferentia” (lat- the short testes to the kidney. Amia (Junger- eral ducts) connect the longitudinal testis sen, 1900)has numerous vasa efferentia that duct to nephrons over the enti% length of the conduct sperm from the testis duct to a lon- kidney. The testis does not have discrete gitudinal kidney canal, then to the nephrons, sperm-producing and vesicular components. and finally to the archinephric duct and the Thus the archinephric duct evacuates urine, cloaca. Jesperson (’69)concluded that Neocer- and also sperm during the breeding season, atodus, at least, showed a primitive ostei- from the entire extent of the kidney. Sperm chthyan pattern similar to that of primitive packing the nephrons and the archinephric actinopterigians. Clearly, sperm transport in duct are shown in Figure 6A,C,D. Lepidosi- dipnoans differs significantly from that of ren has sperm transported from a tubular amphibians, probably due to the constraint longitudinal testis duct extending through of the pattern of mesonephric reduction as the vesicular part of the testis, where 5-6 noted above. lateral ducts lead to nephrons of the posterior part of the kidney (Fig. 1; Kerr, ’01, ’02). The oviducts Protopterus had a reduced “testicular net” in The oviducts are Miillerian duct deriva- which a single lateral duct extends from the tives that extend from near the heart to their longitudinal testis duct to several posterior entrance in to the cloaca dorsal to the uri- nephrons (Fig. 1; Parker, 1892; Kerr, ’01, ’02). nary caecum. The full sequence of develop- The dipnoan situation differs significantly ment has not yet been traced for any dipnoan, from that in amphibians. In gymnophiones though Semon (’01) described several stages. the archinephric duct transports both sperm Members of all three genera that I examined and urine and lateral ducts conduct sperm have slightly dilated infundibula (Fig. 1D- from the longitudinal testis duct to the me- F). In all genera examined, adult females with yolky ovarian ova have ducts that are highly convoluted, hyperemic, and lined with a multi-layered secretory epithelium (Fig. Fig. 5. Dipnoan ovaries. A) Protopterus aethiopicus. 7B,C). The regressed duct of a specimen with Ovary closely adheres to oviduct. Note stages of ovum very few large ova has a luminal epithelium development. Nuclei have numerous peripheral nucleoli. B) Lepidosiren paradoxa Note ova in two different stages without secretory cells and a much reduced of atresia. The upper one (arrows) still contains some diameter. yolk; the lower is multicellular and the follicle is nearly The primitive vertebrate state has the em- disintegrated. C) Neoceratodus forsteri Note large, yolky, bryonic Mullerian ducts becoming the adult (postvitellogenic) ova, a second class of vitellogenic ova, and a third class of slightly-yolked ova. Abbreviations: oviducts. Among osteichthyans, this pattern ca, corpus atretecum; m, melanocyte; n, nucleus; od, is retained by dipnoans, actinistians, chon- oviduct; y, yolk. Scale bar, 50 pm. drosteans, and some holostean neopterygi- 2 10 WAKE

Fig. 6. Cloaca, archinephric duct, Miillerian duct, and duct. Note mass of sperm in lumen. D) Posterior kidney. posterior kidney of a male Neocerutodus forsterz. A) Note glomerulus and tubules packed with sperm (ar- Transverse section of periphery of cloaca with ducts rows). Abbreviations; ad, archinephric duct; c, cloaca; ct, bound to cloaca by connective tissue. Section taken just connective tissue; g, glomerulus; md, Miillerian duct; nt, above juncture of ducts with cloaca. B) Miillerian duct. nephric tubule; s and arrow, sperm. Scale bar in A, 0.4 Epithelium of lumen is not secretory. C) Archinephric mm; in B,C,D, 50 pm. DIPNOAN UROGENITAL SYSTEM 211 ans. The condition in which most neoptery- Abdominal pores gians do not develop Mullerian ducts (their Despite the reports of a single abdominal “oviducts” are therefore not homologous to pore in Lepidosiren (Owen, 1841) and paired those of other vertebrates) is a derived state pores in Neoceratodus (Gunther, 18711, it (see Wake, ’85). The presence of oviducts of seems reasonable that the large numbers of dipnoans therefore gives no positive support mature ova of all genera are borne to the to hypotheses of relationship using presence cloaca via the convoluted, glandular oviducts of Mullerian duct derivatives as a character. and extruded via the vent. The pores are The abdominal pores of Protopterus opening located at the vent opening just beyond the by a single aperture was likened by Goodrich cloaca (but may be variable according to (’30)to the condition in “primitive osteichth- Owen, 1841), and the oviducts open into the yans,” but this conclusion seems very cloaca anteriorly. Bles (1898) noted that Lep tenuous. idosiren lacks pores, in contradiction to Ow- en’s 1841 report. The abdominal pores are The male Mullerian ducts therefore likely not associated with ovum Males of all three genera of lungfish retain transport as Goodrich (’30) also stated. Bles Mullerian ducts, at least to some extent. (1898) argued that the abdominal pores are Those of Neoceratodus extend from the ante- excretory, alluding to data on kidney-gonad rior end of the testis to the cloaca, where they development that are unclear to me. They fuse. They do not open to the cloaca (Jesper- may be vestigial, or involved in other fluid son, ’69),as confirmed in my dissections (Fig. flow, as Goodrich (‘30) suggested. 1C) but contra Gunther (1871). Jesperson’s (’69) Figure 4 shows the duct with a dilated The cloaca infundibulum; I did not find such a dilation. All three genera of dipnoans retain a sim- The ducts are thin, non-functional, connec- ple cloaca (Fig. 1) with the urinary ducts tive tissue strips. opening by a single aperture (Jesperson, ’69, In Protopterus, adult males retain vestiges Neoceratodus; Kerr, ’01, Lepidosiren and l? of the infundibulum at the anterior end of annectens). All three genera have a urinary the kidney, and a few millimeters of each caecum or bladder. Kerr (‘01) considered the duct posteriorly. The ducts unite in the wall caecum to be an anteriorly projecting dila- of the cloaca. My dissection of l? aethiopicus tion of the fused urinary ducts, or the urogen- revealed a condition similar to that described ital sinus. The amphibian bladder is an by Kerr (’01) for l? annectens, but the ducts outpocketing of the cloaca. In males the ar- do not reach the urogenital papilla in my chinephric (testis) ducts open separately on specimen (Fig. 1A). The ducts are even more the dorsal wall of the cloacal caecum at its reduced in males of Lepidosiren (Fig. 1B). juncture with the cloaca proper (Fig. 1A-C). Only the anterior infundibula are present Kerr (‘01) noted that each aperture is marked (the funnels of Kerr, ’01). Kerr (’01)reported by a prominent papilla during the breeding that additional vestiges of the ducts were season in l? annectens and Lepidosiren. I ob- present in a second-year male. served slight prominences at the openings in In amphibians, if Mullerian ducts are re- spermatogenic specimens of Lepidosiren, l? tained at all by males, they are represented aethiopicus, and Neoceratodus, but I do not only by connective tissue strips of varying know if these were in breeding condition. lengths beside the kidneys or, as in gymno- Goodrich (‘30) illustrates genital and urinary phiones, by connective tissue strips ante- papillae in a female l? annectens. My dissec- riorly followed by several millimeters of tions of females of all three genera of dip- glandular tissue posteriorly. The lumen of noans revealed slight rings of tissue at these the glandular portion opens into the cloaca. apertures but not pronounced papillae. The Wake (‘81) suggested that this secretory ac- rectal portion of the intestine is dilated and tivity in the male Mullerian duct of amphib- narrows slightly at its juncture with the ians is correlated with internal fertilization cloaca. The vent opens to the left of the tail and terrestriality. I find no indication of se- fin in the specimens I examined. cretory cells in the posterior ducts of Neocer- Lagios and McCosker (‘77) report the pres- atodus (Fig. 5B) and l? aethiopicus. The ence of a cloacal gland in l? aethiopicus and retention of anterior components and the lack l? dolloi, and its absence in Neoceratodus of effective association with the cloaca by and Lepidosiren. The gland is dorsal and pos- lungfish is distinctly different from the situ- terior to the urinary caecum and opens into ation in amphibians that retain the ducts. the cloaca at the juncture of the caecum with

DIPNOAN UROGENITAL SYSTEM 213 the cloaca. Lagios and McCosker (‘77) sug- Stasko-Concannon, quoted in Chester Jones gest that the gland may be cation-secreting, and Mosley, ’80). Acipenser has a similar in- functionally similar to the rectal glands of terrenal distribution along the cardinals and elasmobranchs, holocephalans, and Lati- their tributaries beside and within the kid- meria, though the anatomical relationship ney (Youson and Butler, ’76a); corpuscles are differs from that of the latter groups. The found throughout the kidney, but primarily sizes of Lagios and McCosker’s (‘77) speci- in the reduced anterior two-thirds of the mens were not indicated; I cannot determine kidney. Corpuscles lie in the anterior two- whether they looked for age or sex differ- thirds of the kidney of the brachiopterygian ences in the morphology of the gland. Polypterus (Youson and Butler, ’85). A simi- Presence of a cloaca is of course the prim- lar situation obtains in Polyodon (Lagios and itive vertebrate pattern, and all bony fish Stasko-Concannon, quoted in Chester Jones that lack a cloaca are derived. and Mosley, ’80) and in the neopterygian “holostean” Amia (Youson and Butler, ’76b). The “adrenal” In the neopterygian Lepisosteus, corpuscles Several workers have cited the “amphib- are in the anterior half of the kidney (Bhat- ian characteristics” of the interrenal or ad- tacharyya et al., ’81). As Youson and Butler renocortical homolog tissue of dipnoans (’85)point out, the distribution of adrenocor- (Chester Jones and Mosley, ’80; Janssens et tical corpuscles offers little to support (or ne- al., ’65; Call and Janssens, ’75). Such tissue gate) current hypotheses of the taxonom- has been identified in Protopterus (Gerard, ic status of the “primitive” osteichthyans. ’54; Janssens et al., ’65) (species not listed) Among teleosts, interrenal tissue is confined and in Neoceratodus (Call and Janssens, ’75) to the head kidney, so far as is known, which based on morphology and histochemistry. is largely lymphoid and hemopoietic tissue. I Cord-like cell groups located on the postcar- found no information about the interrenal dinal veins and their tributaries between tissue of teleosts that: 1) lack a head kidney renal and perirenal tissue converted proges- (sensu Nandi, ’62); or 2) retain a functional terone to corticosterone when tested in uitro, pronephros throughout life, as do some tele- and tested positively for cholesterol (Call and osts (Zoarces, for example). When one exam- Janssens, ’75). No chromaffin tissue was ines the data on interrenal tissue for am- found in either genus (Call and Janssens, phibians, one quickly realizes that there is ’75). The position and histochemical charac- no common amphibian pattern, despite as- teristics of the interrenal tissue in Protopte- sertions in the literature. Dittus (’36) consid- rus and Neoceratodus were considered to be ered the gymnophione condition primitive for very similar to those of interrenal tissue in amphibians. The interrenal tissue starts an- adult Pleurodeles (Certain, ,611, a urodele, terior to the kidney and is associated with and the larvae of certain frogs (Chester Jones the aorta. Masses of interrenal tissue extend and Mosley, ’80). posteriorly in the entire extent of the kidney An examination of the position and cellu- associated with post-cardinal and renal ves- lar characteristics suggests that the similar- sels. Tissue may also lie medial to the kid- ity of dipnoan and amphibian interrenals is neys or embedded in the medial kidney tis- a tenuous one. The interrenal of Latimeria sue, especially ventrally (see also Gabe, ’71). has a large number of corpuscles in the walls Urodele interrenal tissue shows considerable and along the extent of the postcardinals and variation, from association with blood ves- their tributaries in the kidney (Lagios and sels only in Siren, to being embedded in the kidney along the renal vessels in Am- phiuma, and embedded and concentrated an- Fig. 7. Dipnoan oviducts. A) Regressed duct in Protop teriorly in Necturus (Hartman and Brownell, terus aethiopicus. The ovary of the specimen lacked large ’49) and several other diverse species (Hanke, yolky ova; thus I infer that it is post-breeding. B) LepC ’78). Anurans usually have interrenal tissue dosiren puradozu oviduct bound to posterior part of the concentrated as a longitudinal mass on the kidney. The epithelium of the lumen is proliferated and secretory. C) Neocerutodus forsteri oviduct in a hyper- ventral side of each kidney associated with emic, secretory state. The ovary contained large ova; branches of the renal vein. thus I infer that ovulation is imminent, and the oviduct The literature is contradictory with regard ready to transport ova. Little appears to be known of to dipnoans. Chester Jones and Mosley endocrine regulation of breeding in dipnoans. Abbrevia- tions: k, kidney; 1, lumen of oviduct; se, secretory epithe- (‘80:422) state that “the adrenocortical hom- lium. Note that all three specimens are photographed to ologue consists of small cells closely associ- the same scale. Bar, 50 pm. ated with the post-cardinal veins and their 2 14 WAKE tributaries where these vessels pass through which information is available; and 4) an the kidneys.” Balment et al. (’80:529) claim almost dogmatic assumption that amphibian that “jawed fish, teleosts and elasmo- morphology provides information crucial to branchs” share a common association with understanding dipnoan biology. Some very the cardinal vein and pronephric-mesone- general but similar aspects of dipnoan and phric units, but they state that dipnoans have amphibian biology suggest that bases for an amphibian-type interrenal associated with similarity should be explored, but that explo- kidney tissue. Hanke (‘78) defines the am- ration has rarely been done on an appropri- phibian characteristic as a “close association ate scale. Further, comparisons with am- [of interrenal tissue] with kidney cells, sepa- phibians have usually been made with highly rated from the tubules only by a basement selected species, not considering whether the membrane and a thin layer of connective species were primitive and representative of tissue.” The “amphibian characteristic” does the entire amphibian grade, or exhibiting not obtain for many amphibians and appears states derived for particular lineages and to be a derived state among some urodeles therefore indicative of convergence if and most frogs. This is probably a conver- “shared” by dipnoans and one or more am- gence within amphibians, since some frogs phibian species. As indicated above, analysis show a more primitive condition. It appears of urogenital morphology almost invariably then that urodeles and anurans (and gymno- indicates one of three conclusions: 1) Dip- phiones) followed independent evolutionary noans share a primitive vertebrate pattern courses to their particular states, although it with fishes and with amphibians; 2) dip- should be noted that information is available noans share general features with many os- only for primitive gymnophiones (Dittus, ’36). teichthyans, and various teleostean lineages The interrenal morphology of Protopterus (see are derived; and 3) the derived features Janssens et al., ’65) approaches the “amphib- shared by dipnoans and some amphibian spe- ian characteristic” less closely than does cies are not those of the order, let alone the Neoceratodus (see Call and Janssens, ’75) and class, and often not even the family of am- is clearly associated with postcardinal and phibians being compared. Therefore, conver- renal veins. Dipnoan interrenals, while re- gence, not relationship, must be inferred. In sembling those of selected amphibians, also general, the data do not support hypotheses resemble to some degree those of chondros- of dipnoans being the sister group of tetra- teans and Latimeria. Chester Jones (‘57) sug- pods. Urogenital system morphology is more gested teleost and crossopterygian affinities similar to that of actinistians, chondros- before he emphasized the “amphibian” teans, and primitive teleosts, but there are features. few shared derived characters of the urogen- I concur with Lagios and Stasko-Concan- ital system to ally even these groups (Fraser, non (in Chester Jones and Mosley, ’80) that ’27, lists some). This is not to say definitively the diffuse distribution of interrenal tissue that dipnoans are not the sister group of te- along the cardinal veins and their branches trapods, but that support for that hypothesis is a primitive condition in vertebrates. A (and most others) must come from other number of lineages often have modified that systems. pattern in different ways, apparently inde- Urogenital morphology does, however, pro- pendently. There are no shared derived char- vide support for the familial allocation of dip- acters of the interrenals that ally dipnoans noans, in agreement with osteology with amphibians as a group, or with urodeles (Marshall, this volume), myology @emis, this in particular. There are, however, some con- volume), and numerous other features (Good- vergences among derived genera in several rich, ’30). Lepidosiren and Protopterus have fish and amphibian lineages. four synapomorphies; Protopterus and Nee ceratodus each have an autapomorphy (see CONCLUSIONS Fig. 8). These data support the inclusion of It is apparent that analysis of the compar- Lepidosiren and Protopterus in a family sep- ative biology and evolution of the dipnoan arate from Neoceratodus. Even these data urogenital system is incomplete for several are incomplete. For example, Neoceratodus reasons: 1)lack of direct comparison of mem- has uniflagellate sperm and Protopterus bi- bers of all three genera; 2) lack of informa- flagellate, as noted above. However, there is tion for many primitive osteichthyans and no comparable information about the sperm for an effective representation of the diver- of Lepidosiren; thus the difference between sity of teleosts; 3) lack of any attempt to Lepidosiren and Neoceratodus is not a useful compare dipnoans with other fish taxa for character. DIPNOAN UROGENITAL SYSTEM 2 15 Examples of paucity of complete informa- California Academy of Sciences and Kristen tion for most aspects of urogenital morphol- Nygren for sectioning the material. I am ogy have been indicated in several instances pleased to acknowledge the research support above. Therefore my first goal, that of provid- of the National Science Foundation (BSR 83- ing a direct comparison of all three genera of 05771). dipnoans, is not yet completely fulfilled. First, more basic data on structure and de- LITERATURE CITED velopment of the urogenital system of dip- Agar, W.E. (1911) The spermatogenesis of Lepidosiren noans, non-teleostean osteichthyans, various paradoxa. Q. J. Microsc. Sci. 571-44. Ayers, H. (1885)Beitrage zur Anatomie und Physiologie teleosts, and a greater diversity of amphibi- der Dipnoer. Z. Naturwiss. Jena 18:479-527. ans of all three orders are needed. These Ballantyne, F.M. (1928) Note on the male genito-urinary should be provided systematically, using organs of Ceratodus forsteri. Proc. Zool. SOC.Lond. modern technology. Second, an objective 1928:697-698. cross-comparison of many taxa must be un- Balment, R.J., I.W. Henderson, and I. Chester Jones (1980) The adrenal cortex and its homologues in verte- dertaken. Third, hypotheses of relationship brates; evolutionary comparisons. In I. Chester Jones must be structured rigorously, and alterna- and I.W. Henderson (eds): General, Comparative and tive hypotheses also considered. Identifica- Clinical Endocrinology of the Adrenal Cortex. London: Academic Press, pp. 525-562. tion of character states should be done Beddard, F.E. (1886a) Observations on the ovarian ovum according to current methods, with assess- of Lepidosiren (Protopterus). Proc. Zool. SOC.Lond. ment of polarities and analysis of grades and 1886:272-292. clades, not by comparisons of just randomly Beddard, F.E. (188613) Observations on the development and structure of the ovum in the Dipnoi. Proc. Zool. (or worse, subjectively) chosen species. With Sac. Lond. 1886502-527. these three criteria met, analysis of the uro- Bhattacharyya, T. K., D. G. Butler, and J. H. Youson genital system should provide insight into (1981) Distribution and structure of the adrenocortical the functional biology of the system and its homolog in the garpike. Am. J. Anat. 160231-246. pattern of diversification, thereby giving Bles, E. J. (1898) The correlated distribution of abdominal pores and nephrostomes in fishes. J. Anat. Physiol. clues to the evolutionary relationships of dip- 32:484-512. noans to other vertebrates. Boisson, D. (1961)La spermatogenese de Protopterus annectens (Dipneuste) du Senegal. Ann. Fac. Sci. univ. ACKNOWLEDGMENTS Dakar 6133-144. Soisson, C. (1963) La spermiogen&sede Protopterus an- I thank William E. Bemis for insisting that nectens (Dipneuste) du Senegal Qtudiee au microscope I undertake this survey, and for providing optique et quelques details au microscope electronique. specimens and the Annotated Bibliography Ann. Fac. Sci. Univ. Dakar 10:43-72. Boisson, C., and X. Mattei (1965) A propos de l’acrosome compiled by Elizabeth Conant. 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