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The genus Xiphophorus is found from northeastern Mexico (Coahuila) for about 2200 Km as far as Honduras. There are 26 species, of which 21 occupy headwaters on the eastern slope of the Sierra Madre Oriental and continuing Cordillera to the southeast. Virtually all the species in the headwaters occupy limited ranges, often in rivers traversing karst country that are separated from lowland streams by underground passages. Only the three forms in the coastal plain are more widely distributed. Nineteen taxa occur within 400 Km of the Mexican Trans Volcanic Belt, suggesting that the genus may have evolved in this region. In many localities two species are sympatric, but natural hybrids are only known from three or four sites. Four monophyletic groups have been identified: the northern platyfish and the northern swordtail groups, north of the Mexican Trans Volcanic Axis, and to the south the helleri and the clemenciae swordtail groups. The status of the three southern platyfish is still not resolved and the phylogenetic relationship of the different groups to each other is still not fully understood.

The swordtail, Xiphophorus clemenciae(Poeciliidae), has been considered a species of special concern because of its apparent limited range. Although described in 1959, it is officially still known only from three locations in the Rio Coatzacoalcos basin, Mexico. Zoogeographic studies have now shown that this species is widespread and abundant but restricted to the uplands of the Rio Coatzacolacos basin where it replaces in many areas the common swordtail, X. helleri. Two new swordtail taxa, X. mixei and X. monticolus, are described from headwater streams of the Rio Jaltepec, a major Rio Coatzacoalcos tributary, Oaxaca, Mexico. The new forms are sympatric in part of their range and replace both X. clemenciae and X. helleri. Morphometric and molecular analyses revealed that X. clemenciae and the two new species constitute a monophyletic clade that exhibits a closer evolutionary affinity to the ‘‘northern’’ swordtails and the ‘‘platyfish’’ group of the genus rather than to X. helleri and the other ‘‘southern’’ swordtails. The evolutionary relationships of these taxa are discussed.

The Genetic Stock Center can trace its origin to Myron Gordon’s doctoral research at Cornell University. He began his scientific work in 1924 while still an undergraduate student with 6 platyfish of a domesticated stock, and the following year he added some swordtails. Platyfish and swordtails were not new to him because already as a teenager he had kept many tropical fish. But a great enthusiasm for genetics was aroused by Professor A.C. Fraser in the Department of Plant Breeding. Other professors at Cornell who took a great interest in his work were H.D. Reed (Zoology), who was his official sponsor, and G.C. Embody (Limnology) and R.A. Emerson (Plant Breeding). Dr. Gordon concentrated first on platyfish, working out the genetics of various pigment patterns, and then on swordtails. He coined the terms micromelanophore and macromelanophore (Gordon, 1926). It is an incredible coincidence that at the same time, Dr. Curt Kosswig in Germany also started research on swordtails. Neither Gordon nor Kosswig knew of the other’s existence. But their initial contributions to the Xiphophorus melanoma problem were not the same (Atz, 1941). Kosswig (1928) pointed out that the melanoma was hereditary and in some way associated with melanophore patterns from the platyfish, while Gordon (1931) clearly identified the macromelanophore gene of X. maculatus as being responsible for the tumor. The complementary relationship between laboratory and field research that has made the study of Xiphophorus so exciting and successful was initiated early in Gordon’s career, and one hopes it will be continued in the future. Whereas the development of melanoma in certain platyfishswordtail hybrids was predictable, the sporadic appearance of such growths in some platyfish presented a problem. While working on his thesis at Cornell, Gordon reasoned that platyfish were known only from the hobby and had been bred for many years under domestication and hybridized with swordtails. He could not determine whether platyfish of natural populations also developed pigment cell abnormalities or whether the occurrence of melanoma in the domesticated platyfish was due to the introgression of helleri genes. The sample of 83 platyfish from Mexico in museum collections was too small to provide the answer. He also thought it possible that platyfish and swordtails might hybridize in nature, because Meek (1904) had found them together in one location. He must go to Mexico himself! None of the modern collecting paraphernalia were available to Gordon. There were no maps or rental cars, and only a few paved roads. He went in a model T Ford, loaded with camping equipment, shotguns, letters of introduction to every conceivable agency, and with milk and oil cans for shipping fish back to the United States. In 1930 only 6 of the 22 presently described species of Xiphophorus were known, and virtually nothing was known about their habitat and distribution. Platyfish were assigned to Platypoecilus and swordtails were known as Xiphophorus. Gordon knew that 3 scientific collections of platyfish had been made. The first consisted of 2 fish collected by La Salle prior to 1866 in “Central America,” a locality description utterly useless if you are trying to find the fish (Gordon, Received January 31, 2001; accepted March 30, 2001. E-mail Xiphkallman@aol.com Mar. Biotechnol. 3, S6–S16, 2001 DOI: 10.1007/s10126-001-0022-5

Allelic variation at the autosomal tail spot pigmentation locus in Xiphophorus variatus is correlated with variation in oxygen consumption, condition factor, growth rates, and other physiological parameters. To test whether the locus is also associated with effects on energy storage, we measured triglyceride and glycogen reserves in two sibships derived from test crosses. One sibship contained cut-crescent and wild-type genotypes, while the other contained crescent and wild-type genotypes. Cut-crescent males had significantly less muscle glycogen than wild types, while crescent males had significantly lower body fat levels than wild types. Liver fats and glycogen and gonad sizes did not differ among the three genotypic classes. Thus, diferences among genotypes may be compartment specific. Variation in muscle glycogen levels, growth rates, and oxygen consumption may reflect a common cause, such as mitochondrial respiratory efficiency. Our data cannot test whether the associations between tail spot classes ...

1. 1. Individuals from independent sibships of Xiphophorus variatus were maintained at defined feeding levels from birth to sexual maturity, and then killed for analysis of triglycerides and glycogen. 2. 2. Significant variation was observed among sibships in adult sizes, triglyceride and glycogen storage, condition factor and ovary weights. 3. 3. Reduced rations had major effects on growth and energy storage in one sibship (“steep” response), but no noticeable effect in another (“flat” response). 4. 4. Flat and steep responses may be phenotyped in the laboratory and their genetics analysed by reciprocal crosses among lines. 5. 5. Females had higher liver glycogen and lower liver fats than males and appeared to cycle fats between the liver and the ovaries.

There is great interest in the sex chromosomes of Xiphophorus fishes because both WY/YY and XX/XY sex-determining mechanisms function in these species, with at least one taxon possessing all three types of sex chromosomes, and because in certain interspecific hybrids melanoma arises as a consequence of inheritance of the sex-linked macromelanophore determining locus (MDL). Representational difference analysis (RDA) has been used to clone two sequences from the sex-determining region of X. maculatus, including a cholinergic receptor, nicotinic, delta polypeptide (CHRND) orthologue. Allele-specific assays for these sequences, as well as for the sex-linked XMRK1 and XMRK2 genes, were developed to distinguish W, X, and Y chromosomes derived from a X. maculatus (XX/XY) strain and a X. helleri (WY/YY) strain. Linkage mapping localized these markers to linkage group (LG) 24. No recombinants were observed between XMRK2 and MDL, confirming a role for XMRK2 in macromelanophore development. Although the master sex-determining (SD) locus certainly resides on Xiphophorus LG 24, autosomal loci are probably involved in sex determination as well, as indicated by the abnormal sex ratios in the backcross hybrids that contrast theoretical predictions based on LG 24 genotyping. Marker development and allelic discrimination on the Xiphophorus sex chromosomes should prove highly useful for studies that utilize this genus as an animal model.

The inheritance of 23 protein polymorphisms was compared with the inheritance of a DNA restriction fragment length polymorphism (RFLP) of a strongly cross-hybridizing erb B-related sequence, epidermal growth factor receptor-like-1 (EGFRL1), in Xiphophorus clemenciae X X. milleri-derived backcross hybrids. Two polymorphic bands were noted in this cross with a v- erb B probe after Pst I digestion: a 10-kilobase (kb) band in X. clemenciae and a 9-kb band in X. milleri. Joint segregation analysis of the RFLPs and protein polymorphisms indicate that this erb B-related sequence can be assigned to Xiphophorus linkage group VI, which also comprises genes coding for glutamine synthetase ( GLNS ), nucleoside phosphorylase-2 ( NP2 ), and transferrin ( TF ). We have designated this RFLP as alleles at a locus called EGFRL1 because of very strong cross-hybridization with the v- erb B probe, known to be homologous to the mammalian EGFR gene. This mapping assignment is the first autosomal linkage of an oncogene sequence reported in fish, which provide a large number of genetically controlled experimental tumor models.

Fish gene mapping studies have identified several syntenic groups showing conservation over more than 400 million years of vertebrate evolution. In particular, Xiphophorus linkage group IV has been identified as a homolog of human chromosomes 15 and 19. During mammalian evolution, loci coding for glucosephosphate isomerase, peptidase D, muscle creatine kinase, and several DNA repair genes ( ERCC1, ERCC2 , and XRCC1 ) appear as a conserved syntenic group on human chromosome 19. When X. clemenciae and X. milleri Pst I endonuclease-digested genomic DNA was used in Southern analysis with a human ERCC2 DNA repair gene probe, a strongly cross-hybridizing restriction fragment length polymorphism was observed. Backcrosses to X. clemenciae from X. milleri × X. clemenciae F 1 hybrids allowed tests for linkage of the ERCC2 -like polymorphism to markers covering a large proportion of the genome. Statistically significant evidence for linkage was found only for ERCC2L1 and CKM (muscle creatine kinase), with a total of 41 parents and 2 recombinants (4.7% recombination, χ 2 = 35.37, P GPI and PEPD in linkage group IV was detected. The human chromosome 19 synteny of ERCC2 and CKM thus appears to be conserved in Xiphophorus , while other genes located nearby on human chromosome 19 are in a separate linkage group in this fish. If Xiphophorus gene arrangements prove to be primitive, human chromosome 19 may have arisen from chromosome fusion or translocation events at some point since divergence of mammals and fishes from a common ancestor.

Male platyfish, Xiphophorus maculatus, ranging in age from 12 to 54 months (normal life span is 30 months), were examined for age-related changes in the distribution of immunoreactive gonadotropin (ir-GTH) and luteinizing hormone releasing hormone (ir-LHRH) in their pituitary glands, and for changes in the histology of their testes. Males sacrificed at 13 and 16 months of age served as controls. Immunocytochemical methods demonstrate that even in the oldest fish, some almost twice the average platyfish life span, ir-GTH and ir-LHRH are localized in the same pituitary cell types in the caudal pars distalis and pars intermedia as in younger sexually mature fish. The testes of old fish continue to contain all stages of spermatogenesis; however, there are age-related increases in the amount of intertubular connective tissue and the relative number of spermatozeugmata, and distortions in the organization of acini. Our observations indicate that GTH and LHRH production and spermatogenesis continue, and thus a potential for reproductive capability persists, in male platyfish up to the time of their death resulting from old age.

In natural populations of platyflsh, Xlpbophorus maculatus, there are three sex chromosomes—W, X, and Y. Females may have one of three genotypes: WY, WX, or XX, and males may have either XY or YY. The W chromosome can be considered a modified X that blocks the male-determining function of the Y. The platyfish may represent an evo- lutionary stage at which female heterogamety arises through a single mutation. In the midst of a male heterogametlc system. Our serological analysis revealed presence of H-Y antigen In XY and YY males, but not In XX, WX, or WY females, Indicating that H-Y antigen may not be associated Invariably with the heterogametlc-type gonad—especially in transi- tional systems.

The onset of sexual maturity in fishes of the genus Xiphophorus depends upon a multiple allelic series at the P locus. X. helleri of genotype PhPh initiate sexual maturity at 10.4 weeks and X. maculatus of genotypes P3P3 or P4P4 at 8.7 or 20 weeks, respectively. Hybrids between the two species of genotype PhPh and possessing some autosomes of X. maculatus in homozygous condition exhibit precocious maturation at 5.3 weeks and are correspondingly small. PhPh hybrids of the backcross generations to X. helleri mature at the same age and size as X. helleri. The P3Ph and P4Ph backcross hybrids, however, exhibit greatly delayed maturation, and the delay becomes more severe as the number of X. helleri autosomes increases. The phenotype results from the interaction of the P locus with a system of regulatory genes. Both the P factors and the regulatory systems are species-specific. Synthetic luteinizing hormone-releasing hormone (LH-RH) was effective in inducing proliferation and functional activity in the gonadotrops and ultimately sexual maturity in all genotypes, including PhP3 hybrids. Whereas the response of different genotypes to gonadotropin was essentially the same, their response to LH-RH was significantly different. A positive correlation exists between the rapidity of the response to LH-RH and the genetically determined age at which spontaneous maturation takes place. The maturity induced by LH-RH was not permanent. After cessation of LH-RH administration the gonadotrops became inactive and regressed. These observations suggest that the P locus is not concerned with the production of LH-RH. Rather, the P locus appears to control the physiological function or the fate of LH-RH.

Data are presented from studies on heritable melanoma formation in certain hybrids of Xiphophorus fishes. In the best investigated case, benign and malignant melanomas were caused primarily by hemizygous and homozygous loss, respectively, of a tumor suppressor gene that promoted final pigment cell differentiation, although additional genetic events may be of importance. In other melanoma cases, different genes, most of them presumably also tumor suppressor genes, have been implicated in the development of melanomas and may interact at other levels of pigment cell development, such as commitment, migration, or homing of pigment cell precursors. Importantly, genetic factors have been identified that do not allow melanoma formation despite the loss of tumor suppressor genes. Taken together, these findings stress the complexity of tumorigenesis and the heterogeneity of pathways that lead to cancers of the same histological type.

1. By use of flow cytometry we measured nuclear DNA content in cells from 16 stocks representing 9 species of the genus Xiphophorus. 2. Significant differences were detected between certain stocks and species with respect to DNA content. 3. Male-female differences were apparent in 5 of 7 stocks in which males and females were studied. 4. Estimation of nuclear DNA content is of potential significance in connection with the genetics of sex determination and the study of taxonomic relationships.

The highly inbred Coatzacoalcos (Cp) strain of the platyfish, Xiphophorus maculatus, was noted for a high percentage of infertile females (XX). The ovaries of approximately one-quarter of all females regress. The time of gonadal atrophy varied from before sexual maturation up to 11 months of age. The gonadotropic zone of the pituitary was hypertrophied in regressed females. Transplants of immature testes and ovarian tissue into the caudal musculature of regressed females and the subsequent maturation of the grafts demonstrated that the ovarian degeneration was not due to pituitary or hypothalamic malfunction or an autoimmune disease. The cause of the gonadal degeneration was apparently localized to the ovary itself. This phenomenon was never observed in males (XY). Regressed ovaries fell into two categories, designated types I and II, with all being characterized by the presence of ductlike structures which resembled male efferent ducts, lined by Sertoli cells. Type I ovaries bore a marked similarity to certain mammalian dysgenetic gonads, while type II ovaries contained many proliferating germ cells and could be compared to the human neoplasm termed gonadoblastoma. It is suggested that the physiological lesion responsible for the ovarian regression syndrome involves the processes that control the determination and differentiation of the germ cells similar to those found in human 46,XY gonadal dysgenesis.

Ten species of teleosts belonging to three families of Atheriniformes (Poeciliidae, Cyprinodontidae, Goodeidae) have been tested for their ability to survive in fresh water without a pituitary gland. The fish of the current report respond similarly to Atheriniformes previously investigated. Hypophysectomized fish enter into osmotic shock and generally die within three weeks when placed into fresh water but they will survive in dilute sea water (1.2% solution of commercial sea salts). Fish belonging to Poecilia survive for the shortest period. By comparison the mean survival time for Xiphophorus is longer and more variable. No correlation exists between the number of days hypophysectomized fish are held in one third sea water prior to testing and the length of time they survive in fresh water. This is also true for fish challenged a second time after recovering from osmotic shock. Presumably, osmotic difficulty commences upon exposure to fresh water but can be alleviated when fish are returned to dilute sea water. Five hypophysectomized fish survived in fresh water. Each of these exceptional animals had suffered brain damage during surgery. The significance of these observations is discussed as it is related to the natural habitat and salt tolerances of the fish studied.

The capacity of the pars distalis to secrete its hormones when removed from connexions with the hypothalamus has been assessed in the teleost Poecilia formosa. Mortality, integumentary characters, body growth, fin regeneration, freshwater adaptation, liver reserves, fat stores, haematology, and the histology of the thyroid gland, interrenal tissue and ovary, have been studied in intact fish, in hypophysectomized fish, and in hypophysectomized fish bearing a pituitary homotransplant in the musculature of the caudal peduncle. Growth, fin regeneration, liver reserves, fat stores, interrenal and ovary, have been studied also in sham-hypophysectomized fish. It is concluded that the ectopic pituitary transplant in this fish secretes thyrotrophin (TSH) at a higher rate than normal, adrenocorticotrophin (. at a subnormal rate, and growth hormone (GH) only in very small amounts. The transplant secretes the prolactin-like hormone essential for freshwater adaptation, but does not secrete gonadotrophin. The extensive literature on the ectopic pituitary has been surveyed, and the present results were compared with those obtained for other vertebrate groups. The hypothalamic influence on TSH secretion in Poecilia appears to be inhibitory, in contrast to the stimulatory relationship in mammals, and the pituitary of this teleost seems to have greater autonomy in the secretion of ACTH than the mammalian gland. The hypothalamic control of GH, prolactin and gonadotrophin, however, is probably essentially the same in Poecilia as in mammals.

Two clones of the hermaphroditic fish Rivulus marmoratus Poey, identified by their intraclonal histocompatibilities and interclonal histoincompatibility, were kept through successive uniparental generations, each fish in isolation. These hermaphrodites emit internally self-fertilized eggs incubated up to 2 1/2 days intraparentally and thereafter extraparentally. By extraparental incubation at low temperature, primary males can be produced at will from eggs otherwise yielding hermaphrodites. Secondary males originate from hermaphrodites, usually late in life, if the ovarian component of the ovotestis involutes. There are no females. Eggs from an aberrant hermaphrodite chronically emitting un-self-fertilized eggs were cross-fertilized artificially and also naturally by sperm from a primary male. A fin graft from the clone of the male parent was accepted (autograft reaction) by the interclonal hybrid, showing that amphimixis had occurred and that the clone of the graft donor is homozygous for the histocompat...

1. 1. In five species of teleosts hepatic activities of glucose-6-phosphate dehydrogenase were extremely high, indicating an active pentose shunt. 2. 2. Other enzyme parameters in these fish were not remarkable. 3. 3. In lower vertebrates the liver may be a major site of fatty acid synthesis and evolutionary implications of these findings are discussed.

X. maculatus is polymorphic for sex-linked macromelanophore patterns. Fish of different river systems exhibit identical markings, but they have a different genetic basis. The macromelanophore factors give rise to a population or species-specific pattern only within their own gene pool. Crosses involving XsdYsr males of the Jamapa stock (Jp) gave rise to 3305 progeny — all but one either Sd or Sr. In the exceptional male Sd (spotted-dorsal) became linked to Sr (stripe-sided), SrSd. The dorsal fin of this fish and all of its SrSd descendants (5 generations) was almost solid black and heavy spotting was present in the caudal fin. There was no change in expression of Sr. Jp fish with Xsd Ysr, by comparison, possess from one to six well defined spots in the dorsal fin and a clear caudal fin. After introduction into the Hondo stock of X. maculatus Sd of Jp exhibits zero penetrance; the expression of Sd linked to Sr becomes greatly reduced. Thus the changes in gene expression go in the same direction. However, Sd and SrSd behave differently after introgression into X. couchianus. Sd is completely suppressed while Sd linked to Sr gives rise to melanotic and melanomatous dorsal and caudal fins. A similar alteration in gene expression towards a more extreme phenotype has also been observed among the offspring of a Jp Xsp Ysr male in which Sp and Sr became apparently linked. It is postulated that a modifier is closely linked to the macromelanophore factor that regulates its expression. The new phenotype is due to the separation of the two genetic elements. According to this view the phenotype that is expressed when a macromelanophore gene is introduced into a foreign genotype, is primarily determined by the modifier.

SUMMARYPlatyfish,Xiphophorus maculatus, are polymorphic for the patterns red-dorsal (Dr) and spotted-dorsal (Sd) fins, both controlled by closely linked loci on theXchromosome of Jamapa strain, Jp 163A. The intensity of red pigment looks the same in males and females, but spectrophotometric analysis of dorsal fin extracts showed that heterozygous intact males have significantly more red pigment (drosopterin) than homozygous or heterozygous females or castrated males. The mechanism of Dr expression in Jamapa is, thus, similar to the one present in the Belize stock, where a sex difference is readily apparent that is known to be under androgenic control. The Sd phenotype is identical in both sexes.SdandDrare not restricted to theXchromosome, and no evidence for gene dosage compensation has been obtained. Within the Jamapa stock the expression of Sd and Dr are best described in terms of dominance and recessiveness. Dr is strongly augmented by a testicular hormone.DrandSdhave been separated by crossing-over. In natural populations both genes can occur by themselves, linked to each other or to other pigment genes. The development of the Sd macromelanophores is not contingent upon the presence of pterinophores (Dr) in the dorsal fin or elsewhere in the body.

The teleosts Xiphophorus maculatus, Xiphophorus hellerii, Xiphophorus milleri , and Xiphophorus variatus xiphidium are native to freshwater streams. Following hypophysectomy their ability to survive in fresh water is abolished and they must be maintained in 1/3 sea water. When hypophysectomized fish are returned to fresh water they succumb within 16 days. The mean survival time of these fish in fresh water is: X. maculatus , 8.7 days; X. hellerii , 8.6 days; X. milleri , 5.5 days; X. v. xiphidium , 4.3 days. Fish in osmotic shock, when returned to dilute sea water, generally show gradual recovery. Injections of saline, neurohypophysial hormones (vasopressin and oxytocin), growth hormone, TSH, and ACTH failed to maintain hypophysectomized X. maculatus in fresh water. Only prolactin (ovine) was effective. Platyfish, injected with 10 μ g of prolactin per gram weight of fish, survived the duration of hormone administration. However, following the withdrawal of hormone, all completely hypophysectomized fish died within 16 days. Prolactin in quantities of less than 1.0 μ g is totally unsuccessful in promoting freshwater survival of fish without their pituitary glands. Compared with uninjected controls, the administration of saline or ineffective hormones considerably shortens the mean survival time of hypophysectomized fish in fresh water. In a few fish, hypophysial tissue in amounts sufficient to support them in fresh water, regenerated in four to six weeks. In some of these individuals the glands regenerated without connection to the hypothalamus.

A sex-linked gene controlling the age at which the gonadotropic zone of the adenohypophysis differentiates and becomes physiologically active has been identified in the teleost Xiphophorus maculatus . Within the Belize stock, the factor P e , for early differentiation, is linked to a pigment gene Ir (red iris) whereas P l , for late differentiation, is linked to Br (red body). Males homozygous for Ir (Y- Ir Y- Ir ) become sexually mature between 10 and 16 weeks and heterozygous males (Y- Ir Y- Br ) between 14 and 25 weeks. No overlap in maturation times is found when the two kinds of males are raised under identical conditions. A cross of a heterozygous Belize male, Y- Ir Y- Br , with an XX female of the Jamapa stock homozygous for early maturation, yielded 13 Ir males that matured between 12 and 15 weeks and 15 Br males that differentiated sexually between 18 and 24 weeks. At 60 days the gonadotropic zone of the pituitary gland of males homozygous for Ir is well developed while that of Ir Br male heterozygotes is composed of only a few chromophobes. No other differences in pituitary structure were detected. At 105 days the gonadotropic zone of Ir Br males is fully formed. Presence or absence of the gonadotropic zone is well correlated with the differentiation of the testis. Androgen from the developing gonad, in turn, controls the metamorphosis of the anal fin into a gonopodium. Because the growth rate of males decreases markedly at the time of sexual maturity, an effect also mediated by androgenic hormone, early-maturing males (Ir) are significantly smaller than late-maturing ones (Br). Up to that time the growth rate of the two kinds of males is the same. Size is thus the ultimate permanent record of the differences between the two kinds of males. The action of the P genes in females has not yet been investigated. Preliminary evidence shows that males homozygous for P l mature between 20 and 32 weeks and grow correspondingly larger. the polymorphism effecting gonadotrop differentiation is a natural component of wild populations and of laboratory stocks derived from them.

1. Since self-fertilization constitutes the ultimate basis of inbreeding, a species or race of self-fertilizing hermaphrodites should consist of clones, all members of which possess identical genotypes and are homozygous. This prediction has been tested on a recently discovered population of Rivulus marmoratus from the east coast of Florida. All wild-caught specimens of this population tested so far have proved to be hermaphrodites. Those kept in isolation in aquaria, and their her maphrodite descendants kept in isolation ab ovo, have reproduced by self-fertilization.2. The transplantation test has been used to determine whether fish that had descended from the same wild-caught progenitor possess identical genotypes. Fins spleens and hearts were transplanted in 36 different host-donor combinations involving six different lines (sib to sib, parent to offspring, offspring to parent). Only two transplants failed to survive, but their loss may have been due to mechanical reasons. These results are in accordan...

A sex-linked gene controlling the age at which the gonadotrops of the pituitary gland differentiate has been discovered in Xiphophorus maculatus. Males homozygous for early differentiation become sexually mature between 10 and 16 weeks; those homozygous for late maturation between 22 and 40 weeks. Heterozygotes are intermediate. Since growth rate decreases when testes mature. the two classes of males differ significantly in adult size.

A sex-linked gene controls the age at which the gonadotropic zone of the pituitary gland develops and becomes physiologically active. The alleles for early (Pe) and late(Pl) differentiation are linked to pigment genes that serve as genetic markers. Pe may be marked by Sp, Dr, Sr, Ir and + and Pl by Br and N. Ir females (PePe) matured between 10 to 15 weeks of age and Br (PePl) females between 16 to 20 weeks. Dr (PePe) females had ripe ovaries six to eight weeks earlier than NDr (PePl) females (8 to 12 weeks versus 16 to 20 weeks). The average age of sexual maturation was 12.5 weeks for IrIr (PePe) males, 20 weeks for IrBr (PePl) and 26.5 weeks for BrBr (PlPl) males. DrSr (PePe) males reached maturity at 13.5 weeks and NSr (PePl) males at 27.5 weeks. The transformation of the anal fin into a gonopodium which is under androgenic control, takes longest in the late maturing fish (IrIr, 3.8 weeks; IrBr, 4.8; BrBr, 6.5; DrSr, 4.2; NSr, 6.4). Similarly, the rate of development of the gonadotropic zone and of the gonad is protracted in the late genotypes. Eventually, the gonadotropic zone of early and late maturing fish are indistinguishable. The Pl factor linked to N delays sexual maturity to a greater extent than the one linked to Br suggesting that not all Pl factors are identical and that important allelic interactions may exist. The growth rate of immature males and females is the same, however it declines sharply in males (but not females) at the time of sexual maturity. This leads to significant differences in adult size between male genotypes. Female genotypes do not exhibit size differences and eventually become larger than all males. The development of the gonadotropic zone precedes, and is essential for, complete gonadal maturation. In the absence of a functional gonadotropic zone, oocytes developed through the oil droplet stage and were surrounded by active follicle cells and a prominent zona pellucida. Spermatogenesis proceeded up to the spermatocyte stage and the cells lining the efferent ducts displayed a modicum of activity. Some of our data suggest that low levels of androgens are present in all males as early as five weeks of age prior to the development of gonadotrops. The genes regulating the development of the pituitary-gonadal axis may be an important factor in explaining the evolution of poeciliid fish.

A sex-linked gene, P, controls the onset of sexual maturity in the platyfish, Xiphophorus maculatus. The activity of this gene is correlated with the age and size at which the gonadotropic zone of the adenohypophysis differentiates and becomes physiologically active. Immature fish of all genotypes grow at the same rate; however, as adults, males with "early" genotypes are significantly smaller than males of "late" genotypes, since growth rate declines strongly under the influence of androgenic hormone. Five alleles, P(1)... P(5), have been identified from natural populations that under controlled conditions cause gonad maturation between eight and 73 weeks. P(1)P(1) males become mature at eight weeks and 21 mm, P(2)P(2) and P(3)P(3) males between eleven and 13.5 weeks and 25 to 29 mm, and P(4)P(4) males at 25 weeks and 37 mm. Since P(5) is X-linked, no males homozygous for P(5) could be produced. The difference between P(2) and P(3) is largely based upon their interaction with P(5). P(3)P(5) males mature at 17.5 weeks and 33.5 mm and P(2)P(5) males at 28 weeks and 38 mm. The rate of transformation of the unmodified anal fin into a gonopodium, which is under androgenic control, is directly related to the age at initiation of sexual maturity, ranging from 3.2 weeks in P(1)P(1) males to seven weeks in P(2)P( 5) males. These differences may reflect different levels of circulating gonadotropic and androgenic hormones.-In two genotypes of females, initiation of vitellogenesis was closely correlated with size and this critical size was independent of age (e.g., 21 mm for P(1)P(1 )). In a third genotype (P(1)P(5)) the minimum size for vitellogenesis decreased with increasing age, so that females would mature as early as eleven weeks, provided they had attained at least 29 mm, but at 25 weeks even females as small as 23 mm possessed ripe gonads. For P(5)P(5) females, which become mature between 34 and 73 weeks of age, there is no correlation between size and initiation of vitellogenesis. In all four genotypes of females examined, egg number is strongly correlated with size, but the regression of egg number on standard length is distinct for each genotype. Late maturation of P(5)P( 5) females is not offset by an increased number of eggs; for this genotype there is a strong negative correlation between age and number of eggs. Heterozygous fish always mature later than those homozygous for the "earlier" allele. The site of action of the P locus could be the pituitary gland, the hypothalamus or higher centers of the brain where peripheral information is transduced into an appropriate signal required for the activation of the hypothalamus-pituitary-gonadal axis. The P gene could also control the peripheral information. The platyfish may be a useful model to test theories concerning the evolution of life history strategies.

Poeciliid fishes are one of the dominant groups in Middle America and the West Indies (Rosen and Bailey, 1963). They are relatively small forms with a highly developed sexual dimorphism. Interspecific differences between males are very pronounced, but females differ relatively little. Some species (e.g., Xiphophorus maculatus) often occur in dense aggregates in small ditches or pools. These fishes are ovoviviparous and females may store sufficient sperm to fertilize the ova of several successive broods at monthly intervals (Dzwillo, 1959; Hildemann and Wagner, 1954; van Oordt, 1928; Zander, 1962). Males of many species exhibit incessant and highly visible courtship activity, but virtually nothing is known about the mating system of these forms under natural conditions. In general it is believed to be promiscuous. Hubbs (1964) reported that in a population of Poecilia latipinna and P. formosa virtually all females were gravid, although females outnumbered males by a ratio of 100: 1. Laboratory observation and some progeny analyses of gravid females from natural populations indicate that males of the guppy, Poecilia reticulata, and platyfish, Xiphophorus maculatus, may mate with more than one female and females mate with more than one male (Gandolfi, 1971; Gordon, 1947; Haskins et al., 1961; Kallman, 1965, 1970; Rosenthal, 1952). Territoriality has been reported for only one poeciliid, Poeciliopsis lucida, and its mating system is basically polygynous (Moore and McKay, 1971). Observations on natural populations of X. variatus, however, indicate that males of this species are not territorial and that the mating system is promiscuous (Borowsky, 1969 and unpubl.). In this paper we present the results of an indirect analysis of reproductive patterns in natural populations of the platyfish, Xiphophorus maculatus. This species is highly polymorphic for color patterns the genetic basis of which is known in some detail (Kallman, 1970; Kallman and Atz, 1966). Gravid females were collected and brought to the laboratory for progeny analyses. From the color patterns of offspring and P1 females, we determined the number and genotypes of inseminating males, the existence of sperm succession, fitness differences among genotypes, and whether mating is at random with respect to color patterns. We believe this information to be of interest both to evolutionists and to students of poeciliid fish.

Fishes of the genus Xiphophorus (Poeciliidae, Atheriniformes) are found on the Atlantic slope of Mexico and adjacent parts of Central America.

The sex-determining mechanism of swordtails (Xiphophorus, Poeciliidae, Pisces) is poorly understood, because many laboratory strains of these species are characterized by biased sex ratios in either the male or female direction. Because of these ratios sex determination is said to be controlled by numerous male and female factors scattered over many chromosomes. However, direct experimental evidence for the polygenic theory is absent. Three strains of X. alvarezi were derived from natural populations in Guatemala, two of which (Dolores, Candelaria) always exhibit a ratio of 1:1, whereas the third one (San Ramon) shows a preponderance of males. Males of X. alvarezi, like those of other species of swordtails, develop a caudal appendage or sword at maturity. Chromatophores with carotenoid pigment (orange) are present in the sword of Dolores males and pigment cells containing sepiapterin (green) are present in the swords of males and the other two stocks. F1 and backcross hybrids were produced between the Dolores and Candelaria stocks. The sex ratios of all hybrid pedigrees was always in statistical agreement with unity. The pattern of inheritance of the two pigmentary traits demonstrated sex-linkage with female heterogamety (WY female female-YY male male).

The recent increased interest in sex-determining mechanisms can be traced directly to Wachtel’s concept that the H-Y antigen is the primary testis organizer. There have been several comprehensive reviews dealing with genetic mechanisms of sex determination and sex differentiation, but all of these deal largely with the mammalian literature and pay only lip service to the other classes of vertebrates (J.W. Gordon and Ruddle, 1981; Haseltine and Ohno, 1981; McCarrey and Abbot, 1979; Ohno, 1979; Wachtel and Ohno, 1979). Of particular interest to geneticists are several cases of atypical sex determination in mammals, including humans, in which an autosomal gene causes the testicular differentiation of XX individuals. This has raised the question as to the location of the male-determining gene.

THE Amazon molly, Poecilia formosa (Girard), is an all-female species native to southern Texas and north-eastern Mexico1. In the northern part of the range, P. formosa is fertilized by males of P. latipinna; in the southern part by a species of the P. sphenops complex. Reproduction is usually by gynogenesis; no introgression of paternal characters into P. formosa stocks has been observed in any of the several laboratories in which they have been maintained during the past 36 yr1–7. With no genetic material being transferred from the male parent to the offspring, all P. formosa which have descended from a single progenitor should possess the same genotype and constitute a clone. The existence of several clones in natural populations has been demonstrated by tissue transplantation4,5. In rare instances some offspring of P. formosa females exhibit paternal characters and thus are hybrids6,8,9. In the Genetics Laboratory of the New York Aquarium where several clones of P. formosa have been maintained since 1960 by either mating them to males of P. vittata or P. sphenops (black molly stock), eighteen hybrids (about 1 per cent) have been obtained. Of these, three were sired by P. vittata and fifteen by P. sphenops.

Amounts of DNA per nucleus were estimated by microphotometry in Feulgen-stained tissue sections from several species of poeciliid fish and their hybrid offspring. In terms of average nuclear volumes as well as relative DNA levels, the all-female gynogenetic species P. formosa was indistinguishable from the related bisexual species P. sphenops, confirming the essentially diploid nature of the amazon molly, P. formosa. Although DNA values from the less closely related species P. vittata were about 15% higher than those from P. sphenops, both species showed expected patterns of DNA behavior in several somatic tissues and in developing male sex cells. Somatic nuclei from two types of interspecific hybrids contained more DNA than corresponding types of nuclei from typical maternal P. formosa, the additional amount being equivalent to the haploid DNA value characteristic of the participating paternal species. The results strongly suggest that these hybrids are genetic triploids, probably containing diploid maternal and haploid paternal chromosome contributions. The high incidence of chromosomal stickness, bridging, and fragmentation observed during metaphase I of spermatogenesis, and the occurrence of both “giant” and “micro” spermatids in testis of the hybrid P. formosa X P. vittata may well account for the apparent sterility of amazon molly offspring which show transmission of paternal characteristics. Production of triploid interspecific hybrids in poeciliid fish and corollary implications for interpreting previous tissue transplantation studies are discussed.

Differences in adult male size and age at sexual maturity in the Río Coy (Mexico) population of Xiphophorus nigrensis (Pisces; Poeciliidae) are controlled by genetic variation at a Y-linked locus. Four genetic size-classes have been identified. The mating behavior of the males of the three largest size-classes consists exclusively of an elaborate courtship display, whereas that of the genetically small males ranges from display to a sneak-chase attempt at copulation. In the presence of large males, small males switch to the sneak-chase behavior. Females prefer the display of large males. In mating-competition experiments (two females with one large male and one small male), the large males are dominant and deny the small males access to females. From 20 such experiments, 601 large-male and 200 small-male progeny were obtained, indicating that the switch to sneak-chase behavior by small males is not particularly effective in overcoming the large-male advantage. By using the largest males of the genetically smallest size class and the smallest males of the genetically next-larger size-class, size was kept constant, whereas genotype was varied. When these males were tested in competition with genetically large males, only the males of the genetically smallest size class showed sneak-chase behavior. These observations suggest that the difference in mating behavior is not an indirect developmental effect of size but, rather, is under direct genetic control.

In order to elucidate the relationship between age, weight, genotype and sexual maturation, we raised male platyfish, Xiphophorus maculatus, of two "maturation" genotypes under a wide variety of environmental conditions. In spite of enormous variation in the ages and weights at sexual maturation, the two maturation genotypes produced phenotypes that were almost completely distinguishable on the basis of their ages and weights at maturation. The demographic correlates of maturation within a genotype were an inseparable combination of age and weight.

reversal in unisex grass carp fed methyltestosterone, p. 194-199. In: Culture of exotic fishes symposium proceedings. R. O. Smitherman, W. L. Shelton and J. H. Grover (eds.). Fish Cult. Sec., Amer. Fish. Soc., Auburn, Alabama. 9, W. W. MILEY, II, AND D. L. SUTTON. 1978. Reproductive r'equirements and likelihood for naturalization of escaped grass carp in the United States. Trans. Amer. Fish. Soc. 107:118-128. STROMSTEN, F. A. 1931. The development of the gonads in the goldfish Carassius auratus (L.). Univ. Iowa Stud. Nat. Hist. 13:3-45.

The olivaceous background coloration of the platyfish, Xiphophorus maculatus, results from the presence of micromelanophores interspersed among small xanthophores in the epidermis of the scales and from the arrangement of the micromelanophores in a reticular pattern in the dermis of the flank. Two autosomal loci, St and R, with two alleles each have been identified which control the presence or absence of these pigment cells. St - R - fish have the wild-type coloration. The St - r r genotype, "gray fish," lack the xanthophores, but the micromelanophore pattern is indistinguishable from that of St fish. Fish of genotype st st R are yellow all over. They are virtually free of micromelanophores in the scales and dermis, but the number of xanthophores is increased manyfold in the scales. The dermis and the perineural lining of the brain are densely and uniformly populated by xanthophores. By contrast, xanthophores are absent from these two tissues in the St fish. The double recessive fish, st st r r, have a whitish appearance, "ghost," and lack micromelanophores and the background xanthophores throughout the integument. In st st R - and st st r r fish, melanophores are also absent from the perivascular lining of the blood vessels, the gill arches and the wall of the swim bladder. Neither the St nor the R locus have any detectable effect on the retinal pigment layer or on the melanophore pigmentation of the connective tissue sheath of the spinal cord. Dopa positive cells could be demonstrated in the scales and in the dermis of st st R - and in the scales of st st r r fish. The dermis of the double recessive was Dopa negative. The St locus has at best a minor effect on the expression of the macromelanophore patterns which are controlled by sex-linked genes. Similarly, the R locus has no effect on the expression of the sex-linked xanthophore and xantho-erythrophore patterns. The existence of several main lineages of pigment cells can be discerned in X. maculatus and the action of St and R loci are restricted to only one of these lines. The St locus of X. maculatus may be homologous to the B locus of Poecilia reticulata and the R locus of X. maculatus may be homologous to the R locus of P. reticulata and the Gr locus of Poeciliopsis viriosa. No gene homologous to the G locus of P. reticulata, which also effects the background melanophores and xanthophores, has been identified in X. maculatus.

The color and sex chromosome polymorphisms of the poeciliid fishes present classical problems in ecological genetics. (See Gordon, 1947, 1951; Gordon and Gordon, 1957; Haskins et al., 1961, 1970; Kallman, 1973, 1975.) One such polymorphism is the presence of three sex chromosomes in the platyfish, Xiphophorus maculatus. In all but two natural populations of this species, there are three types of females: WY, WX, and XX, and two types of males: XY and YY (Kallman, 1973). This species is also polymorphic at several sex-linked and autosomal pigment loci. The sex chromosomes have not been identified cytologically but the many visible markers segregating with the segments determining gender and the constancy of the sex ratios in broods produced by the various possible matings attest to their existence. Although recombination in low frequency (1%) is observed between the W and Y (Gordon, 1937) and the X and Y (MacIntyre, 1961) chromosomes in the laboratory, the W chromosomes of females from natural populations invariably possess the dull colored wildtype alleles at the sex-linked pigment loci (Kallman, 1970, 1973), while the X chromosomes possess both wildtype and colored -alleles at such loci. Kallman argues that predators can more easily. see brightly colored individuals and that there is natural selection against the XX females carrying colored alleles. In addition, wildtype X chromosomes will be selected against in males, as brightly colored poeciliid males have higher mating success than dull colored males (Fisher, 1930; Sheppard, 1953; Haskins et al., 1961; and Endler, 1978). Such selection for coloration never occurs against wildtype W chromosomes as they do not appear in males. Subsequently, Borowsky and Kallman (1976) found that dull colored, wildtype WY females had more offspring than such females with sex-linked color patterns which suggests that there may be an intrinsic sex limited disadvantage to the colored alleles. On the basis of these observations it is reasonable to conclude that the WY and WX females have higher fitnesses than both colored and wildtype XX females. Kallman (1970) suggested that the sex chromosomal polymorphism is transitory due to the higher fitnesses of WY and WX females and thus that the X chromosome would disappear from natural populations. However, this hypothesis ignores the effects of different male fitnesses, which were not investigated. By analysis of a mathematical model we have derived the necessary and sufficient conditions for the maintenance of the sex chromosomal polymorphism. Our model assumes non-overlapping generations and random mating, with the possibility that some males mate more often than others being reflected in higher fitness values. Let Pwy, Pwx, and Pxx be respectively the frequencies after selection of the WY, WX, and XX females (as fractions of the total population of males and females). Similarly let the frequencies of the XY and YY males be qxy and qyy so that Pwy + Pwx + Pxx + qxy + qyy = 1. Each genotype also has an associated fitness value

The males of the Rio Coy population of Xiphophorus nigrensis, San Luis Potosi, Mexico, exhibit a vertical bar pattern on the flank. During aggressive encounters there is an intensification of the bars as the pigment becomes dispersed throughout the melanophores whereas in submissive individuals the pigment becomes concentrated in the center of the cells and the pattern fades. The Rio Choy population of X. nigrensis, X. pygmaeus and an undescribed species from the Rio Ojo Frio, all in San Luis Potosi, lack this pattern. Within the Rio Coy population there are four size genotypes of males controlled by variation at a Y-linked locus. The pattern is strongly expressed in all males of the three larger size genotypes, but virtually absent from the small size class. In the area of size overlap between the largest males of the genetically small size class and the smallest males of the genetically next larger size class, the expression of the pattern is correlated with genotype not size. The inheritance of the pattern in F, and backcross hybrids with the Choy population and with X. pygmaeus indicates that the trait has a polygenic basis and that the Y chromosome of the Coy stock with the factor for small size has a suppressor gene for bars. The suppressor is absent from all other X and Y chromosomes. Similar patterns are present in 14 of the 19 species of Xiphophorus, and species with and without this trait are found in each of the three major subdivisions of the genus. It is suggested that the pattern has evolved independently in different species.

Populations of platyfish, Xiphophorus maculatus, inhabiting the Rio Jamapa, Mexico, and the Belize River, British Honduras, are polymorphic for three sex-linked patterns: red anal fin, Ar; red dorsal fin, Dr; spotted dorsal fin, Sd. Apart from minor differences than can be recognized when a large series of fish is examined, pigment patterns of the two populations look alike. Within their own gene pool dominance is virtually complete for the pigment factors of the two populations. Only Ar and Dr of Belize are sometimes not expressed in female Belize fish. When the pigment factors of Jamapa are introduced through backcrossing into Belize, expression becomes suppressed. Preliminary experiments with the Sd factor of Jamapa indicate that this is not due to a loss of dominance but to low penetrance. The expression of Belize pigment factors in the F1 of Jamapa X Belize is exaggerated and further enhanced in the backcross generations to Jamapa. Patterns in the two populations must result from the interaction of a primary pigment gene with a system of modifiers. Since the patterns of the F1 and backcross generations differ from that of either parental stock and also depend upon the geographical origin of the pigment factors, not only the modifiers of each population must be different, but the primary pigment factors as well. Each factor together with its modifiers constitutes a coadapted genetic system. Since identical patterns in the two populations have a different genetic basis, the patterns must have evolved independently and must possess a high selective value.