Inheritance of a Dominant Spotted Melanic Mutation in the Livebearing Fish Phalloceros Caudimaculatus Var

© 2007 The Japan Mendel Society Cytologia 72(2): 131–139, 2007 Inheritance of a Dominant Spotted Melanic Mutation in the Livebearing Fish Phalloceros caudimaculatus var. reticulata from “Bañados del Este” Reserve of Biosphere Site in Uruguay María Laura Gutiérrez* and Graciela García Sección Genética Evolutiva, Facultad de Ciencias, Udelar, Iguá 4225, CP 11400 Montevideo, Uruguay Received November 13, 2006; accepted November 28, 2006 Summary An alternative chromatic variation to the wild-type phenotype of the livebearing fish Phalloceros caudimaculatus (Phalloceros caudimaculatus var. reticulata) was detected in a single female from “Bañados del Este” Reserve of Biosphere Site, in Uruguay. The founder mutant female, which presented a melanic spotted pattern overlapping the wild-type pigmentation, produced laboratory breed strains. Four phenotypes showing different degrees of spotted patterns were obtained. In order to determine the inheritance of this chromatic mutation, experimental crosses and cytogenetic analyses of these laboratory strains were carried out. The breeding experiments among the spotted phenotypic classes and backcrosses suggest the dominant and non-sex linked inheritance of this mutation. Statistical tests demonstrated that the inheritance mechanism of this mutation does not corre- spond to a 2-gene independent segregation hypothesis as it was proposed in Poecilia sphenops, or to a single dominant gene hypothesis. Additionally, cytogenetic analyses detected a partially hete- rochromatic biarmed chromosome associated to the inheritance of the pigmentation pattern in laboratory breed strains. Key words Inheritance, Livebearing, Phalloceros caudimaculatus, Spotted mutation. The livebearing fish Phalloceros caudimaculatus Hensel 1868, popularly known as “madrecitas de agua” in Uruguay and “Guaru” in Brazil, belongs to order Cyprinodontiformes, to family Poe- ciliidae and to tribe Cnesterondontinii. This extensive Neotropical family of viviparous fishes in- cludes approximately 200 species (Rosen and Bailey 1963). The monotypic genus Phalloceros ex- hibits a vast distribution. It could be found in different areas of Brazil, Argentina, Paraguay and Uruguay (Fig. 1a). Its members are non-migratory fishes that present sexual dimorphism in size, where females (65 mm long) are longer than males (35 mm long) (Lucinda 2003). The uniform light-grey wild-type pigmentation, the most frequent phenotypic class, is due to a differential distribution of micromelanophores over the body and head, with a major concentration on dorsal surfaces. The presence of a rear and elliptical bar vertically elongated reaching the dorsal and ventral side near the caudal peduncle, is observed. Sometimes a second spot near the caudal peduncle is present and it represents the main chromatic and exomorphological character for species recognition. In addition to this, the wild-type phenotypic class shows translucent fins sharing melanophores along fin-ray edges (Fig. 1b). An alternative chromatic variation to the wild-type pigmentation was described in Phalloceros caudimaculatus var. reticulata Hensel 1868. As in other poeciliids (Schartl et al. 1995), their pigmentation is due to cell-type specific macromelanophores, easily distinguishable from micromelanophores because of their enormous size. Macromelanophore pigmentation is encoded by domi- * Corresponding author, e-mail: [email protected] 132 María Laura Gutiérrez and Graciela García Cytologia 72(2) Fig. 1. (a) Distribution of Phalloceros caudimaculatus Fig. 2. Ontogenetic melanism evolution of phenotypic natural populations (Rosen and Bailey 1963). (b) classes in P. caudimaculatus var. reticulata labo- Wild-type pigmentation in males (above) and fe- ratory breed strains. Juvenile melanic individuals males (below) (www.fishbase.net). (c) Phallo- reach directly T3 phenotype (a) or show a T2 ceros caudimaculatus var. reticulata female from phenotype intermediate step (b). Adult fish show “Bañados del Este” in Uruguay. T3 or T4 phenotypes. Barϭ1 cm. nant genes. In P. caudimaculatus this dominant gene could arise from low frequency spontaneous mutation events because it has been found in a single spotted female in natural populations from “Bañados del Este” Reserve of Biosphere Site, in Uruguay (Fig. 1c). The morphologic analysis of this single female revealed that there are no other characters that distinguish it from the wild-type (Azpelicueta and García 2001). This founder mutant female presented a melanic spotted pattern overlapping the wild-type pigmentation and it was used in breeding experiments with wild-type males belonging to the same natural population. The resulting offspring showed a 1 : 1 backcross ratio. Through laboratory crosses among F1 spotted males and females, new phenotypic classes corresponding to highly spotted fish were generated. Four phenotypes showing different degrees of spotted pattern were obtained, namely: class I (T1ϭwild-type pigmented) formed by uniform light grey fish which shows no black pattern at all; class II (T2) corresponding to slightly spotted fish (not present in adult fish); class III (T3) including heavily spotted fish whose spots are isolated and do not form large patches of black pigmentation, and class IV (T4) integrated by highly spotted fish whose spots blend into condensed black patches (Fig. 2). The same color strains were genetically characterized in another related livebearing fish, Poe- cilia sphenops (Schröeder 1976). In this species, 3 color strains were used in experimental crosses to analyze the inheritance of melanism. In this case, 2 dominant pairs of genes, which are additive in their effect and located in different chromosomes, were described. These genes can act to pro- duce 5 phenotypic classes with an increasing degree of pigmentation. Other genetic mechanisms to explain the melanic pattern were proposed in Poecilia formosa. Cytogenetic analyses carried out on this species showed that melanic pigmentation genes are incorporated in gynogenetic female genomes and transported by microchromosomes from a bisexual host species (Schartl et al. 1995). In the present study, we first conducted mating breeding experiments using the different phenotypic classes to test the 2-gene independent inheritance hypothesis. Secondly, we performed cytogenetic analyses to find any karyotypic structural evidence that could be associated to the melanic mutation. Both approaches were taken to assess the genetic inheritance mechanisms of the spontaneous melanic mutation in P. caudimaculatus. Materials and methods Experimental crosses Three strains of P. caudimaculatus (T1, T3 and T4) from the founder mutant female and wild- type males were used in laboratory crosses. All fish remained in a single-sex freshwater aquarium at 2007 Inheritance of a Dominant Spotted Melanic Mutation 133 room temperature (17–23°C on 10 : 14 light : dark cycle) until the experiment was conducted. Fish were fed ad libitum daily with commercial pellets. Each breed experiment was placed in a 220 l aquarium under the same conditions described above. Mating crosses including 3 phenotypic classes (T1ϫT1, T3ϫT3 and T4ϫT4) and 2 backcrosses (/ T1ϫ?T3 and / T3ϫ?T1) were performed using P. caudimaculatus T1, T3 and T4 laboratory strains. The adult fishes of the resulting offspring were classified and recorded according to sex and pigmentation degree. Each cross experiment was replicated twice. Statistical analyses In order to evaluate the statistical significance of the breeding results we performed a Chi- square test and a G test (Sokal and Rolf 1979) implemented for small size samples. A statistical significance of aϭ0.05 was assumed to test the null hypothesis. Cytogenetic analyses Among 67 individuals included in cytogenetic analyses, 21 were selected due to the best quali- ty of the chromosome spreads. Parents and offspring from 3 strains (T1, T3 and T4) and non-in- breeding individuals of P. caudimaculatus were analyzed. Tissue and voucher specimens were de- posited in the Sección Genética Evolutiva, Facultad de Ciencias, Universidad de la República, Mon- tevideo, Uruguay. Mitotic chromosomes were obtained by using conventional techniques (Kligerman and Bloom 1977). At least 20 metaphases per individual were examined. Chromosomes were classified according to Levan, Fredga and Sandberg (1964) using the modifications introduced by Denton (1973) for fish cytogenetics. The short arms of biarmed (Mϭmetacentric, SMϭsubmetacentric) and evident small arms of subtelo/acrocentric (ST/A) chromosomes were considered for the calculation of FN. Metaphase plates were C-banded according to Sumner (1972) and Ag-stained by the Howell and Black (1980) method. A Fisher exact test was performed to evaluate statistical associations between P. caudimaculatus phenotypes and karyotype structure. Results Experimental crosses All breeding experiments resulted in a successful and viable offspring. At birth, all fish presented wild-type phenotype. However, crosses within phenotypic classes T3ϫT3 and T4ϫT4, as well as backcrosses have provided offspring that reached a melanic form after 1 month. After this period, young spotted fish were classified as T2 and T3 individuals. However, when they were adults, all T2 and some T3 were re-located in the T3 or T4 phenotypic classes. None of the T1 young fish evolved to a spotted form (Fig. 2). Due to the absence of T2 adult phenotypic class, the original hypothesis of 2 dominant pair of genes was reformulated in to a simpler one, considering a single-gene hypothesis

Load more