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

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 correspond 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 heterochromatic 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. Bar1 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 (T1wild-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 (T1T1, T3T3 and T4T4) and 2 backcrosses (/ T1?T3 and / T3?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 sig- niﬁcance of a0.05 was assumed to test the null hypothesis.

Cytogenetic analyses Among 67 individuals included in cytogenetic analyses, 21 were selected due to the best quality 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 (Mmetacentric, SMsubmetacentric) 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 T3T3 and T4T4, 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 of melanism inheritance. Experimental corsses and statistical tests data are presented in Table 1. According to this interpratation, wild-type (T1) individuals shared the homozigous receive genotype (mm), the genotype class T3 correspond to heterozygous fish (Mm) and the phenotype class T4 to the homozigous dominant ones (MM). The offspring analyses allowed us to corroborate the dominant condition of this melanic mutation in P. caudimaculatus (Table 1). As expected based on the above-mentioned hypothesis, only wild-type progeny was obtained from T1T1 mating crosses. The T3T3 crosses progeny corre- sponded to the expected T1, T3 and T4 phenotypic classes. Only melanic progeny resulted from T4T4 crosses. 134 María Laura Gutiérrez and Graciela García Cytologia 72(2) 0.2 0.2 0.05 0.001 p p p p p p labora- G 50 3.974 0.1 50 801.7 ? ? 50/ 50/ Phalloceros caudimaculatus Phalloceros / / T1) using ? 50 50 Expected values (%) Expected values ? ? T1 T3 T4 T1 Melanic G T3 50/ 50/ / / / T3 and Mm Mm ? MM 0 0 100 — — ? ? mm 100 0 0 — — MM 0 50 50 2.481 0.1 Mm 25 50 25 8.642 T1 entative entative T T genotype genotype / mm mm Mn / / n n T4) and backcrosses ( T3 and T4 T1, T3 -values are shown. Inferred genotypes and expected values in each cross under a 1-gene hypothesis of melanism in- in each cross under a 1-gene hypothesis Inferred values genotypes and expected are shown. -values Melanic p Observed values Males Females 4000029mm 52 Males Females Males Females Males Females Offspring from different phenotypic crosses (T1 from different Offspring tory strains (T1, T3 and T4). G test results heritance are displayed T1 13 16 14 7 50 T3 19 35 5 9 68 T4 T3 14 13 16 20 5 0 68 Mn T4 0 0 10 19 20 4 53 MM T1 ? ? Table 1. T3 T3 T4 T1 Crosses T1 T3 T4 T1 T3 / / Backcrosses T1 2007 Inheritance of a Dominant Spotted Melanic Mutation 135

Fig. 4. Conventional karyograms of somatic cells in the Fig. 3. Conventional karyograms of somatic cells in the T3 phenotypic class of Phalloceros caudimacula- T1 phenotypic class of Phalloceros caudimaculatus. (a) Male. (b) Female, both parents in the cor- tus. (a) Male. (b) Female, both parents in the corresponding crosses. 2n48, FN49. Biarmed responding crosses. 2n48, FN48. chromosomes of pair 2 are shown in the box. Bars10 mm. Bars10 mm.

Statistical tests showed discordance between the expected and the observed offspring of the T3T3 breeding experiments results, because we obtained more T1 than T4 individuals (Table 1). Remarkably, although in the T4 breeding experiments we would have expected a 100% T4 progeny, we obtained T3 and T4 phenotypic progeny. This unexpected result could be attributed to an error in parental individuals choice, perhaps including T3T4 crosses instead of T4T4 crosses. To avoid this experimental mistake, the statistical analysis was carried out considering an alternative T3T4 cross (Table 1). Under this interpretation, p-values from G test do not show statistical sig- niﬁcance. At the same time, through reciprocal backcross experiments we obtained melanic and wild- type male and female progeny in all cases, allowing us to discard a sex linked inheritance of this mutation. However, the statistical test (Table 1) demonstrated that there are major differences between expected and observed results from the / T1?T3 backcross experiments, which showed more T1 phenotypic progeny than spotted offspring. Since similar results were obtained by using G test and Chi-square test, here we present the former, because it shows greater robustness in experimental designs for small samples. The low non-signiﬁcant p-values (Table 1) could be attributed to the small size of the sample obtained in experimental crosses.

Cytogenetic analyses Chromosome analyses were performed in the well-spread metaphases. In all cases, a diploid number of 48 chromosomes was found in P. caudimaculatus laboratory strains. Differences between males and females were not detected indicating absence of sex chromosome heteromorphism. 136 María Laura Gutiérrez and Graciela García Cytologia 72(2)

The karyotype of T1 phenotypic class presented an FN48 and is constituted by small subtelo/acrocentric chromosomes (Fig. 3). However, the karyotype of T3 and T4 phenotypic classes presented an FN of 49 and 50, re- spectively. Individuals of T3 class presented 47 small subtelo/acrocentric chromosomes and a partially heteropycnotic biarmed chromosome in pair 2 (Fig. 4), while T4 individuals presented 46 small subtelo/acrocentric chromosomes and 2 biarmed heteropycnotic chromosomes in pair 2, one of them wholly heteropycnotic (Fig. 5). Fisher exact test (p0.0001) supports a strong association between the presence of 1 or 2 of these biarmed chromosomes and the spotted pigmentation (Table 2). The pattern was re- peatable in diverse chromosome spreads from the same ﬁsh (74%); the quality of the remain- ing metaphases prevented accurate counting of biarmed chromosomes. Remarkably, this cytogenetic evidence is concordant with the afore- mentioned 1-gene hypothesis of the melanism Fig. 5. Conventional karyograms of somatic cells in the inheritance in P. caudimaculatus. T4 phenotypic class of Phalloceros caudimaculatus. (a) Male. (b) Female. 2n48, FN50. Two The C-banding technique reveals the pres- biarmed chromosomes of pair 2 are shown in the ence of pericentromeric and telomeric hete- box. (c) C-banding of biarmed chromosome pair. rochromatic blocks in all subtelo/acrocentric Bars 10 mm. chromosomes (data not shown). Biarmed chromosomes have heterochromatic short arms and a large heterochromatic block on sub-telomeric region (Fig. 5c). Preliminary Ag-stained banding experiments suggest that biarmed heterochromatic blocks were Ag-positive (data not shown). Nev- erthless, more experiments are necessary to corroborate this fact.

Discussion The present work is the ﬁrst genetic characterization of the melanic chromatic mutation in laboratory strains of P. caudimaculatus. All experimental crosses provided the evidence of the domi-

Table 2. Cytogenetic data in P. caudimaculatus. Fisher exact test (p0.0001) supporting a strong association between the presence of biarmed chromosomes and the spotted pigmentation. M/SMMeta- centric/Submetacentric

N° metaphases N° metaphases Phenotype N° individuals Karyotype* examined with M/SM

T1 8 156 0 Melanic 13 252 186 Total 21 408

* The minus sign () refers to biarmed chromosome absence, while the plus sign () corresponds to biarmed chromosome presence. 2007 Inheritance of a Dominant Spotted Melanic Mutation 137 nant and non-sex linked inheritance of this mutation. Statistical tests (Table 1) demonstrated that the inheritance mechanism of this mutation does not correspond to a 2-gene independent segregation as it has been proposed in Poecilia sphenops (Schröeder 1976) or to a single dominant gene hypothesis. Therefore, considering the discordance between expected and observed values which emerge from crosses within strains and backcross experiments, the existence of other underlying mechanisms acting in the inheritance of this chromatic mutation could be postulated. In addition to this, cytogenetic analyses (Figs. 3–5) shed light on this issue, because heteropycnotic biarmed chromosomes were detected in the spotted T3 and T4 phenotypic classes, while they were absent in wild-type T1 individuals. Fisher exact test corroborated the association of these pe- culiar biarmed chromosomes with the inheritance of the pigmentation pattern in the laboratory breed strains of P. caudimaculatus (Table 2). In the present work, we have found a 2n48 for these laboratory breed strains of P. caudimaculatus. The same diploid number was described by Wasko et al. (1990) in natural populations of Brazil. These authors found a chromosome formulae constituted by 46 telo/acrocentric chromosomes and 2 subtelocentric ones. Foresti (1974) proposed a 2n48 too, whereas other authors have reported a diploid number of 46 in P. caudimaculatus (Arkhipchuk 1999, Vasil’ev 1980, Wickbom 1943). Contrary to our findings, these studies have not shown the presence of biarmed chromosomes. The origin of biarmed chromosomes from acrocentric ones could be related to some types of chromosome rearrangements such as pericentric inversions and/or heterochromatin addition. These chromosome rearrangements were previously reported in other Cyprinodontiformes fishes (García et al. 1995), in reef fishes from the genus Centropyge (Affonso and Galetti Jr. 2005), in the genus Hypostomus (Artoni and Bertollo 1999) and in “bitterlings” of the family Cyprinidae (Ueda et al. 2001). The partial or whole heteropycnotic biarmed chromosomes found in our analyses were also heterochromatic by means of C-banding. These additional segments could be amplified or accumu- lated by unequal chromosome exchanges, transpositions and/or regional duplications as it was proposed in the fish Leporinus desmotes by Margarido and Galetti (2000). Thus, pericentric inversions and heterochromatin addition could be a plausible explanation for the origin of biarmed elements in P. caudimaculatus. Other interesting issues remain open, since faculative heterochromatic mechanisms, like the dosage effect, could be acting when 2 biarmed chromosomes are present in the T4 phenotypic class (Fig. 5). Remarkably, Sola et al. (1990) reported the possible occurrence of transposition and/or amplification events associated with Ag-NOR polymorphisms in many species of Poecilia.

Further experiments including Ag-NOR and CMA3 staining techniques will be performed to elucidate a possible association of heterochromatic segments detected in P. caudimaculatus to the presence of size NOR-polymorphisms. At present, B chromosomes in the laboratory breed strains were not detected, as it was found in melanic morphs of Poecilia formosa (Schartl et al. 1995). Statistical results from experimental crosses and cytogenetic analyses, let us hypothesize on the possible existence of other kinds of selfish genetic elements, like Transposable Elements (TE), extensibly reported in fish genomes (Volff et al. 1999, 2000) and producing genomic instability also in P. caudimaculatus. Interestingly, differential expression of melanism described in the genus Xiphophorus was associated to the presence of the TX-1 transposon, which is also widespread among other Poeciliids (Schartl et al. 1999). Hence, it is possible that transposable elements could be acting in the P. caudimaculatus genome, producing a bias in the phenotypic ratio from experimental crosses results, as well as the reversion of this mutation in breed laboratory strains. Further analyses could clarify the association between chromosome rearrangements, hete- 138 María Laura Gutiérrez and Graciela García Cytologia 72(2) rochromatin addition, possible Ag-NORs amplification or transposition supporting the supposed TE activity in the Phalloceros caudimaculatus genome.

Acknowledgements We would like to thank G. Speranza, D. Carnevia and E. Cabruja for technical assistance and W. Norbis for the statistical analysis. We thank V. Gutiérrez and P. Gaiero for manuscript sugges- tions. This work was supported by grants to M. L. G. supplied by Comisión Sectorial de Investi- gación Cientíﬁca (CSIC), Uruguay. The authors are grateful to the Japanese Government for equip- ment donation.

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