Supernumerary chromosomes in the pufferfish Sphoeroides spengleri-First occurrence in marine Teleostean Tetraodontiformes fish

Cytogenetic analyses carried out in eight specimens of Sphoeroides spengleri revealed the presence of 2n = 46 chromosomes (20 M/SM and 26 ST/A). Besides the standard karyotypical set, the presence of B microchromosomes was observed in two individuals, ranging from 0 to 2 microchromosomes per cell. A karyotype composed by 2n = 46 chromosomes with occurrence of M and SM chromosomes is considered basal for the species from the clade comprising the families Tetraodontidae, Balistidae, and Diodontidae, although it represents a derived condition for the order Tetraodontiformes, whose basal karyotype would be composed by 2n = 48 acrocentric chromosomes. The occurrence of B microchromosomes in marine Tetraodontiformes fish was not known, and this represents the first report of such a chromosomal type.

The order Tetraodontiformes comprises nearly 340 species, divided into five families (Nelson, 1994).Although typically marine, a few species are found in freshwaters, such as Colomesus asellus in the Amazon Basin, and are regarded as freshwater invaders (Figueiredo and Menezes, 2000).Phylogenetically, the Tetraodontiformes are considered one of the most derived clades within Teleostei, representing a sister group of Perciformes (Lauder and Liem, 1983).The genus Sphoeroides (Tetraodontidae) is regarded as basal for the family (Arai, 1983;Brum et al., 1995;Brum, 2000), evidencing its importance for evolutionary studies.Sphoeroides spengleri, one of the six species of the genus, presents a small body size of about 15 cm, and it is widespread from Massachusetts (USA) to the southeastern Brazilian shore (Figueiredo and Menezes, 2000).
The occurrence of supernumerary chromosomes in Neotropical fishes seems to be a common event in freshwater species, previously reported in 41 of the 921 analyzed species (Oliveira et al., 2000), characterized by the presence of B microchromosomes (Alves and Martins-Santos, 2002) or B macrochromosomes (Porto-Foresti et al., 1997).In about 81 Neotropical marine species analyzed so far, this chromosomal type was not observed in fishes from the family Tetraodontidae (Oliveira et al., 2000;Galetti et al., 2006).Therefore, aiming to increase the karyotype information in fishes from the family Tetraodontidae, specimens of S. spengleri from the northern coast of the State of São Paulo, Brazil, were cytogenetically analyzed.
The cytogenetic analyses were performed in eight specimens (3 females, 4 males, and 1 sex undetermined individual) of S. spengleri collected at Ubatuba Bay, São Paulo, Brazil.The specimens were identified and deposited in the fish collection of the Laboratório de Biologia e Genética de Peixes, LBP-UNESP, Botucatu, SP, Brazil.Chromosome preparations were obtained from gill and kidney tissues using the technique described by Foresti et al. (1993).Chromosome morphology was determined on the basis of arm ratio as proposed by Levan et al. (1964), and the chromosomes were classified as metacentric (M), submetacentric (SM), subtelocentric (ST), and acrocentric (A).
Although the diploid number is conserved in Sphoeroides species, the karyotypical macrostructure presents differences related to the number of metacentric and acrocentric chromosomes, useful for species identification.Such karyotypical differences are a result of successive chromosomal rearrangements, mainly pericentric inversions.The diploid number of 2n = 46 is regarded as basal for the polyphyletic clade composed by Tetraodontidae, Balistidae, and Diodontidae.The species of this group present highly derived karyotypes for Tetraodontiformes, ranging from 2n = 28 in Canthigaster coronata (Arai, 1983) to 2n = 52 in Chilomycterus spinosus (Brum, 2000), characterizing it as the most karyotypically diverse group of marine teleosteans (Galetti et al., 2000;Galetti et al., 2006).According to Brum et al. (1995), these derived karyotypes might have arisen from Triacanthidae species, considered the basal group of Tetraodontiformes, which present primitive karyotypes composed of 2n = 48 acrocentric chromosomes (Lauder and Liem, 1983).
The occurrence of supernumerary microchromosomes in Sphoeroides spengleri represents the first report of such a chromosomal type inmarine Tetraodontiformes fish.The absence of supernumerary chromosomes in this fish group could be important in the future to understand the probable origin of this type of chromosome in fishes.However, the group of marine fishes needs more cytogenetical studies to find new cases of supernumerary chromosomes.The occurrence of supernumerary chromosomes in S. spengleri reinforces the hypothesis that marine teleosteans do not represent a group that is highly karyotypically conserved, as previously thought.In addition, chromosomal polymorphisms and sex chromosomes have been identified in several marine fish species (Galetti et al., 2006).
The origin of such supernumerary chromosomes is controversial.A hypothesis for the origin of the B macrochromosome in Astyanax scabripinnis is a putative adaptative advantage determined by these chromosomes in individuals from high altitude (Néo et al., 2000) and inhabiting headwaters (Porto-Foresti et al., 1997).However, there is no adaptative hypothesis associated with B microchromosomes, and their low frequency might suggest a re- cent origin in freshwater fishes (Alves and Martins-Santos, 2002).Furthermore, the differences related to intraindividual frequency, morphology and size of supernumerary chromosomes among freshwater teleosteans suggest different origins of these chromosomes.Similar to freshwater teleosteans, the origin of supernumerary chromosomes amongst marine fishes is still unknown, but the origins of B microchromosomes in freshwater and marine fishes do not seem to be phylogenetically related, representing independent evolutionary pathways.However, further analyses in other species are required to the understanding of the origin of supernumerary chromosomes within this fish group.

Figure 1 -
Figure 1 -Karyotype of Sphoeroides spengleri (Tetraodontidae) in (a).In (b) one B microchromosome is shown in detail, and in (c) two B microchromosomes are shown in detail.