Multiple sex chromosome system in penguins (Pygoscelis, Spheniscidae)

Abstract Penguins are classified in the order Sphenisciformes into a single family, Spheniscidae. The genus Pygoscelis Wagler, 1832, is composed of three species, Pygoscelis antarcticus Forster, 1781, P. papua Forster, 1781 and P. adeliae Hombron & Jacquinot, 1841. In this work, the objective was to describe and to compare the karyotypes of Pygoscelis penguins contributing genetic information to Sphenisciformes. The metaphases were obtained by lymphocyte culture, and the diploid number and the C-banding pattern were determined. P. antarcticus has 2n = 92, P. papua 2n = 94 and P. adeliae exhibited 2n = 96 in males and 2n = 95 in females. The difference of diploid number in P. adeliae was identified as a multiple sex chromosome system where males have Z1Z1Z2Z2 and females Z1Z2W. The C-banding showed the presence of a heterochromatic block in the long arm of W chromosome and Z2 was almost entirely heterochromatic. The probable origin of a multiple system in P. adeliae was a translocation involving the W chromosome and the chromosome ancestral to Z2. The comparison made possible the identification of a high karyotype homology in Sphenisciformes which can be seen in the conservation of macrochromosomes and in the Z chromosome. The karyotypic divergences in Pygoscelis are restricted to the number of microchromosomes and W, which proved to be highly variable in size and morphology. The data presented in this work corroborate molecular phylogenetic proposals, supporting the monophyletic origin of penguins and intraspecific relations.


Introduction
In the class Aves, penguins are classified in the order Sphenisciformes in a single family Spheniscidae. The 18 extant species are divided in six genera, Aptenodytes Miller, 1778 (2 species), Eudyptes Vieillot, 1816 (6 species), Pygoscelis Wagler, 1832 (3 species), Spheniscus Brisson, 1760 (4 species), Megadyptes Milne-Edwards, 1880 (1 species) and Eudyptula Bonaparte, 1856 (2 species) (Stonehouse 1975). The monophyletic origin of penguins has been evidenced in different studies using morphological characters, DNA hybridization and more recently by molecular data with whole-genome analysis (Sibley and Ahlquist 1990, Giannini and Bertelli 2004, Baker et al. 2006, Jarvis et al. 2014. Using mitochondrial and nuclear sequences, Baker and co-workers (2005) reconstructed the phylogeny of penguins and solved divergences observed in an intra genera relationship. The molecular dating estimated that ancestral penguins originated about 71 million years (Mya) ago in Gondwanaland while current species shows an Antarctic origin about 40 Mya. Aptenodytes (king and emperor penguins) was the first genus to diverge as basal lineage about 40 Mya. Pygoscelis branched about 38 Mya diversifying to P. adeliae Hombron & Jacquinot, 1841 in 19 Mya, P. antarcticus Forster, 1781 and P. papua Forster, 1781 at 14.1 Mya. The common ancestry of other genera was estimated at 27.8 Mya, followed by the division between genus Spheniscus and Eudyptula at approximately 25 Mya, followed by penguins Megadyptes and Eudyptes, about 15 Mya.
Related to sex chromosomes in Aves, it is known the chromosome system of type ZZ in males and ZW in females. The Z chromosome is relatively conserved among different orders, varying in size between the third and fourth pair of macrochromosomes (Takagi and Sasaki 1974), whereas the W chromosome presents significant differences, being of size similar to Z in Ratites to a small and heterochromatic chromosome in Passeriformes (Takagi andSasaki 1974, Correia et al. 2009).
In this work, the goal was to describe the karyotype of P. antarcticus, P. papua and P. adeliae, contributing to the karyotypic knowledge about the order Sphenisciformes and to compare it with species already described.

Blood cultures
Blood samples were used for lymphocyte culture, according to Moorhead et al. (1960). Briefly, cultures were prepared using approximately 1ml of peripheral blood in 10ml of RPMI 1640 culture medium, supplemented with 20% fetal bovine serum, 0.25ml penicillin/streptomycin and 0.2ml phytohemaglutinin. Cultures were incubated at 39°C for 72 hours. One hour before the incubation end, cells were treated with 0.1ml of colchicine solution (0.05%). After incubation, samples were treated with 10ml of 0.075M KCL for 20 minutes and fixed in a methanol and acetic acid (3:1) solution.

Chromosomal analysis
For chromosomal analysis, the metaphases were stained with Giemsa solution. For each specimen, 40 metaphases were observed and photographed to assemble the karyotypes. Morphological classification of each chromosome pair was made according to Guerra (1986). CBG banding was conducted in sequential analysis, it was used slides preparations with a conventional staining and procedures of C-banding according to Sumner (1972).

Results
Pygoscelis antarcticus has 2n = 92 chromosomes (Fig. 1a). The karyotype is formed by seven pairs of macrochromosomes and the remaining were 38 pairs of microchromosomes. Sex chromosome Z was identified as submetacentric corresponding to the size of the 4 th pair and W is acrocentric corresponding to size of the 8 th pair.
Pygoscelis papua showed 2n = 94 (Fig. 1b). The karyotype exhibited the same morphological characteristic such as in P. antarcticus, with seven autosomal pairs of macrochromosomes and the divergence was observed in the number of microchromosomes, 39 pairs. The Z chromosome was submetacentric and W was metacentric.
For the P. adeliae it was observed that there is a difference in the diploid number between males and females. The males had 2n = 96, while the females showed 2n = 95 (Fig. 1c). The autosomal complement was formed by seven pairs of macrochromo- somes highly similar at P. antarcticus and P. papua, the remaining were microchromosomes. The sex chromosomes in males were identified as one pair of submetacentric chromosomes (Z 1 Z 1 ) and one small pair of telocentric denominated as Z 2 Z 2 . In females P. adeliae the sex chromosome system Z 1 Z 2 W was identified, and the W chromosome was telocentric.
The classification of chromosome morphology in Pygoscelis confirmed the conservation of seven macrochromosomes and the Z chromosome between species. The pairs 1, 2, 4, 6, 7 and Z are submetacentric, pair 3 is acrocentric and pair 5 is metacentric. The W chromosome is acrocentric in P. antarcticus, metacentric in P. papua and telocentric in P. adeliae (Table 1).
The sequential Giemsa and C-banding analysis in P. antarcticus (Figs 2a-a') showed centromeric C-positive heterochromatin blocks in macro and microchromosomes; the Z chromosome was C negative. The W chromosome was slightly reactive to the banding. For the female P. papua (Figs 2b-b'), centromeric markings on macro and microchromosomes were observed. In the species P. adeliae, both sexes, female (Figs 2c-c') and male (Figs 2d-d') were analyzed. The macro and microchromosomes and the Z1 chromosome were little reactive and not showed marking in the centromere. The W chromosome has a characteristic heterochromatin block in the terminal region and the Z2 chromosome is completely heterochromatic.
The diploid number of Pygoscelis is slightly elevated in relation to the values observed in more than 60% of the known Avian karyotypes, which correspond to 2n = 74-86 according to Rodionov (1997). Nevertheless, the conservation of macrochromosomes morphology suggests that karyotypes differences among these species are due to variations which occurred in microchromosomes. Considering the proposed phylogeny by Baker et al. (2006), in which P. adeliae (2n = 95-96) was the first species of the genus to diverge 19 Mya, it is suggested that a diploid number reduction occurred in P. papua (2n = 94) and P. antarcticus (2n = 92). This reduction can be attributed to chromosomal fusions involving microchromosomes.
The numerical and morphological conservation of the macrochromosomes in Pygoscelis is shared with Spheniscus and Aptenodytes species (Table 1). Ledesma et al. (2003) was the first to identify that, despite slight variations in the diploid number, Sphenisciformes showed high chromosomal homology. This homology extends not only to autosomal macrochromosomes, but also to the Z chromosome which is observed as submetacentric in seven of the eight species with known karyotypes. The Table 1. Comparison of the diploid number and morphology of the macrochromosomes and sex chromosomes of Sphenisciformes species.  small variations that can be observed in the morphology of the macrochromosomes correspond to a thin line dividing a chromosome to be classified as metacentric or submetacentric, such as in par 6 and 7 of the king and emperor penguins according to Cardozo et al. (2003). These homologies can be certainly confirmed by chromosome painting with fluorescent in situ hybridization experiments.

Species
Furthermore, on the basis of these observations, in this work we propose an ancestral karyotype for the present penguin species (Figure 3), based on the frequency of macrochromosomes and the sex chromosomes Z and W (Table 1). According to Baker (2006) penguins from the genus Spheniscus diverged from the Eudyptula penguins, thus it is quite probable that karyotypes highly similar are observed in Eudyptula minor Forster, 1781 and E. albosignata Finsch, 1874, whose karyotypes are unknown.
The W chromosome seems to play an important role in chromosomal evolution of Sphenisciformes. It presented morphological variations (Table 1) especially in the species from the genus Pygoscelis ranging from to acrocentric in P. antarcticus, to metacentric in P. papua and telocentric in P. adeliae. The analysis of C-banding made possible the differentiation of P. antarcticus and P. papua (Figure 2 a' and b') karyotypes in relation to P. adeliae (Figure 2 c'). However, it was not possible to differentiate the karyotypes of the first two species. In fact, the male karyotypes can only be recognized by their different diploid number, while in females the distinction is facilitated by the presence of the W chromosome that presented variations in morphology.

Multiple sex chromosome system in Adelie penguin
The most interesting result of this work was seen in a multiple sex chromosome system in P. adeliae, where males have Z 1 Z 1 Z 2 Z 2 and female has Z 1 Z 2 W. In birds this observation is unpublished, in higher animals multiple systems are rare exceptions and can be found in fishes, reptiles (some snakes), in some mammals Monotremes (platypus and echidna), in marsupials, in some Neotropical Primates, in eight species of bats, in two species of antelope (White 1977, Yonenaga et al. 1979, Tucker and Bickham 1986, Rens et al. 2007, Steinberg et al. 2014. According to White (1973), multiple sex chromosome systems have arisen as a result of rearrangements involving sex chromosomes and autosomes, by centric fusion, reciprocal translocation, centric fission or tandem fusions. Cioffi et al. (2011) emphasizes the importance of repetitive sequences in multiple forms of sex chromosomes from fish, generating differences in morphology and size among them.
In the case of P. adeliae our hypothesis is that the multiple systems originated from a translocation involving the ancestral heterochromatic Z2 chromosome, with the terminal portion of the long arm of the W chromosome ( Figure 4a). This hypothesis explains the presence of differentiated heterochromatic segment in the W chromosome (Fig. 2c'). In addition, the absence of this segment in the W chromosomes of P. papua and P. antarcticus suggests that this translocation occurred after their separation from P. adeliae or it was lost. A fairly similar heterochromatic labeling pattern was described by Toledo and Foresti (2001) on the X chromosome from the fish Eigenmannia virescens Valenciennes, 1842 and according to the authors these represent an early evolutionary stage of sex chromosome differentiation.
It is important to consider that the pairs Z2Z2 and Z1Z1 chromosomes, in males segregate normally during meiosis forming balanced gametes (Fig. 4b). In females, the formation of the trivalent Z1-W-Z2 must occur. The Z1 and Z2 segregate independently of the W chromosome. The W chromosome carries the translocated portion homologous to the Z2 chromosome (Fig. 4b). These hypotheses can be proven or refuted with the performance of meiotic analysis in females of P. adeliae.
In this work for the first time the karyotypes of P. adeliae, P. papua and P. antarcticus were presented. The presence of conserved macrochromosomes suggests that differences in diploid number 2n = 95-96, 94 and 92 are due to fusions between microchromosomes, reducing the diploid number. The results also point to a significant role of the W chromosome in speciation, with the first record of a multiple sex chromosome system in birds such as observed in P. adeliae. In addition, the comparison showed a high karyotype homology in Sphenisciformes which can be seen in the morphological conservation of macrochromosomes and in the chromosome Z.