Laboratório de Citogenética, Instituto de Ciências Biológicas, Universidade Federal do Pará, b

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1 Original Article DOI: / Comparative chromosome painting between chicken and spectacled owl (Pulsatrix perspicillata): implications for chromosomal evolution in the Strigidae (Aves, Strigiformes) E.H.C. de Oliveira a S.P. de Moura b L.J.S. dos Anjos b C.Y. Nagamachi a, c J.C. Pieczarka a, c P.C.M. O Brien d M.A. Ferguson-Smith d a Laboratório de Citogenética, Instituto de Ciências Biológicas, Universidade Federal do Pará, b PIBIC-UFPA, Belém, PA, and c Bolsista de Produtividade Científica, CNPq (Brazil); d Cambridge Resource Centre for Comparative Genomics, Cambridge University Department of Veterinary Medicine, Cambridge (UK) Accepted in revised form for publication by M. Schmid, 17 June Abstract. The spectacled owl ( Pulsatrix perspicillata ), a species found in the Neotropical region, has 76 chromosomes, with a high number of biarmed chromosomes. In order to define homologies between Gallus gallus and Pulsatrix perspicillata (Strigiformes, Strigidae), we used chromosome painting with chicken DNA probes of chromosomes 1 10 and Z and telomeric sequences. This approach allowed a comparison between Pulsatrix perspicillata and other species of Strigidae already analyzed by chromosome painting ( Strix nebulosa and Bubo bubo, both with 2n = 80). The results show that centric fusions and fissions have occurred in different chromosomal pairs and are responsible for the karyotypic variation observed in this group. No interstitial telomeric sequences were found. Although the largest pair of chromosomes in P. perspicillata and Bubo bubo are submetacentric, they are homologous to different chicken chromosomes: GGA1/GGA2 in P. perspicillata and GGA2/ in B. bubo. Copyright 2008 S. Karger AG, Basel The gross organization of the genome in birds is a characteristic feature of this group, consisting of diploid numbers close to 80, in combination with a large number of microchromosomes (Griffin et al., 2007). The relative lack of variation in the karyotypes of birds is remarkable, with about 63% of birds showing diploid numbers of chromosomes (Christidis, 1990) and conserved synteny at least for macrochromosomes (Shetty et al., 1999; Raudsepp et al., 2002; Guttenbach et al., 2003; Kasai et al., 2003; Shibusawa This work was financially and logistically supported by CNPq (Edital Universal, / and Post-Doctoral Scholarship, /2006-6), Universidade Federal do Pará and the Wellcome Trust. Request reprints from Prof. Dr. Edivaldo Herculano C. de Oliveira Laboratório de Citogenética, Instituto de Ciências Biológicas Universidade Federal do Pará, Rua Augusto Correa 01 Belém, PA (Brazil), CEP telephone: ; fax: ehco@ufpa.br et al., 2004a, b). However, there are some groups of birds which show substantial chromosomal variation, not only in diploid number, but sometimes also in the organization of chromosomes. Although the most remarkable examples with atypical karyotypes comprise diurnal birds of prey (Accipitridae and Falconidae) and stone curlews or thick knees (Burrhinidae) with diploid numbers as low as 40, some degree of variation is also found among owls (Schmutz and Moker, 1991; Rebholz et al., 1993; Schmid et al., 2000; Guttenbach et al., 2003). Owls belong to the order Strigiformes, which consists of 134 species in 25 genera (Burton, 1973). According to the most recent analyses, including DNA DNA hybridization methods (Sibley and Ahlquist, 1990), allozyme divergence (Randi et al., 1991) and cytochrome b gene sequencing (Wink and Heidrich, 1999), barn owls and typical owls are segregated into different families, Tytonidae and Strigidae, respectively. Chromosomal analysis corroborates this division: While Strigidae species show karyotypes with diploid Fax karger@karger.ch S. Karger AG, Basel /08/ $24.50/0 Accessible online at:

2 Fig. 1. Karyotype of the spectacled owl ( Pulsatrix perspicillata ), with 2 n = ZW numbers close to 80, similar to the standard avian complement with a few pair of macrochromosomes and many small chromosomes, Tytonidae show karyotypes made up of chromosomes, with no clear distinction between macro- and microchromosomes and only one pair of biarmed elements (Renzoni and Vegni-Talluri, 1966; Belterman and De Boer, 1984; Bhunya and Mohanty, 1987; Rebholz et al., 1993). Most species of Strigidae share the first five chromosome pairs, which led to the assumption that these pairs were found in the putative ancestral karyotype (Renzoni and Vegni-Talluri, 1966; Belterman and De Boer, 1984). In this ancestral karyotype, chromosomes 1 3 and 5 are acrocentric, chromosome 4 is metacentric and 6 9 telocentric. Only three species of Strigidae ( Bubo bubo, Strix nebulosa and Athene noctua ) have been studied by FISH with telomeric or whole chicken chromosome probes (Schmid et al., 2000; Nanda et al., 2002; Guttenbach et al., 2003). The spectacled owl ( Pulsatrix perspicillata ), a species found in the Neotropical region, has 76 chromosomes (Sasaki et al., 1984; Yamada et al., 2004) with a high number of biarmed chromosomes. Like Bubo bubo, Pulsatrix perspicillata has a karyotype which differs from other Strigidae in having a submetacentric chromosome as the largest in the karyotype, while other species of this family show an acrocentric/subtelocentric pair as chromosome 1. Based on chromosomal morphology, it was suggested that the P. perspicillata karyotype could be derived from the chromosome complement found in Athene brama, Athene noctua and Surnia ulula (Schmutz and Moker, 1991). This hypothesis was rejected by Rebholz et al. (1993), who argued that pairs 1 and 2 of Pulsatrix and Athene are not identical, as postulated previously. In order to identify chromosome rearrangements that occurred in the differentiation of the karyotype of P. perspicillata (PPE), we used chromosome painting with Gallus gallus (GGA) probes. The results allowed us to construct a comparative map between this species and the other two owls already analyzed by FISH, from North America and Eurasia, and infer the chromosome evolution in this family. Materials and methods Chromosome preparations and banding techniques Metaphase chromosomes were obtained from two individuals, one male and one female, of Pulsatrix perspicillata, using tissue culture from feather pulp (Sasaki et al., 1968, with modifications). Metaphase chromosomes were obtained by standard arrest with colcemid (Gibco), hypotonic treatment with M KCl, cell fixation in methanol:acetic acid (3: 1) and air-dried chromosome preparation on glass slides. For diploid chromosome definition and karyotype ordering, chromosome preparations were stained with 5% Giemsa (Sigma) solution in 0.07 M phosphate buffer (ph 6.8) and 50 metaphase spreads were analyzed for each specimen. Further, the preparations were trypsin G-banded according to Seabright (1971). Images of 30 banded metaphase spreads were captured, of which ten were karyotyped. Fluorescent in situ hybridization using Gallus gallus chromosome specific probes and telomeric sequences Chromosome specific paints of Gallus gallus, comprising chromosomes 1 10 and Z, were generated from flow-sorted chromosomes amplified by DOP-PCR. Probes were labelled with biotin (Roche) or fluorescein (Roche) and used in single or dual colour experiments. For detection of telomeric sequences, biotinylated (TTAGGG) n probe was applied. Briefly, slides were pepsinized for 3 min, dehydrated and incubated for 2 h at 65 C. Standard techniques were used for denaturation, hybridization, stringency washes and detection using avidin- CY3 and Alexa 488. Chromosomes were counterstained with DAPI. 158

3 Fig. 2. Representative FISH experiments with Gallus gallus (GGA) painting probes hybridized onto Pulsatrix perspicillata metaphases. In each metaphase the respective probe color assignment is given and the PPE chromosomes to which each probe hybridizes is indicated. Analysis and image capturing Slides were analyzed under a 100! objective. Both Giemsa stained chromosomes and hybridization images were captured with a cooled CCD camera coupled to a Zeiss Axiophot microscope. Camera control, digital image acquisition and pseudocolour assignment were performed using Smartcapture VP 1.4 software (DigitalScientific, Cambridge, UK). Image processing and karyotype ordering were performed using Adobe PhotoShop 7.0. R e s u l t s The diploid number found in Pulsatrix perspicillata was 76, confirming previous reports. Pair 1 is submetacentric and much larger than pair 2, which is subtelocentric. Pairs 4, 7, 9, 11 and 14 are metacentric, as well as pairs 17 26, 29, 30, 34 and 36. Pairs 3, 5, 6, 8, 10, 12, 13, 27, 33 and 35 are submetacentric, while pairs 15, 16, 28, 31, 32 and 37 are acrocentric. Both Z and W are submetacentric ( Fig. 1 ). Chicken paints 1 10 and Z produced 13 distinct signals in Pulsatrix perspicillata. Several chromosome rearrangements were revealed. Paints 1, 4 and 5 hybridized to two chromosome pairs each, while paints 2, 3 and 6 10 produced one signal each. Some paints hybridized to the same chromosome pair: GGA1/GGA2, /GGA5, GGA6/ GGA7, /GGA9 and GGA5/GGA8, hybridized to PPE1, PPE3, PPE5, PPE6 and PPE7, respectively, revealing the occurrence of centric fusions. The largest chromosome pair of P. perspicillata was painted by chicken paints 1 and 2 ( Fig. 2 ). The homologies between PPE and GGA are shown in Fig. 3. Signals produced by telomeric probes were restricted to telomeres of chromosomes. No interstitial signals were observed ( Fig. 4 ). 159

4 GGA1 GGA2 GGA3 GGA5 GGA1 GGA6 GGA GGA9 GGA5 GGA8 GGA10 GGAZ GGAZ Z W Fig. 3. Partial G-banded karyotype of Pulsatrix perspicillata together with the assignment of Gallus gallus chromosome-specific painting probes. Horizontal bars mark the borders of homologous regions. Fig. 4. In situ hybridization of a telomere-specific (TTAGGG)n probe (red) to a Pulsatrix perspicillata metaphase (grey) produced hybridization signals on chromosome ends, but not in interstitial chromosome regions. 160

5 GGA2 Fig. 5. Proposed comparative homology maps between the Strigidae Ancestral Karyotype (SAK) and Gallus gallus (GGA), based on the results of in situ hybridization using Gallus gallus chromosome paints on three different species of owls: Bubo bubo, Strix nebulosa and Pulsatrix perspicillata. 1 GGA3 GGA1 GGAZ GGA6 GGA1 GGA7 GGA5 GGA8 GGA Z Discussion Although most karyotypes of Strigiformes still exhibit the main characteristics of the unique dichotomous organization of avian complements, comprising a few macrochromosomes and many pairs of microchromosomes, cytogenetic data have been of great importance in this order, supporting the division of the species into two different groups, with distinct chromosomal characteristics. Considering solely the chromosome morphology of different species of owl, it was suggested that the Strigidae ancestral karyotype (SAK) comprised ten pairs of macrochromosomes, with pairs 2, 3, 4 and Z biarmed and 1 and 5 10 acrocentric. This karyotype resembled those observed in 11 species from five different genera, although the morphology of some pairs is different: Asio flammeus, Bubo lacteus, B. poensis, B. virginianus, Ketupa blakistoni, K. ketupa, K. zeylonensis, Nyctea scandiaca, Otus bakkamoena, O. leucogatis and O. scop (Rebholz et al, 1993). Moreover, Strix nebulosa and Strix aluco differ from this ancestral karyotype by an inversion in pair 5 (Schmutz and Moker, 1991). On the other hand, Bubo bubo and Pulsatrix perspicillata have karyotypes which differ from this standard Strigidae formula. In both species chromosome 1 is submetacentric. However, the arm ratio of this pair is different in each, suggesting the occurrence of different rearrangements. Our results show that GGA chromosome probes 1 10 produce the same number of signals in both species (13 distinct signals), but some are involved in different rearrangements. Chromosome painting performed with Gallus probes onto Bubo bubo confirmed the hypothesis based on conventional staining, that chromosome 1 originated from a fusion between chromosomes 1 and 5 of the Strigidae ancestral karyotypes (GGA2 and, respectively). Apart from this difference, other BBU chromosomes resemble the karyotype of Bubo virginianus (Guttenbach et al., 2003). The combination of the results of in situ hybridization of Strix nebulosa, Bubo bubo and the additional information from our analysis suggest homology between the Strigidae ancestral karyotype (SAK) and Gallus gallus, as shown in Fig. 5. Robertsonian fusions seem to have predominated during avian karyotype evolution (Nanda et al., 2002). The karyotype of Pulsatrix perspicillata is derived from the ancestral karyotype by a series of centric fusions, none of which involve the same segments observed in BBU1. Hence, PPE1 originated from the fusion of SAK1 and 3 (whereas BBU1 corresponds to SAK1 and 5). In addition, four other pairs of chromosomes of P. perspicillata were painted with two distinct probes of Gallus : PPE3 (/GGA5), PPE5 (GGA6/ GGA7), PPE6 (GGA9/) and PPE7 (GGA5/GGA8), none of which are found together in BBU. Interestingly, no interstitial signals were observed in any of the chromosomes of Pulsatrix perspicillata that originated by centric fusion. This fact may indicate a complete loss of telomeric sequences at the fused ends of telocentric elements, which implies the occurrence of breaks within or close to telomere-associated heterochromatin. Finally, considering the karyotype stability in most species of birds, the analysis of only a few species of owls has already shown interesting patterns of genomic reorganization. Robertsonian fusions and fissions seem to be the most common rearrangements involved in this variability. So far, the analysis of chromosomal organization has revealed some important information concerning the phylogenetic relationships among the different species of owl, being concordant with phylogenies proposed by other approaches. However, it would be necessary to analyze the karyotypes of other species of owls with GGA probes, in order to clarify the relationship with Pulsatrix, as well as to confirm whether the biarmed elements found in different species correspond to homologous segments or involve different chromosomes of the group s ancestral karyotype. Acknowledgements The authors are grateful to Parque Zoobotânico do Museu Paraense Emilio Goeldi (Belém, PA, Brazil) for the supply of biological material for this study. References Belterman RHR, De Boer LEM: A karyological study of 55 species of birds, including karyotypes of 39 species new to cytology. Genetica 65: (1984). Bhunya SP, Mohanty MK: Distribution of constitutive heterochromatin in the collared scops owl. J Hered 78: (1987). Burton JA: Owls of the World (EP Dutton, New York 1973). Christidis L: Aves, in John B (ed): Animal Cytogenetics, Vol 4, Chordata 3 (Gebrüder Bornträger, Berlin 1990). 161

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