The Congo African grey parrot (Psittacus erithacus, PER) is an endemic species of Central Africa, valued for its intelligence and listed as vulnerable due to poaching and habitat destruction. Improved knowledge about the P. erithacus genome is needed to address key biological questions and conservation of this species. The P. erithacus genome was studied using conventional and molecular cytogenetic approaches including Zoo-FISH. P. erithacus has a ‘typical' parrot karyotype with 2n = 62-64 and 8 pairs of macrochromosomes. A distinct feature was a sharp macro-microchromosome boundary. Telomeric sequences were present at all chromosome ends and interstitially in PER2q, the latter coinciding with a C-band. NORs mapped to 4 pairs of microchromosomes which is in contrast to a single NOR in ancestral type avian karyotypes. Zoo-FISH with chicken macrochromosomes GGA1-9 and Z revealed patterns of conserved synteny similar to many other avian groups, though neighboring synteny combinations of GGA6/7, 8/9, and 1/4 were distinctive only to parrots. Overall, P. erithacus shared more Zoo-FISH patterns with neotropical macaws than Australian species such as cockatiel and budgerigar. The observations suggest that Psittaciformes karyotypes have undergone more extensive evolutionary rearrangements compared to the majority of other avian genomes.

The Congo African grey parrot (Psittacus erithacus,Linnaeus, 1758, PER) is an endemic species of the forests of Central Africa. It is closely related to the Timneh African grey parrot (P. timneh), and until recently, the 2 were considered as African grey parrot subspecies [Clements et al., 2015]. However, due to distinct morphology, size, and range, in 2012 the Congo and Timneh grey parrots were recognized as separate species [Taylor, 2012].

The 2 species of African grey parrots belong to the family Psittacidae (New World and African parrots) which is distributed in Africa and South America. Psittacidae form together with Psittaculidae (Old World parrots; Africa and Australia), Cacatuidae (cockatoos; Australia), and Strigopidae (New Zealand parrots) the order Psittaciformes [Christidis and Boles, 2008; Clements et al., 2015]. Relationships between the ∼350 Psittaciformes (parrots, parakeets, and cockatoos), especially the African taxa, and their evolutionary connections to other avian orders are, as yet, poorly understood [Cracraft, 2001; de Kloet and de Kloet, 2005; Schweizer et al., 2010; Seabury et al., 2013]. Due to this, inconsistencies in parrot taxonomy exist between publications, though many, including citations used in this report, follow a rather conservative avian classification, known as ‘Clements Checklist' [Clements et al., 2015].

The family Psittacidae has been estimated to contain the highest number of threatened or endangered bird species [see Seabury et al. 2013], and the Congo African grey parrot is not an exception. As a forest species, its populations are threatened due to increased forest loss. Also, African grey parrots are considered among the most intelligent birds [Huber and Gajdon, 2006], making them subject for extensive pet trade which further reduces their numbers in the wild [Amuno et al., 2007]. Currently, the Congo African grey parrot is listed as vulnerable, with global population estimated at 0.56-12.7 million individuals and listed in CITES 2 [IUCN, 2014]. However, their numbers are rapidly declining, making the study of the species a high priority for future conservation efforts.

The over 300 divergent parrot species are characterized by substantial geographic, phenotypic, cognitive, and behavioral variation [Seabury et al., 2013]. As a result, the study of individual species is of interest for many fields of biology, including phylogenetics, population genetics, natural history, behavioral studies, nutrition, and conservation biology [Seabury et al., 2013], but also disease biology, virology, and immunology [Katoh et al., 2010]. Despite their significance, only limited information is available for Psittacid genomes and chromosomes, thereby limiting the application of genomic approaches for addressing key biological questions [Seabury et al., 2013].

To date, whole genome sequence assemblies are available for only 2 true parrots - the scarlet macaw (Ara macao) [Seabury et al., 2013] and the Puerto Rican parrot (Amazona vittata) [Oleksyk et al., 2012], both representing the New World group. The majority of cytogenetic studies in parrots, including the African grey parrot, were performed in the 1970s and 1980s [De Boer and Belterman, 1980; Van Dongen and De Boer, 1984] and might thus need revisiting with advanced imaging tools for karyotyping. Improved karyotype information, on the other hand, will permit investigation of Psittacid genomes using comparative chromosome painting, also known as Zoo-FISH [Chowdhary and Raudsepp, 2001]. This is one of the most efficient approaches for the rapid determination of molecular relationships between chromosomes of different species. In the case of birds, the reference genome/chromosomes are typically from the chicken (Gallus gallus domesticus; GGA) - a species with the most complete genome sequence and map information [see Romanov et al., 2014]. Zoo-FISH allows indirect transfer of the map and genome sequence data of chicken to the species of interest and provides information about comparative genome organization and karyotype evolution [Chowdhary and Raudsepp, 2001]. To date, Zoo-FISH karyotypes are available for 6 parrot species: budgerigar, cockatiel, peach-faced lovebird [Nanda et al., 2007], scarlet macaw [Seabury et al., 2013], red-and-green macaw, and hyacinth macaw [de Oliveira Furo et al., 2015]. Of these, only the 3 macaw species are members of the family Psittacidae.

In this study we carried out detailed cytogenetic and Zoo-FISH analysis of the Congo African grey parrot using whole chromosome paints for chicken macrochromosomes. The aim was to improve our knowledge of its karyotype and comparative chromosome organization as compared to the chicken, related parrots, and other birds.

Animal and Samples

Regenerating feathers were obtained from a 7-year old male P. erithacus(Band number HCA8268) housed in the Schubot Exotic Bird Health Center, Texas A&M University, USA. Feather collection was carried out according to an approved animal use protocol (AUP 2011-136).

Cell Cultures and Chromosome Preparations

Culturing of the parrot cells was performed as described in detail elsewhere [Raudsepp et al., 2002; Raudsepp and Chowdhary, 2008]. Briefly, fibroblast cultures were initiated under sterile conditions from feather pulp which was minced in EBSS (Gibco, Life Technologies) and digested with 0.5% Collagenase B (Roche). These fibroblasts were incubated in MEM-alpha containing nucleosides and GlutaMax (Life Technologies), supplemented with 10% fetal bovine serum and antibiotic-antimycotic (Life Technologies) at 40°C with 5% CO2. Metaphase chromosomes were obtained by arresting cells with demecolcine (Sigma Aldrich; final concentration: 0.1 µg/ml) followed by hypotonic treatment with Optimal Hypotonic Solution (Rainbow Scientific) and fixation in methanol:acetic acid (3:1). Metaphase spreads were prepared on pre-cleaned wet glass slides at room temperature.

Karyotyping and Conventional Cytogenetic Analysis

For chromosome counting and initial karyotyping, the slides were stained with 5% Giemsa (Sigma) in 0.07 M phosphate buffer (pH 6.8). Macrochromosomes were identified and arranged into pairs according to G-banding [Seabright, 1971] which included treatment with 0.05% trypsin (Life Technologies) in Mg- and Ca- free Hank's balanced salt solution, HBSS (Life Technologies), and staining with 4% Giemsa in Gurr buffer (Life Technologies). Constitutive heterochromatin was visualized by C-banding [Arrighi and Hsu, 1971] which included hydrolysis in 0.1 N HCl and 2.5% Ba(OH)2, treatment with 2× SSC at 60°C, and staining with 5% Giemsa in phosphate buffer (pH 6.8). The results were analyzed under an Axioplan2 microscope (Zeiss), and images were captured and processed with Ikaros (MetaSystems GmbH) software. At least 50 Giemsa-stained and 20 G- and C-banded cells were captured and analyzed for karyotyping and chromosome counting.

Mapping Telomeric and rDNA Sequences by FISH

Telomeric sequences were detected with a Cy3 labeled commercial (TTAGGG)n PNA probe (DAKO) following the manufacturer's protocol. Ribosomal RNA genes (rDNA), also known as the NOR, were mapped using a digoxigenin-labeled probe containing human 18S and 28S rDNA [Maden et al., 1987] cloned in pHr13 plasmid vector. Probe labeling and FISH were carried out according to our standard protocol [Raudsepp and Chowdhary, 2008].

Zoo-FISH with Chicken Macrochromosomes

Probes for chicken (G. gallus) macrochromosomes GGA1-9 and Z were generated by flow sorting at Cambridge Resource Centre for Comparative Genomics, UK [Raudsepp et al., 2002]. The probe DNA was amplified and labeled with biotin-16-dUTP (Roche Diagnostics) or digoxigenin-11-dUTP (Roche Diagnostics) by DOP-PCR as described in detail elsewhere [Raudsepp and Chowdhary, 1999]. Preparation of the hybridization mix, in situ hybridization, post-hybridization washes, and signal detection were carried out according to standard Zoo-FISH protocols [Raudsepp et al., 2002]. All probes were also tested for origin and quality on chicken metaphase spreads.

Microscopy and Analysis of FISH and Zoo-FISH Results

The chromosomes were counterstained with 4′-6-diamidino-2-phenylindole (DAPI), and a minimum of 10 metaphase spreads were analyzed for each experiment. Metaphases were examined and images captured and analyzed with a Zeiss Axioplan2 fluorescence microscope equipped with Isis image analysis software v5.2 (MetaSystems GmbH).

Karyotype of the Congo African Grey Parrot

The diploid chromosome number of the Congo African grey parrot was 2n = 62-64 (fig. 1), as inferred from examination of 50 Giemsa-stained metaphase spreads of a single individual. Criteria for a metaphase spread to be considered suitable for counting were the absence of excessive chromosome overlaps or over-spreading and the presence of 16 macrochromosomes (fig. 1a). Diploid numbers in the 50 cells analyzed were as follows: 2n = 62 (24%), 2n = 64 (22%), and 2n < 62 (54%). The proposed diploid number was confirmed by additional counts obtained from G- and C-banded and DAPI-stained cells. While the number of chromosomes varied between cells due to difficulties in counting overlapping or poorly stained microchromosomes, there were no cells with a diploid number >64.

Fig. 1

The karyotype (2n = 62-64) of African grey parrot (P. erithacus). a Giemsa-stained metaphase spread showing an example of a type of cells that were used for chromosome counting. b Metaphase spread with C-bands. Note the presence of C-bands in 3 pairs of macrochromosomes (solid arrows) and in most of the microchromosomes. Dotted arrows show weak interstitial C-bands that coincide with telomeric signals in PER2q. c G-banded karyotype with 8 pairs of macrochromosomes and 46-48 microchromosomes. The chromosome count in this particular cell was 2n = 62.

Fig. 1

The karyotype (2n = 62-64) of African grey parrot (P. erithacus). a Giemsa-stained metaphase spread showing an example of a type of cells that were used for chromosome counting. b Metaphase spread with C-bands. Note the presence of C-bands in 3 pairs of macrochromosomes (solid arrows) and in most of the microchromosomes. Dotted arrows show weak interstitial C-bands that coincide with telomeric signals in PER2q. c G-banded karyotype with 8 pairs of macrochromosomes and 46-48 microchromosomes. The chromosome count in this particular cell was 2n = 62.

Close modal

Distinct but not prominent C-bands were observed in the majority of microchromosomes and in 2-3 pairs of macrochromosomes (fig. 1b). Among the latter, C-bands in PER2 and 3 were consistently seen in most cells analyzed, while the C-band in acrocentric PER6 was observed in just a few cells. The Z chromosome showed no C-banding. In addition to centromeric C-bands, a weak but consistent C-positive region was observed in PER2q (fig. 1b) and overlapped with an interstitial telomeric DNA array (see below; fig. 2).

Fig. 2

Localization of telomeric and rDNA sequences in African grey parrot chromosomes by FISH. a Telomeric sequences are located at the termini of all chromosomes (red signals). Arrows indicate interstitial telomeric sequences in PER2q which is also shown in the inset (inverted DAPI with red telomere signals). Bold arrows point at mega-telomeres. b rRNA genes localize to 4 pairs of microchromosomes (arrows).

Fig. 2

Localization of telomeric and rDNA sequences in African grey parrot chromosomes by FISH. a Telomeric sequences are located at the termini of all chromosomes (red signals). Arrows indicate interstitial telomeric sequences in PER2q which is also shown in the inset (inverted DAPI with red telomere signals). Bold arrows point at mega-telomeres. b rRNA genes localize to 4 pairs of microchromosomes (arrows).

Close modal

The karyotype comprised of 16 macrochromosomes which included 7 pairs of autosomes and the sex chromosomes and 46-48 microchromosomes (fig. 1c). The boundary between macro-and microchromosomes was sharp and the 2 groups of chromosomes distinct. Macrochromosomes were arranged into the karyotype according to G-banding, size, and centromere position. Our arrangement largely followed the one proposed by de Boer and Belterman [1980]. Based on centromere position, PER1 was metacentric, PER2-5 and 7 submetacentric, PER6 acrocentric, and the Z chromosome submetacentric (fig. 1c). Since cell cultures were from a male individual, no information for the W chromosome was obtained.

Localization of Telomeric and rRNA Sequences by FISH

Telomeric repeat sequences were present at the termini of all chromosomes, and an interstitial telomeric site was detected in PER2q (fig. 2a). The latter overlapped with a weak C-positive band (fig. 1b). In general, telomeric signals were more prominent in microchromosomes compared to macrochromosomes, and several microchromosomes carried ultra-long telomeric arrays, also known as mega-telomeres [Delany et al., 2007], as inferred from FISH signal intensity (fig. 2a). Ribosomal 5.8S, 18S, and 28S RNA genes were mapped to 4 pairs of microchromosomes (fig. 2b).

Chicken-Congo African Grey Parrot Zoo-FISH

The genome of the Congo African grey parrot was further characterized by Zoo-FISH using chromosome paints generated from flow-sorted chicken macrochromosomes GGA1-9 and Z. This allowed the identification of homologous chromosome segments between the 2 avian species. All chicken chromosome paints were exclusive to their GGA chromosome of origin, as verified by their application to chicken metaphase spreads (fig. 3). When applied to Congo African grey parrot metaphase spreads, paints for GGA2, 3, 5, 9, and Z hybridized to a single chromosome or a chromosomal segment; GGA6, 7, and 8 each hybridized to 2 syntenic segments, and GGA1 and 4 hybridized to 2 different parrot chromosomes. Neighboring synteny combinations were observed for GGA1/4 in PER1, GGA6/7 in PER6, and GGA8/9 in PER7 (fig. 3). Notably, dual-color FISH with paints for GGA6 and 7 and GGA8 and 9 showed an alternating pattern of homologous segments in PER6 and PER7, respectively (fig. 3). A summary of the chicken-Congo African grey parrot Zoo-FISH results is presented in figure 3.

Fig. 3

Congo African grey parrot Zoo-FISH with GGA1-9 and Z. Microscope images showing test-hybridization of chicken paints to chicken metaphases (left). Partial metaphases of P. erithacus showing hybridization signals with GGA paints (middle). P. erithacus macro-karyotype with homology segments to individual GGA chromosomes seen as green or red painting signals (right).

Fig. 3

Congo African grey parrot Zoo-FISH with GGA1-9 and Z. Microscope images showing test-hybridization of chicken paints to chicken metaphases (left). Partial metaphases of P. erithacus showing hybridization signals with GGA paints (middle). P. erithacus macro-karyotype with homology segments to individual GGA chromosomes seen as green or red painting signals (right).

Close modal

Here, we present the first detailed description of the Congo African grey parrot (P. erithacus) karyotype and the second chromosome study in grey parrots. The first report describes a Giemsa-stained karyotype of a grey parrot [De Boer and Belterman, 1980]. Obviously, this prior study could not define the precise species involved, because the Congo (P. erithacus) and the Timneh (P. timneh) grey parrots were recognized as separate species only recently [Taylor, 2012].

Our findings are in good agreement with this prior study regarding the number and morphology of macrochromosomes (8 pairs including the Z chromosome) (fig. 1). However, due to difficulties in accurately counting avian microchromosomes, our estimate of the diploid number 2n = 62-64 is more cautious compared to the 2n = 66 proposed by De Boer and Belterman [1980]. Also, we cannot exclude the possibility that the 2 African grey parrot species have slightly different diploid numbers and that De Boer and Belterman [1980] studied the Timneh grey parrot. In addition, we characterized the PER karyotype for the presence and location of heterochromatin (fig. 1): the C-banding pattern of PER did not have any unusual or prominent features and was similar to that observed in several other parrot karyotypes [Caparroz and Duarte 2004; Christidis et al. 1991].

The Psittaciformes are a cytogenetically fairly well studied avian group. Of the ∼350 extant species [de Kloet and de Kloet, 2005], over 70 have been karyotyped. The majority of these are New World parrots (∼40 species), and the remaining are parrots, parakeets, and cockatoos from Australasia [Van Dongen and De Boer, 1984; Christidis et al., 1991; Caparroz and Duarte, 2004; Seabury et al., 2013; de Oliveira Furo et al., 2015]. In contrast, karyotype information is available for just 2 African species, namely the grey parrot (Psittacus sp; 2n = 62-66) [De Boer and Belterman, 1980; this study] and the peach-faced lovebird (Agapornis roseicollis;2n = 46) [Christidis et al., 1991; Nanda et al., 2007], and 1 Afro-Asian species - the rose-ringed parakeet (Psittacula krameri;2n = 70) [Ray-Chaudhuri et al., 1969]. These 3 species belong to 2 different families (Psittacidae - grey parrot; Psittaculidae - lovebird and parakeet) [Clements et al., 2015], show variation in diploid number and the morphology of their macrochromosomes, and thus provide limited comparative cytogenetic information for the African group. However, compared to the many studied karyotypes of Australasian and New World parrots, where most diploid numbers range from 62 to 76 and the karyotypes usually contain 6-9 pairs of macrochromosomes [Van Dongen and De Boer, 1984; Christidis et al., 1991; Caparroz and Duarte, 2004; Nanda et al., 2007], the Congo African grey parrot with 2n = 62-64 and 8 pairs of macrochromosomes is a ‘typical' parrot karyotype. Perhaps, the only distinct feature of the PER karyotype is a sharp distinction between the macro-and microchromosomes (fig. 1). A similar macro-microchromosome boundary has been observed in some Psittaculidae species, such as rose-ringed parakeet (P. krameri) and red-breasted parakeet (P. alexandri) [Ray-Chaudhuri et al., 1969] but not in other Psittaciformes karyotypes [Van Dongen and De Boer, 1984; Christidis et al., 1991; Caparroz and Duarte, 2004; Seabury et al., 2013; de Oliveira Furo et al., 2015].

Compared to conventional cytogenetics, considerably less information is available for molecular features of parrot chromosomes. So far, telomeric (TTAGGG)n DNA arrays have been studied by FISH in 4 species: scarlet macaw [Seabury et al., 2013], budgerigar [Nanda et al., 2002], peach-faced lovebird, and cockatiel [Nanda et al., 2007]. Our primary aim was to gain information about chromosome evolution, since telomeric sequences located at non-telomeric sites (interstitially) might signify ancestral chromosome fusions. Such interstitial (TTAGGG)n repeats have been detected in cockatiel macrochromosomes [Nanda et al., 2007] and were found in PER2q in this study (fig. 2a). However, in both species the interstitial telomeric sequences coincided with C-bands, suggesting that these are not ancestral telomeres but rather sequences in constitutive heterochromatin that share similarity with telomeric repeats [Nanda et al., 2002]. This is supported by Zoo-FISH which does not indicate any evolutionary fusion sites in PER2q. This PER chromosome corresponds one-to-one to GGA2 (fig. 3), and the latter represents an ancestral configuration (table 1).

Table 1

Comparison of the number of conserved synteny segments and neighboring synteny combinations corresponding to individual chicken macro-autosomes (GGA1--GGA9) in Psittaciformes and selected species from other avian orders

Comparison of the number of conserved synteny segments and neighboring synteny combinations corresponding to individual chicken macro-autosomes (GGA1--GGA9) in Psittaciformes and selected species from other avian orders
Comparison of the number of conserved synteny segments and neighboring synteny combinations corresponding to individual chicken macro-autosomes (GGA1--GGA9) in Psittaciformes and selected species from other avian orders

In addition, our FISH results indicated that some PER microchromosomes might possess mega-telomeres or ultra-long telomeric arrays (fig. 2a) - similar to those described in chicken [Delany et al., 2007] and Japanese quail [McPherson et al., 2014]. These findings, however, need further validation by examining more individual birds and cell types and applying additional methods, such as Southern hybridization and quantitative PCR. Overall, there is continuing interest in avian telomeres, because birds in general, but social, intelligent, and long-living parrots in particular, are good models for the studies of telomere dynamics associated with genome stability, longevity, and stress [Swanberg et al., 2010]. For example, a recent study in Congo African grey parrots showed that telomere shortening is significantly associated both with ageing and stress resulting from social isolation [Aydinonat et al., 2014].

Another characteristic molecular feature of a genome is the number and location of 18S-5.8S-28S rRNA gene clusters or NORs as they are directly related to ribosome biosynthesis and, thus, the translational activity of the cell. In the Congo African grey parrot, NORs were mapped to 4 pairs of microchromosomes (fig. 2b) which is in agreement with prior studies showing that NORs are typically located on microchromosomes [Raudsepp et al., 2002; Seabury et al., 2013]. Though in a few species, such as griffon vulture [Schmid et al., 1989], magpie [Roslik and Kriukov, 2001], and South American woodcreepers [de Oliveira Barbosa et al., 2013], this rRNA gene cluster is located on macrochromosomes. Regardless of the location, the number of NOR-bearing chromosomes varies between species. For example, there are 3 pairs of NOR microchromosomes in scarlet macaw [Seabury et al., 2013] and Japanese quail [McPherson et al., 2014] but only 1 pair in chicken [Delany et al., 2009], California condor [Raudsepp et al., 2002], and all palaeognathous birds [Nishida-Umehara et al., 2007]. Because the latter are the most basal avian species, the presence of 1 pair of NOR-bearing chromosomes is considered ancestral [Nishida-Umehara et al., 2007]. Thus, 4 NOR chromosomes as observed in P. erithacus is a derivative characteristic and a likely consequence of amplification and translocation of ribosomal genes during evolution [Kretschmer et al., 2014].

Zoo-FISH homology for chicken macrochromosomes is to date available for over 30 avian species [Nanda et al., 2007, 2011; Kretschmer et al., 2014; Romanov et al., 2014; de Oliveira Furo et al., 2015], including 7 parrots from different geographic locations (fig. 4) [Forshaw, 2010]): the Congo African grey parrot [this study] and peach-faced lovebird [Nanda et al., 2007] from Africa; scarlet macaw [Seabury et al., 2013], red-and-green macaw and hyacinth macaw [de Oliveira Furo et al., 2015] from South America, and budgerigar and cockatiel [Nanda et al., 2007] from Australia. In the hyacinth and the red-and-green macaw Zoo-FISH was further refined using chromosome painting probes from the white hawk [de Oliveira et al., 2010]. Because homology to chicken macrochromosomes is the common denominator for all avian Zoo-FISH studies, the P. erithacus karyotype can now be compared at the molecular level with those of related Psittaciformes species (fig. 4) as well as with other avian groups (table 1).

Fig. 4

Comparative chicken Zoo-FISH in 7 Psittaciformes species. Colors in chromosome ideograms denote homology to corresponding chicken macrochromosomes. The centromere positions are marked with a black bar, and the geographic range of each species [Forshaw, 2010] is shown at the left. Classification of the 7 species follows the ‘Clements Checklist' [Clements et al., 2015].

Fig. 4

Comparative chicken Zoo-FISH in 7 Psittaciformes species. Colors in chromosome ideograms denote homology to corresponding chicken macrochromosomes. The centromere positions are marked with a black bar, and the geographic range of each species [Forshaw, 2010] is shown at the left. Classification of the 7 species follows the ‘Clements Checklist' [Clements et al., 2015].

Close modal

Conserved whole chromosome synteny of GGA2, 3, 5-9 and the correspondence of GGA4 to 2 conserved segments in the P. erithacus karyotype closely resemble the pattern observed in many other avian groups (table 1), including the paleognathous species [Nishida-Umehara et al., 2007] and the putative ancestral avian karyotype [Kretschmer et al., 2014; de Oliveira Furo et al., 2015]. Thus, synteny of these segments of avian genomes has been preserved for about 100 million years (MYR) preceding the divergence of neo- and paleognathous birds [Jarvis et al., 2014]. Fission of GGA1 into 2 segments seems to be an evolutionary event shared by Psittaciformes and Passeriformes which diverged about 55 MYR ago [Jarvis et al., 2014]. Further, while whole chromosome synteny of GGA6, 7, 8, and 9 is conserved in most avian species, fusions in Psittaciformes karyotypes have brought GGA6/7 and GGA8/9 together as neighboring synteny segments (fig. 4). Additional inversions in these segments, as first described by Nanda and colleagues [2007], have produced a characteristic alternating pattern of GGA6/7 in PER, peach-faced lovebird, cockatiel, and all 3 macaws, and of GGA8/9 in PER, budgerigar, scarlet macaw, and red-and-green macaw (fig. 4). Such an alternating pattern of neighboring segments has not been observed in other avian species, even though conserved synteny of GGA6/7 and GGA8/9 as separate arms of the same chromosome is present in coot and Eurasian nuthatch, respectively [Nanda et al., 2011]. Additional minor differences in these patterns distinguish the 2 Australian species from African and South American parrots, thus dating back for about 45 MYR ago, which is the proposed divergence time for the cockatiel and budgerigar [Schweizer et al., 2010]. Furthermore, conserved synteny between GGA1/4 is characteristic only for African and South American parrots and has not been found in Australian parrots (fig. 4) or in other avian groups (table 1). Instead, in budgerigar and cockatiel GGA4 has fused with the segment homologous to GGA8/9 (fig. 4). Overall, within Psittaciformes the Zoo-FISH pattern of P. erithacus very closely resembles those of the 3 neotropical macaws insisting that no major chromosomal rearrangements have taken place since the split of Arini-Psittacus lineages about 40 MYR ago [Schweizer et al., 2010].

The Zoo-FISH karyotype of the Congo African grey parrot, as well as those of other Psittaciformes, shows distinct patterns of neighboring synteny combinations that have not been observed in most other avian groups and are not present in the putative ancestral karyotype [de Oliveira Furo et al., 2015]. These observations are in line with previous studies, suggesting that the karyotypes of the Psittaciformes have undergone more extensive evolutionary rearrangements compared to the majority of other avian genomes. The only other group with even more rearrangements are the birds of prey which have lost the macro- and microchromosome arrangement in their karyotypes [Nanda et al., 2007; de Oliveira et al., 2010; de Oliveira Furo et al., 2015].

In order to reveal the full extent of chromosome/genome evolution in Psittaciformes, Zoo-FISH studies in more species should be combined with the emerging sequence data for avian genomes [Romanov et al., 2014]. For example, a recent comparative sequence analysis of 20 diverse avian species including budgerigar [Romanov et al., 2014] showed that GGA5, which is an ancestral-type chromosome (table 1), has undergone multiple and diverse intrachromosomal rearrangements during the evolution of avian lineages. Thus, more extensive changes at the DNA sequence level could be expected in chromosomes that have diverged from the ancestral configuration like, for example, PER1, 6, and 7, and their counterparts in other parrots.

This research was supported by CVM Merial Veterinary Scholars Program and the Schubot Exotic Bird Health Center, College of Veterinary Medicine & Biomedical Sciences, Texas A&M University. The authors thank Dr. N.A. Serdyukova for kindly providing a plasmid clone for 18S and 28S rRNA sequences.

The authors have no ethical conflicts to disclose.

The authors have no conflicts of interest to declare.

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