Jurnal Biologi Indonesia 7 (1): 1-11(2011)

Phylogenetic relationships within (Aves: Psittaciformes) Based on DNA Sequences of The Seventh intron of Nuclear β-fibrinogen gene

Dwi Astuti Zoological Division, Research Centre for Biology-Indonesian Institute of Sciences Jl. Raya Jakarta - Bogor Km 46 Cibinong, W. Jawa-Indonesia. Email: [email protected]

ABSTRAK Hubungan Kekerabatan Kakatua (Aves: Psittaciformes) Berdasarkan Sekuen DNA dari Intron ke Tujuh dari Gen β--fibrinogen. Hubungan kekerabatan diantara burung kakatua masih menjadi perdebatan, khususnya menyangkut posisi dari Nymphycus hollandicus. Intron pada gen β-fibrinogen telah diketahui berguna untuk mempelajari filogeni dari beberapa kelompok burung, oleh karena itu penelitian ini menggunakan sekuen DNA dari intron ketujuh pada gen ini (β-fibint7) untuk mengkonstruksi filogeni dari enam genus (, Callocephalon, Eolophus, Calyptorhynchus, Probosciger, dan Nymphicus) yang terdapat di dunia berdasarkan analisis neighbor-joining (NJ) and maximum-parsimony (MP). Ditemukan beberapa indel (insersi-delesi) pada sekuen DNA dari â-fibint7 kakatua, sehingga panjang sekuen DNA bervariasi diantara taxa-taxa yang diteliti. Tidak terjadi saturasi antara substitusi transisi dengan transversi, dan juga antara transversi dengan jarak genetik. Tribe Cacatuini yang terdiri dari tiga genus Cacatua, Callocephalon, dan Eolophus bersifat monophyletic. Meskipun hubungan diantara spesies dari genus Cacatua tidak terungkap dengan jelas, tetapi C. alba, C. galerita, C. goffini, C. sanguinea, C. moluccensis dan C. sulphurea berada di dalam satu group, dan C. leadbeateri relatif terpisah dari species-species congeneric lainnya. Callocephalon secara filogenetik terpisah jauh dari genus-genus lainnya pada tribe Cacatuini. Tribe Calyptorhyncini yang terdiri dari Probosciger dan Calyptorhynchus adalah paraphyletic. Calyptorhynchus tampak monophyletic. Nymphicus (tribe Calopsittacini) menjadi clade basal dari kakatua.

Key words: Kekerabatan, Kakatua, Sekuen DNA, β--fibrinogen

INTRODUCTION cus) which form one of the most characteristic groups (Forshaw 1989). Approximately 340 living species of Some authors classified cockatoos Psittaciformes have a pan-tropical and belonged to the family Cacatuidae (e.g. southern distribution. Of these , the del Hoyo et al. 1997; Dickinson 2003), cockatoos, with 18 –21 species are but (Forshaw 1989) placed cockatoos into member of six genera (Cacatua, subfamily Cacatuinae and into family Eolophus, Callocephalon, Probosci- Psittacidae (Dickinson 2003). The ger, Calypthorhynchus, and Nymphi- cockatoos have always been thought to

1 Dwi Astuti form a natural, monophyletic group within more closely related to black Calypto- the order Psittaciformes. rhynchini or to white Cacatuini. At spe- Cockatoos are characterized by cies level, controversy has arisen over movable crest. They are lack in dyck- the position of Cacatua leadbeateri. texture which produces green in the Several phylogenetic studies had plumage of other (Smith 1975; used sequence data of mitochondrial Forshaw 1989), and have long been DNA, but this study employs nuclear recognized as a unique group of Psitta- DNA sequence data. Contrary to ciformes. They have several characteris- mitochon-drial DNA, nuclear DNA tics that separate them from other parrots sequences were rarely analyzed for the (Adam 1984). Most authors have aligned phylogenetic studies of birds including them into major lineages: the predomi- cockatoos, partly because the substitution nantly black Calyptorhynchini (Calypto- rates of most nuclear genes in rhynchus and Probosciger) and the are too low to examine the phylogeny of predominantly white Cacatuini closely-related birds (Allen & Omland (Eolophus, Callocephalon, and 2003; DeBry & Seshadri, 2001; Johnson Cacatua). Various studies have been & Clayton 2000; Palumbi et. al. 2001). conducted to make grouping of In animals, nuclear introns were cockatoos, however, the branching and initially considered to be too slowly the evolutionary relationships of evolving, susceptible to incomplete cockatoos remain unclear and have not lineage sorting, and overly shued due to been tested and there were several recombination and gene conversion for unsolved taxonomic problem (Smith 1975; resolving interspecific phylogenies when Adam 1984). One of the controversial compared to mtDNA (Allen & Omland problems is the phylogenetic position of 2003; DeBry & Seshadri 2001; Johnson cocktail (Nymphycus). For example, & Clayton 2000; Palumbi et. al. 2001). Smith (1975) and Homberger (1980) Yet, several studies have successfully amassed compelling morphological and used nuclear introns for resolving species behavioral evidence to assume it as a level phylogenies in a diversity of diminutive cockatoos. However, Walters groups (Beltran et. al. 2002; Driskell & and Condon (1975) grouped Nymphycus Christidis 2004; Lavoue et. al. 2003; with the polytelitine or platycercine Peters et. al. 2005). Although there are parrots. More recently taxonomists many benefits for employing nuclear (Adam 1984; Forshaw 1989; del Hoyo intron sequence data for phylogeny 1997; Par 1998) agree that Nymphicus reconstruction, they remain a relatively is belong to cockatoos, but its position unexploited resource because of the within cockatoos is still controversial, and difficulties in isolating orthologous loci belong to monotypic tribe Calopsittacini (Doyle et. al. 2003). (Forshaw 1989). The phylogenetic However, since Prychitko & Moore position of Callocephalon within (1997) pointed out that introns are cockatoos has also been in doubt, it is attractive candidates for phylogenetic

2 Phylogenetic relationships within Cockatoos analysis because of their abundance in and Nymphicus, and 3) determine the the nuclear genome, their convenient monophyly or paraphyly of each tribe length, and potentially easy amplification by the PCR, the utility of introns for MATERIALS AND METHODS phylogenetics studies of has been increasing (Johson &Clayton 2000ab; Blood samples were collected from Prychitko & Moore 1997, 2000, 2003). each individual bird at zoos and captive Nuclear-gene introns sequence data breeding, and preserved in the 99 % of have several properties that would ethanol and used as DNA resources for seemingly make them ideal for phylo- polymerase chain reaction (PCR) and genetic studies, because they evolve DNA sequencing. In total 18 species more rapidly than exons (Prychitko & belonging to six extant genera (Cacatua, Moore 2003). Introns are also attractive Callocephalon, Eolophus, Probos- candidates for phylogenetic analysis ciger, Calyptorhynchus, and Nymphi- because of their abundance in the nuclear cus) of three tribes (Cacatuini, Calypto- genome, their convenient lengths, and rhynchini, and Calopsittasini) of potentially easy amplification by the PCR cockatoos (Tabel 1) were used in this (Prychitko & Moore 1997). study. The nomenclature follows β-fibrinogen is one of the nuclear Forshaw (1989). genes consisting of exons and introns. Genomic DNA was extracted from Single seventh intron of β-fibrinogen gene approximately 5-20 mg of each dry blood is known as a non protein coding gene or tissue sample using Qiamp Mini Kit (Prychitco & Moore 1997) and has been DNA (QIAGEN), according to described with regard to phylogenetic & manufacture’s protocol. One or more Clayton 2000) and family levels (Moyle individuals from each species were & Marks 2006; Dor et. al. 2010; sequenced in order to minimize the Gonzalez et. al. 2009). Analysis of possible effects of intraspecific variation nuclear intron demonstrated the ability of on phylogenetic hypotheses (Smouse et. the seventh intron of β-fibrinogen for the al. 1991). phylogenetic studies (Prychitco & Moore A DNA region of β-fibint7 was 1997). analyzed in this study. The DNA The present study was addressed to fragments were amplified by the reveal the relationships among species, polymerase chain reaction (PCR). A genera, and tribes of cockatoos inferred single fragment of β-fibint7 was from DNA sequence of the seventh amplified using a nucleotide primer pair intron of nuclear β-fibrinogen gene ( β- FIB-B17U and FIB-B17L (Prychitko & fibint7). Objectives of this study were to Moore1997) in the following PCR 1) resolve the phylogenetic relationships conditions: one cycle of 94 °C at 5 minute, within cockatoos (Cacatuinae phylo- 35 cycles of [94 °C- 30 sec., 46 °C-30 geny), 2) determine the position of sec., 72 °C-60 sec], and one cycle of 72 Cacatua leadbeateri, Callocephalon, °C for 7 min.

3 Dwi Astuti

PCR products were electrophoresed tide saturations were tested by plotting in 1.5 % agarose gels, stained with the numbers of transitions (ts) and Ethydium Bromide, and visualized under transversions (tv) against genetic UV light. A single fragment of distances for all pairwise species. amplification products was cleaned by Phylogenetic trees were constructed PEG (Polyethelene glycol) and used for using maximum-parsimony (MP) and DNA sequencing. Sequences of both neighbor- joining (NJ) methods adopting strands for each sample were obtained Accipiter and Columba livia as using ABI 3100 automated sequencer outgroups. Kimura’s 2-parameter with a BigDye Terminator Kit version 1.1 distances were calculated for NJ tree. or version 3.1 (Biosystems). All phylogenetic analyses were assessed In the nucleotide DNA sequence using PAUP* Version 4.0b (Swofford alignment, the presence of indels 2000). In the parsimony analysis, (insertion and deletion) was analyzed by heuristic search option in PAUP* was using Proseq software. Base composi- selected with a random taxon addition tion was assessed using PAUP* Version sequence (100 replications) and three 4.0b (Swofford 2000). PAUP* was also bisection-reconstruction (TBR) branch used to determine the number of variable swapping. The random addition of and informative sites. sequences increases the effectiveness of The number of nucleotide substitu- heuristic searches (Maddison, 1991). tions and genetic distances were The bootstrap values were computed calculated using DNAsa and MEGA2 using 1000 replicates for NJ tree and 100 software (Kumar et. al. 2001). Nucleo- full heuristic replicates for MP tree. Tabel 1: species used in this study. No. Species/subspecies Tribe 1. Cacatua alba Cacatuini 2. C. galerita galerita 3. C. g. triton 4. C. g. eleonora 5. C. goffini 6. C. moluccensis 7. C. sanguinea 8. C. s. sulphurea 9. C.s. citrinocristata 10. C. leadbeateri 11. Eolophus roseicapillus 12. Callocephalon fimbriatum 13 Probosciger aterrimus Calyphtorhynchini 14. Calyptorhynchus banksii 15. C. lathami 16. C. baudinii 17. C. latirostris 18. Nymphicus hollandicus Calopsittacini

4 Phylogenetic relationships within Cockatoos

RESULTS The numbers of transition were plotted against transversion substitutions, Based on aligned sequences, several the graph show a linear that transition indels occurred in β-fibint7 of cockatoos, appeared to be not saturated to ranging from 1 to 9 bp. Indels were more trasnversion (Figure 1), and also when common and concentrated toward the all nucleotide substitutions, including middle of the intron. Due to the presence transition and transversion were plotted of several indels, the fragment length of against genetic distance, there was no the β-fibint7 varied from 808 to 817 bp saturation signal (Figure 2). in cockatoos (Cacatuinae). In comparison with outgroups (Columba Grouping and relationships among and Accipiter), total aligned fragments cockatoos consisted of 817 characters. Phylogenetic relationships among The mean of base frequencies of β- cockatoos examined were presented in fibint7 was highes in thymine 31.45 %, Figure 1 and Figure 2. Both phylogenetic followed by adenine (28.67 %), cytosine trees; neighbor-joining (NJ) and (21.63 %), and guanine (18.25 %). maximum-parsimony (MP) show that Nucleotide substitutions ranged from 1- tree genera (Cacatua, Eolophus, and 474 sites. When all characters including Callocephalon) in which belong to tribe indels were analyzed, there were 507 Cacatuini group together and seem to be monomorphic sites, 310 polymorphic monophyletic, with the bootstrap values sites, and 164 parsimony- informative 71 % in NJ and 60 % in MP trees. sites. Based on the sequence data Six species of white cockatoos obtained, there were variations in the (genus Cacatua); C. sulphurea, C. sequences of the β-fibint7 gene in galerita, C. alba, C. moluccensis, C. cockatoos. goffini, and C. sanguinea group together and form a monophyletic group,

30

25

20

15

Transition 10

5

0 010203040 Transversion Figure 1. Plot of transition across transversion substitutions of seventh intron of â-fibint7) gene in cockatoos.

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40 Transition Fibint7 35 Transversion 30 25 20 15 10 No. of substitutions 5 0 0 0.02 0.04 0.06 Distance Figure 2. Plot of nucleotides substitutions (transition and transversion) across Kimura 2- parameter distances of of â-fibint7 gene in cockatoos. supported by bootstrap values 84 % and banksii, C. lathami, C. baudinii, and 57 % in NJ and MP tree, respectively, C. latirostris group together and formed however C. leadbeateri has a relatively a monophyletic group supported by distant relationship to other congeneric bootstrap values 91 % in NJ and 69 % in species, while posistion of others two MP , with C. banskii is close to C. genera; Eolophus and Callocephalon lathami, and C. baudinii is close to C. were unclear. latirostris. Nymphycus as a monotypic Relationships between species of genus of Calypsittacini appear to be a basal genus Cacatua were not well resolved clade of cockatoos. by Fibint7 gene sequence. Both trees show that C. goffini is close to C. DISCUSSION sanguinea supported by 84 % bootstrap value, C. alba and C. moluccensis As reported by previous authors group together, and C. sulphurea closer (Prychitko & Moore 2000; Johnson & to C. galerita than to others congeneric Clayson 2000a), the â-fibint7 sequences species. were rich in thymine and adenine. β-fibint7 supported paraphyly of Overall, the results of their studies were Calypthorhynchus and distant similar to those of the present study. As relationship of Nymphicus and other reported by Lewin (1997), the number and cockatoos. Two genera belong to tribe positions of introns in a gene are usually Calyptorhynchini (Calyptorhynchus highly conserved through evolution, but and Probosciger) formed paraphyletic the length of intron is somewhat variable group, in which Probosciger closer to as the result of indels, and the nucleotide genera of Cacatuini supported by sequence is highly variable. Previous bootstrap values 67 % in NJ and 57 % studies reported that several indels occur in MP, than to Calyptorhynchus. Four in Dove and Pigeon fibint7 sequences, species of Calyptorhynchus; C. ranging from 1 to 125 bp (Johnson &

6 Phylogenetic relationships within Cockatoos

Clayton 2000a) and from 1 to 695 bp (1997), in that nuclear DNA usually (Johnson & Clayton 2000b), respectively, exhibits relatively low substitution rates which are almost consistent with the and no saturation on substitutions. range of 1 to 169 indels in â-fibint7. Phylogenetic relationships of Aligned â-fibint7 sequences contained present study was not congruent to several inferred insertions or deletions previous studies. Biochemical analysis (indels) (Moyle & Marks 2006). The â- conducted by Adams et. al. (1994) has fibint7 gene alignment contained several placed Nymphicus as one group with indels. The Canarian pigeons possess Calypstorhynchus in which both of autapomorphic indels (Gonzalez et. al. them were basal clade of Cockatos. 2009). Unlike woodpeckers (Prychitco Whilts characters analysis conducted by & Moore 2000) and doves (Johson & del Hoyo et. al. (1997) proposed Clayton 2000) in which b-fibint7 is AT Nymphicus was independent to others, rich. and mitochondrial 12S placed Nymphicus No saturation effects on â-fibint7 into one group of Calyptorhynchus and analyzed in this study were also Probosiger (Figure 5). My parsimony supported by Prychitco and Moore and neighbor-joining analyses generated

Cacatua sulphurea citrinocristata 64 Cacatua s. sulphurea Cacatua galerita eleonora Cacatua g. triton 84 Cacatua g. galerita Cacatua alba Cacatua moluccensis Cacatuini 71 Cacatua goffini 91 Cacatua sanguinea 67 Cacatua leadbeateri Eolophus roseicapillus 72 Callocephalon fimbriatum Probosciger aterrimus Calyptorinchus banksii 98 99 91 Calyptorhynchus lathami Calyptorhynchini 98 Calyptorhynnchus baudinii 100 Calyptorinchus latirostris Nymphycus hollandicus Calopsittacini Psittaculirostris Other parrot Columba Outgroup species Accipiter

Figure 3: A neighbor-joining (NJ) tree of six genera of cockatoos based on DNA sequences of â-fibin7 gene. Numbers above indicate bootstrap values > 50 %

7 Dwi Astuti

Cacatua sulphurea citrinocristata 64 Cacatua s. sulphurea Cacatua galerita eleonora Cacatua g. triton 57 Cacatua g. galerita Cacatua alba Cacatua moluccensis Cacatuini 60 84 Cacatua goffini Cacatua sanguinea Cacatua leadbeateri 55 Eolophus roseicapillus Callocephalon fimbriatum Probosciger aterrimus Calyptorinchus banksii 94 Calyptorhynchus lathami Calyptorhynchini 89 69 Calyptorhynnchus baudinii 100 96 Calyptorinchus latirostris Nymphycus hollandicus Calopsittacini Psittaculirostris Other parrot Columba Outgroup species Accipiter

Figure 4: A maximum-parsimony (MP) tree of six genera of cockatoos based on DNA sequences of â-fibint7 gene. Numbers above indicate bootstrap values > 50 % different results with both morphological phalon) was not supported by mitochon- and mitochondrial 12S analysis, however drial 12S and morphological analyses it was likely to biochemical analysis which (Figur 5). presumable Nymphycus was a basal clade of cockatoos. CONCLUSIONS Relationships within genus Cacatua in this study could not be compared to Relationships between species of previous studies, because the species genus Cacatua were not well resolved used were different. However, within the by fibint7 gene sequence. Cacatua tribe Cacatuini, the recent study agrees leadbeateri was relatively distant from that Eolophus was closer Cacatua than congeneric species. Tribe Cacatuini to others genera, and Callocephalon (Cacatua, Eolophus and Callocepha- was phylogeneticaly distance from other lon) was monophyletic. Callocephalon congeneric genera. Whilst the position was phylogeneticaly distant from other of Probosciger in the present study; in congeneric genera of Cacatuini. Tribe which Probosciger is close to Cacatuini Calyptorhynchini (Calyptorhynchus and (Cacatua, Eolophus, and Calloce- Probosciger) was paraphyletic.

8 Phylogenetic relationships within Cockatoos

Nymphicus was presumably a basal REFERENCES clade of cockatoos. Allen, E., & K.Omland. 2003. Novel ACKNOWLEDGEMENTS intron phylogeny supports plumage convergence in orioles (Icterus). The research was supported by Auk 120, 961–969. Centre of Excellent Project, Hokkaido Avise, JC. 1994. Molecular Markers, University, and Japan Society for Natural History and Evolution. Promoting Science, Core University Chapman Hall, New York. (Hokkaido University and LIPI). I would Brown, DM. & CA. Toft 1999. like to thank Prof. Seigo Higashi and Dr. Phylogenetic relationships with in Hitoshi Suzuki (Hokkaido University) for Cockatoos. Molecular systematics and biogeography of the cockatoos kindly supervising me, and to Dr. Ken Aplin (CSIRO) for sharing the samples (Psittaciformes: Cacatuidae). Auk 116(1):141-157.

Biochemical analysis (Adams et al., 1994)

Cacatua leadbeateri Morphologycal Cacatua galerita characters (del Cacatua leadbeateri Cacatua sanguinea Hoyo et al., 1997) Eolophus Cacatua tenuirostris Chalocephalon Nymphicus Eolophus roseicapilus Caluptorhynchus Callocephalo Probosciger Nymphicus All other parrots Origin of Calyptorhynchus parrots

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Mt 12S (Brown & Cacatua Toft, 1999 Cacat ua leadbeateri Eolophus Callopcephalon

Nymphicus

Calyptorhynchus

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Figure 5: Tree topologies of cockatoos relationships based on other characters

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Memasukkan: Juli 2010 Diterima: Desember 2010

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