The Complete Sequence of the Mitochondrial Genome of Buteo buteo (Aves, Accipitridae) Indicates an Early Split in the Phylogeny of Raptors

Elisabeth Haring, Luise Kruckenhauser, Anita Gamauf, Martin J. Riesing and Wilhelm Pinsker

Zoological Department, Museum of Natural History, Vienna, Austria;
Institute of Medical Biology, University of Vienna, Austria


    Abstract
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Supplementary Material
 Acknowledgements
 References
 
The complete sequence of the mitochondrial (mt) genome of Buteo buteo was determined. Its gene content and nucleotide composition are typical for avian genomes. Due to expanded noncoding sequences, Buteo possesses the longest mt genome sequenced so far (18,674 bp). The gene order comprising the control region and neighboring genes is identical to that of Falco peregrinus, suggesting that the corresponding rearrangement occurred before the falconid/accipitrid split. Phylogenetic analyses performed with the mt sequence of Buteo and nine other mt genomes suggest that for investigations at higher taxonomic levels (e.g., avian orders), concatenated rRNA and tRNA gene sequences are more informative than protein gene sequences with respect to resolution and bootstrap support. Phylogenetic analyses indicate an early split between Accipitridae and Falconidae, which, according to molecular dating of other avian divergence times, can be assumed to have taken place in the late Cretaceous 65–83 MYA.


    Introduction
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Supplementary Material
 Acknowledgements
 References
 
Among birds of prey, buzzards and hawks of the genus Buteo are an extremely successful group that is widely distributed, being absent only in Australia, Antarctica, and most parts of the oriental region. The genus currently comprises between 25 (Brown and Amadon 1968Citation ) and 27 species (del Hoyo, Elliott, and Sargatal 1994Citation ), thus representing about 7% of the species in the family Accipitridae. In the present study, we focus on the common buzzard Buteo buteo (Linnaeus, 1758), which is the most abundant raptor species in many parts of Europe (Bijlsma 1997Citation ).

The two largest families of birds of prey are the Accipitridae and the Falconidae. The accipitrids, known colloquially as hawks, kites, harriers, vultures, and eagles, are rather similar in their basic morphological structures, although they show great diversity in size, shape, flying ability, ecology, and predatory habits. The falconids resemble the accipitrids in some characteristics, such as a powerful hooked bill, a fleshy cere straddling the bill, heavy bony brow ridges, and a crop (to store freshly eaten food). The differences between the two families have been summarized by Olsen (1995)Citation in a survey of 25 anatomical and behavioral traits.

According to traditional morphological classifications (e.g., Brown and Amadon 1968Citation ; Storer 1971Citation ; Stresemann and Amadon 1979Citation ; Cracraft 1981Citation ), Accipitridae and Falconidae belong to the order Falconiformes. However, based on detailed morphological studies of several families, Jollie (1976, 1977)Citation concluded that falconids and accipitrids are not closely related. According to his interpretation, the order Falconiformes is polyphyletic, especially with respect to the inclusion of New World vultures (Cathartidae); that view is supported by studies of several behavioral traits (König 1982Citation ) and, more recently, by molecular analyses (e.g., Sibley and Ahlquist 1990Citation ; Seibold and Helbig 1995Citation ; Wink et al. 1998Citation ). Sibley and Ahlquist (1990)Citation estimated the overall genomic similarity by DNA-DNA hybridization and proposed a new classification of birds in which the New World vultures appear as close relatives of the storks (Ciconiidae). In their classification, the falconiform taxa are placed within an expanded order Ciconiiformes, in which they include the infraorders Falconides (including Falconidae and Accipitridae) and Ciconiides (including the family Ciconiidae with the subfamilies Cathartinae and Ciconiinae). Sequence analyses of the cytochrome b gene (cyt b) (Avise, Nelson, and Sibley 1994Citation ; Seibold and Helbig 1995Citation ; Wink et al. 1998Citation ) support this taxonomic position for New World vultures, but the relationships of the Ciconiidae with respect to Accipitridae and Cathartidae have not been resolved unambiguously. So far, no other genetic markers (mitochondrial or nuclear) have been employed to elucidate the phylogenetic relationships of these families.

Analyses of complete mitochondrial (mt) genomes provide not only sequence data for phylogenetic studies, but also information about structural genomic rearrangements which may serve as additional markers. Sequencing of the first complete vertebrate mt genomes suggested that gene content and gene order are highly conserved, but subsequent sequence data have demonstrated that the gene order in vertebrates is not uniform (for review, see Quinn 1997Citation ). A major rearrangement within the mt genome of chickens and other galliform birds has been described by Desjardins and Morais (1990)Citation . It comprises the cyt b gene, the NADH dehydrogenase subunit 6 gene (nd6), and several tRNA genes. Subsequently, this particular gene order has been found in several other avian species, suggesting that this rearrangement could have occurred at the base of the avian branch and thus might be shared by all recent bird species. However, the hypothesis of a universal gene order characteristic for all birds was refuted by the discovery of yet another rearrangement of the mt genes in Falco peregrinus, as well as in birds of four additional orders (Mindell, Sorenson, and Dimcheff 1998Citation ). In an investigation of 137 species, representing 13 orders, Mindell, Sorenson, and Dimcheff (1998)Citation hypothesize that this novel arrangement, which includes the control region (CR) and surrounding sections, must have originated independently four times in avian evolution. In addition to the CR, these species possess a second noncoding (nc) region, probably generated through a duplication process. The same arrangement was recently detected in warblers of the genus Phylloscopus (Bensch and Härlid 2000Citation ). Partial sequence analysis of the B. buteo mt genome (Haring et al. 1999Citation ) revealed the existence of a noncoding section corresponding to the nc region of F. peregrinus, which was designated a pseudo control region ({Psi}CR). Although the position of the functional CR in B. buteo was not determined, this finding suggested that F. peregrinus and B. buteo might share the same gene order.

In this paper, we report the complete sequence of the mt genome of B. buteo. For sequence comparisons, we used the previously published complete mt genomes of nine other bird species, along with that of Alligator mississippiensis as an outgroup, to address the following questions: (1) Do Falconidae and Accipitridae share the same rearrangement within their mt genomes? (2) What are the phylogenetic positions of the genera Buteo, Ciconia, and Falco within the avian tree? (3) Are subsections as useful as complete mt genomes for resolving phylogenies? (Up to now, the majority of avian molecular phylogenies have been based on sequence data of the mitochondrial cyt b gene.) (4) What is the degree of divergence between Buteo and Falco, and can it be related to other splits in the phylogeny of birds to estimate their approximate divergence time?


    Materials and Methods
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Supplementary Material
 Acknowledgements
 References
 
DNA Extraction
DNA from B. buteo was extracted from the blood (stored in EDTA buffer) of a single specimen (specimen b.but-2, common buzzard, B. buteo buteo; Haring et al. 1999Citation ) by overnight incubation at 37°C in extraction buffer (10 mM Tris-HCl [pH 8.0], 10 mM EDTA, 50 mM NaCl, 40 mM dithiothreitol, 1% SDS, 0.5 mg/ml proteinase K). DNA was purified by two phenol/chloroform/isoamylalcohol (25:24:1) extractions and one chloroform/isoamylalcohol (1:1) extraction, followed by precipitation with 1/10 volumes 3 M NaAc, 3 x volumes EtOH.

PCR Amplification
PCR was carried out with an Eppendorf Thermocycler in a volume of 25 µl containing 1 unit Dynazyme DNA polymerase (Finnzymes OY), 1 µM of each primer, 0.2 mM of each dNTP, and 50–200 ng template DNA. The solutions were heated to 95°C (2 min) and then put through 30 reaction cycles: 95°C (10 s), annealing temperature (10 s), and 72°C (1 min/1 kb expected length), followed by a final extension at 72°C (10 min). Primers used for PCR amplification are listed in table 1 . The 11 PCR fragments that were subsequently cloned and sequenced are depicted in figure 1 . These overlapping fragments cover the whole mt genome of B. buteo except the region comprising a part of the nd6 gene, tRNAGlu, {Psi}CR, tRNAPhe, and a part of the 12S rRNA gene (primer pair nd6-1+/12S-1-) determined in a previous study from the same individual of B. buteo (GenBank accession number AF202186). The 11 fragments sequenced in the present study were obtained using the following primer combinations (fig. 1 ): 12S3+/16S3- (910 bp), 12S-2+/16S-2- (1,410 bp), 16S-1+/nd2-2- (2,372 bp), nd2-1+/cox2-1- (2,913 bp), cox2-3+/atp6-2- (1,397 bp), atp6-1+/nd4-4- (1,430 bp), nd3-1+/nd4-5- (1,534 bp), nd4-3+/nd5-2- (1,047 bp), nd5-3+/cytb-2- (1,747 bp), cytb-3+/cytb-4- (899 bp), cytb-1+/nd6-3- (2,071 bp).


View this table:
[in this window]
[in a new window]
 
Table 1 PCR Primers Used

 


View larger version (45K):
[in this window]
[in a new window]
 
Fig. 1.—Mitochondrial genome of Buteo. Overlapping PCR fragments are depicted with their corresponding primers. Arrows at the inner circle indicate orientation of genes. For designation of tRNAs, the three-letter code for amino acids is used. Position 0 of the complete sequence (table 2 ) is indicated

 
Cloning and Sequencing
PCR products were extracted from agarose gels using the QIAquick Gel Extraction Kit (QIAGEN) and cloned (TOPO TA Cloning Kit, Invitrogen). Sequencing of the clones (both directions, M13 primers) was performed by MWG-Biotech (Ebersberg, Germany) with a Li-Cor Sequencer. Due to overlapping clones and overlap of regions sequenced with internal primers, the whole mitochondrial genome was read at least twice (some sections four times). Thus, the complete sequence was determined without any ambiguities.

Sequence Analysis
Alignments of DNA and amino acid sequences were produced with the program CLUSTAL X (Thompson et al. 1997Citation ) and improved manually. Distance-based (the neighbor-joining [NJ] algorithm; Saitou and Nei 1987Citation ) and maximum-parsimony (MP) methods were used to infer the phylogenetic relationships. All dendrograms were calculated with the software package PAUP (test version 4.0b3a; Swofford 1998Citation ) using a heuristic search with the tree bisection-reconnection (TBR) algorithm and a random-taxon-addition sequence. Gaps were treated as "missing," and all characters were weighted equally. The robustness of trees was tested with bootstrap analysis (100 replications). Relative-rate tests were performed with the program Phyltest (Kumar 1996Citation ), which provides the two-cluster test of Takezaki, Rzhetsky, and Nei (1995)Citation . In this test, the constancy of substitution rates is examined for two lineages with a given outgroup lineage. The program allows inclusion of multiple sequences in each of the lineages. If La and Lb are the averages of observed numbers of substitutions per site from the common ancestor of clusters A and B, then La = Lb is the null hypothesis under rate constancy ({delta} = La - Lb = 0). Because the variance of {delta} can be estimated, the deviation of {delta} from 0 can be tested by a two-tailed normal deviate test. For the tests, new alignments including only the sequences used were created, and Kimura (1980)Citation two-parameter distances were used for the calculations.

DNA Sequences
For phylogenetic comparisons, the complete mt genome sequences of the following avian species were retrieved from GenBank: Gallus gallus f. dom., NC001323 (Desjardins and Morais 1990Citation ); Struthio camelus, Y12025 (Härlid, Janke, and Arnason 1997Citation ); Rhea americana, NC000846 (Härlid, Janke, and Arnason 1998Citation ); Vidua chalybeata, NC000880; Aythya americana, AF090337; Falco peregrinus, NC000878; Smithornis sharpei, NC000879 (Mindell, Sorenson, and Dimcheff 1998Citation ; Mindell et al. 1999Citation ); Corvus frugilegus, NC002069 (Härlid and Arnason 1999Citation ); Ciconia ciconia, AB026818 (Yamamotu, unpublished). The sequence of Alligator mississippiensis, Y13113 (Janke and Arnason 1997Citation ), was used as an outgroup for the rooting of phylogenetic trees.

Designation of Sequences
Although the sequences of the various avian mt genomes used in this study are derived from defined taxonomic entities (species/subspecies), e.g., B. b. buteo, the phylogenetic reconstructions were made at the genus level or at even higher taxonomic levels. Therefore, for the sake of brevity, we refer to the taxa only by their genus names in the text. In the tables and figures, the following abbreviations are used: All, A. mississippiensis; Ayt, A. americana; But, B. buteo; Cic, C. ciconia; Cor, C. frugilegus; Fal, F. peregrinus; Gal, G. gallus; Rhe, R. americana; Smi, S. sharpei; Str, S. camelus; Vid, V. chalybeata.


    Results
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Supplementary Material
 Acknowledgements
 References
 
Genome Organization
The complete mitochondrial genome of Buteo is 18,674 bp in size. In comparison with other complete avian mt genomes, which range from 16,591 bp (Struthio) to 18,068 bp (Falco), Buteo has the largest mt genome sequenced so far. This considerable difference in size is due to an expansion of noncoding sequences. The three bird species with the largest portions of noncoding mt DNA (Buteo, 17.1%; Falco, 14.1%; Smithornis, 10.7%) possess, in addition to the CR, a second nc region ({Psi}CR). The gene content of the Buteo mt genome is typical of vertebrates (fig. 1 and table 2 ), consisting of genes for 13 proteins (12 H-strand-encoded, 1 L-strand-encoded), 22 tRNAs, and 2 rRNAs.


View this table:
[in this window]
[in a new window]
 
Table 2 Organization of the Mitochondrial Genome of Buteo buteo

 
The nucleotide composition of the Buteo mt genome (H-strand) is similar to that of other avian species (A = 31.1%, C = 31.7%, G = 13.2%, T = 24.0%). The A+T content of 55.1% is within the range for avian mt genomes (51.6%–55.7%). The usage of translation initiation and termination signals in comparison with other bird species is given in table 3 . The most common start codon is ATG. In Buteo, nonstandard start codons are found in the cox1 and nd5 genes (GTG) and in the nd3 gene (ATC). The unusual ATC start codon in the nd3 gene is also found in Smithornis. As in the mt genomes of the other birds and Alligator, TAA is the most frequent stop codon in Buteo; TAG and AGG are used twice. In cox3 and nd4, a terminal T probably serves as the stop signal after it is completed to UAA by posttranscriptional polyadenylation (Ojala, Montoya, and Attardi 1981Citation ). Buteo and Falco differ in two start codons and in three stop codons. With respect to the length of intergenic spacers and overlaps, Buteo (67-bp spacers) has a rather compact genome compared with Falco (101-bp spacers) (table 4 ).


View this table:
[in this window]
[in a new window]
 
Table 3 Usage of Start and Stop Codons

 

View this table:
[in this window]
[in a new window]
 
Table 4 Spacer and Overlaps

 
Gene Order and Noncoding Regions
In contrast to the standard avian gene order (e.g., Gallus), the CR of Buteo is located between tRNAThr and tRNAGlu. Between tRNAGlu and tRNAPhe, at the position of the CR in the majority of the birds analyzed so far, another nc section is found in Buteo which was designated a {Psi}CR by Haring et al. (1999)Citation . It consists of a short nonrepetitive part (23.2% of its length) followed by 48-bp tandem repeats (76.8%). A similar array of nc sections (designated CR and nc) has been described for the mt genome of F. peregrinus (Mindell, Sorenson, and Dimcheff 1998Citation ). Thus, gene order and content are identical in Buteo and Falco but differ from the standard gene order of many other birds (including Ciconia, Gallus, Aythya, Rhea, Struthio, Vidua, and Corvus) with respect to CR and {Psi}CR (table 5 ). The gene order of Buteo and Falco is similar to that of Smithornis (Mindell, Sorenson, and Dimcheff 1998Citation ) and Phylloscopus (Bensch and Härlid 2000Citation ), but the {Psi}CR regions of these species lack any repetitive parts. An appreciable degree of sequence identity is found between Buteo and Falco in the nonrepetitive sections (fig. 2 ) of the CR (71.4%, 691 bp; uncorrected, gaps >=5 excluded) and the {Psi}CR (58.2%, 189 bp; gaps >=5 excluded). No sequence similarity was detectable in an intragenomic comparison between CR and {Psi}CR of Buteo. Some structural features of the nc sequences and their tandem-repetitive sections are compared in table 6 . The majority of birds listed in this table have tandem repeats within their CRs. The CR of Buteo contains seven different types of repeats; that of Falco contains only two. The mt genome of Buteo has the largest percentage of tandem repetitive sequences (9.6%). We could find no sequence relationships between the repeat units in the CR and the {Psi}CR within or between the mt genomes of Buteo and Falco. In both species, the largest repetitive block is located in the {Psi}CR (table 6 ), but the repeat units differ considerably in length (48 vs. 27 bp). We sequenced the {Psi}CRs of two other accipitrids (Aquila chrysaetos and Haliaeetus albicilla) which had the same gene order as Buteo and Falco. Although the repeat units in these species resemble that of Buteo in length (Aquila, 49 bp; Haliaeetus, 48 bp), there is no apparent relatedness among them at the sequence level.


View this table:
[in this window]
[in a new window]
 
Table 5 Different Gene Orders in Bird and the Alligator

 


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 2.—Comparison of the control regions (CRs) of Buteo, Falco, and Ciconia. Conserved sequence blocks (CSBs) are depicted in black, and repetitive parts are depicted in gray. Designations of CSBs (above CR) are described in the text; designations of repeats (below CR) correspond to table 5 . Nonrepetitive sections used for the calculation of sequence divergence between Buteo, Falco, and Ciconia are marked by gray bars

 

View this table:
[in this window]
[in a new window]
 
Table 6 Comparison of Genome Size and Noncoding (NC) Sections

 
An alignment of the nonrepetitive parts of the CRs of Buteo, Falco, and Ciconia (not shown) revealed several conserved sections (fig. 2 ). Some of them show similarities to conserved sequence blocks (CSBs) of possible functional importance (CSB1, E box, D box) which have previously been described by Walberg and Clayton (1981)Citation , Baker and Marshall (1997)Citation , Randi and Lucchini (1998)Citation , and Clayton (1991)Citation . The section designated "C stretch" is located close to the 5' end and corresponds to the "goose hairpin" in other avian species (Quinn and Wilson 1993Citation ; Randi and Lucchini 1998Citation ), which consists of a stem of seven complementary C's/G's and a loop containing a TCCC motif that may be involved in H-strand termination (Dufresne, Mignotte, and Guéride 1996Citation ). Yet, in the CRs of Buteo, Falco, and Ciconia, the C stretch is not followed by a G stretch, and thus the motif lacks the ability to form a hairpin structure. The predicted TCCC stop motif within the C stretch is found in Buteo and Falco, whereas in Ciconia the corresponding motif is ACCC. Downstream of the C stretch, a 41-bp section corresponding to the consensus sequence of the mammalian ETAS1 (extended termination-associated sequence) described by Sbisà et al. (1997)Citation is found in all three species, but there are no sequences capable of forming hairpin structures. The Buteo ETAS1 contains two termination-associated sequence (TAS) motifs, TACAT and TATAT, whereas in Falco and Ciconia such motifs are absent. Five additional CSBs are found in the three species: (1) a 34-bp section that includes a sequence similar to the E box, although this motif is less conserved than the rest of the section; (2) a 25-bp stretch with high similarity to the D box; (3) a 40-bp section, designated CSB-a, which has no similarity to other described CSBs; (4) a 31-bp section, designated CSB-b, with no similarity to other CSBs (in Buteo, the section surrounding CSB-b is duplicated [97-bp repeat; table 6 ], and therefore this motif is present in two copies); and (5) CSB1, which is well conserved. The following motifs conserved in other avian species cannot be identified in Buteo, Falco, or Ciconia: CSB2 and CSB3 (Walberg and Clayton 1981Citation ), a polyC sequence upstream of CSB1 assumed to represent the origin of H-strand replication (Walberg and Clayton 1981Citation ), and the putative bidirectional promoter (L'Abbé et al. 1991Citation ).

Phylogenetic Analyses
The phylogenetic analysis was carried out using the coding regions of Buteo and the nine bird species listed above and Alligator. Each gene was aligned separately after excluding nonmatching positions at the length-variable 3' ends. Although the Alligator sequence was considerably diverged from the avian sequences, a reasonable alignment could be achieved. In a distance matrix (not shown) calculated from the concatenated coding sequences (15,742 bp), the distances (HKY85; Hasegawa, Kisgino, and Yano 1985Citation ) between all ingroup taxa and Alligator, respectively, are rather homogenous, indicating no major differences in substitution rates. The distances among ingroup taxa range from 18.1% to 27.2%. The distance between Buteo and Falco (21.3%) is on the same order of magnitude as that between the two struthioniform species Rhea and Struthio (19.6%) and between the two galloanserine species Gallus and Aythya (22.3%). Concerning the phylogenetic relationships of Buteo, it is remarkable that with 11 out of 14 genes compared, Buteo appears more closely related to Ciconia than to Falco (table 7 ), an affiliation that does not conform with classical taxonomy. The divergence of the total coding sequence is more extensive between Buteo and Falco than between Buteo and Ciconia ({chi}2 = 34.86, df = 1, P < 0.001).


View this table:
[in this window]
[in a new window]
 
Table 7 Sequence Divergences of Mitochondrial Genes

 
Table 7 shows the uncorrected distances (DNA and proteins) between pairs of related species calculated for different sections of the mt genome. In general, for each region, the values observed for the six species pairs are in the same range. In the Buteo/Falco comparison, the most conserved regions are the tRNAs (with respect to 16S rRNA: {chi}2 = 7.55, df = 1, P < 0.01; with respect to cox1: {chi}2 = 6.72, df = 1, P < 0.01). Among the protein-coding genes, cox(1–3) + cyt b are more conserved than nd(1–6) + atp(8+6) ({chi}2 = 46.63, df = 1, P < 0.001). Among amino acid sequences, COX(1–3) + CYTb are also more conserved than ND(1–6) + ATP(8+6) ({chi}2 = 93.18, df = 1, P < 0.001).

An MP dendrogram based on the complete coding sequence (15,742 bp) is depicted in figure 3 . Three species pairs are supported by high bootstrap values: Gallus/Aythya, Rhea/Struthio, and Corvus/Vidua. In the cluster of the three ciconiiform species, Buteo and Falco appear as sister taxa, although with weak bootstrap support. The passeriform split is at the base of the tree, yet Passeriformes do not form a monophyletic group, since Smithornis branches off as the most basal bird taxon. The corresponding NJ dendrogram (not shown) resembles the MP dendrogram with two exceptions: Buteo clusters with Ciconia (bootstrap value = 97), and Smithornis is not the sister group to all other ingroup taxa. Instead, there is a trichotomy of the three lineages (Smithornis/CorvusVidua/remaining avian taxa). In both the MP and the NJ dendrograms, the passeriform taxa are placed at the base of the birds, and the two pairs Gallus/Aythya and Rhea/Struthio are grouped as one clade that is the sister group of the cluster Buteo/Falco/Ciconia. Dendrograms were also calculated from deduced protein sequences and from DNA sequences using transversions only (data not shown). However, in general, better resolution and stronger bootstrap support were obtained with DNA sequences that included all substitutions, so those were used for subsequent phylogenetic analysis.



View larger version (16K):
[in this window]
[in a new window]
 
Fig. 3.—Single most-parsimonious tree (tree length = 21,916, consistency index = 0.588) for complete mt coding sequences of 10 avian taxa (15,742 bp) generated with a heuristic search using the tree bisection-reconnection (TBR) algorithm and a random-taxon-addition sequence. Gaps were treated as "missing," and all characters were weighted equally. Alligator was used as the outgroup. Bootstrap values (%, 100 replicates) are given at the nodes. Branch lengths are proportional to nucleotide substitutions. The bar indicates 500 substitutions

 
To estimate in detail which clusterings were supported by different sections of the mt genome, MP analyses were performed for each gene separately, as well as for combinations of genes (e.g., rRNAs + tRNAs; nd1–6; complete coding sequence). The three species pairs observed in the majority of these dendrograms are Rhea/Struthio, Corvus/Vidua, and Gallus/Aythya, which is in accordance with traditional classification. However, the position of Smithornis varied considerably. Often, it appeared as the sister taxon of the two other passeriform species Corvus/Vidua, as expected, but in some cases it clustered with other clades or else branched off at the base of the ingroup taxa. The results are summarized in table 8 , where bootstrap values >50 supporting different species pairs are given. One gene, cox3, does not support any of the splits; two others, nd3 and nd6, support only one, namely, Rhea/Struthio and Corvus/Vidua, respectively. Two genes (12S and nd5) support the Buteo/Ciconia clade, but with comparatively low bootstrap values.


View this table:
[in this window]
[in a new window]
 
Table 8 Comparison of Bootstrap Values

 
Altogether, the relationships among the ciconiiform species Buteo/Falco/Ciconia are not clearly resolved. To test whether the use of Alligator as an outgroup taxon might influence the branching pattern of these three species, MP dendrograms with different outgroup taxa (or sets of taxa) were calculated. First, the Alligator sequence was omitted, and the clade Smithornis/Vidua/Corvus was used instead as an outgroup. Trees were calculated from the total coding sequence as well as from a combination of rRNAs and tRNAs. In these dendrograms, the Buteo/Falco cluster is supported by high bootstrap values. When the set of ingroup taxa is reduced to Buteo/Falco/Ciconia and one of the three species Gallus, Rhea, or Vidua is used as outgroup, the Buteo/Falco clade appears in all trees derived from tRNA and/or rRNA genes (bootstrap values = 56–88). In the trees obtained from the total coding sequence, Buteo clusters either with Falco or with Ciconia. Relative-rate tests (Takezaki, Rzhetsky, and Nei 1995Citation ) were carried out to test whether these discrepancies were due to differences in substitution rates among the Buteo/Falco/Ciconia lineages. Since the use of a closely related outgroup makes the statistical test more accurate and powerful, we used Gallus, a taxon of the sister clade, as an outgroup. First, tests were performed with two of the three species forming group A and the third representing group B. In these tests, the relative-rate test Z-statistic (Z = 0.055) rejected rate constancy (P < 0.05) only for the constellation Falco+Buteo/Ciconia. When only two of the three species were tested, rate constancy was always rejected at the 5% level. Falco had the highest rate, followed by Buteo and Ciconia. The Z values for the three tests were 2.69829 (Buteo/Falco), 3.04896 (Buteo/Ciconia), and 5.55873 (Falco/Ciconia).


    Discussion
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Supplementary Material
 Acknowledgements
 References
 
Gene Order and Noncoding Regions
The sequence data revealed that Buteo and Falco share the same mt gene order, one which is different from that of most other bird species. This difference could have arisen in the common ancestor of Falconidae and Accipitridae prior to their split, or it could have arisen independently, as has been postulated for other bird lineages. As a possible mechanism for the rearrangement in Falco, a tandem duplication of the entire region including the CR and tRNAGlu with subsequent deletion of duplicated sequences except parts of the CR has been assumed (Mindell, Sorenson, and Dimcheff 1998Citation ). While in Falco the sequence similarity between CR and {Psi}CR corroborates this hypothesis, in Buteo no apparent sequence homology can be detected between the CR and the {Psi}CR. The CRs of Buteo and Falco can be well aligned, at least in the conserved parts, whereas no reasonable alignment could be achieved between the {Psi}CR of Buteo and the corresponding {Psi}CR region of Falco. Therefore, it cannot be decided from sequence divergence alone whether the similar rearrangements in Buteo and Falco are a shared derived character, nor can we trace unequivocally the events (duplication or transposition) that caused the rearrangements. Nevertheless, a strong argument in favor of a common origin comes from structural similarities between the {Psi}CRs of Buteo and Falco. Both sequences are composed of a short nonrepetitive section followed by a long stretch of tandem repeats. The same structure has been found within the {Psi}CRs of two other accipitrids, A. chrysaetos (Masuda et al. 1998Citation ) and H. albicilla (present study). In contrast, Smithornis (Mindell, Sorenson, and Dimcheff 1998Citation ) and Phylloscopus (Bensch and Härlid 2000Citation ), two passeriform species that have presumably acquired the same gene order independently, do not possess any repetitive sequences in the region corresponding to the {Psi}CR in Buteo. The sequences of the {Psi}CR sections of other species (belonging to the Cuculiformes, Piciformes, and Passeriformes) with rearranged gene orders (described by Mindell, Sorenson, and Dimcheff 1998Citation ) have not yet been published. Thus, an array of tandem repeats in the {Psi}CR section of the mt genome has been detected so far only in falconid and accipitrid species.

Relationships Among Buteo, Falco, and Ciconia
Although we included all avian mt genomes available, our main interest in this study was the group Buteo/Falco/Ciconia. Nevertheless, despite the huge amount of DNA sequence, the relationships within this triad were not clearly resolved. Whereas in the distance-based dendrogram of the complete coding sequence Buteo clusters with Ciconia, the MP analysis yielded the expected group Buteo/Falco. Saturation effects are not the only explanation for the poor resolution, because the topology of the rest of the tree (with the exception of the position of Smithornis) seems to be unambiguous. It is more likely that unequal substitution rates among Buteo, Falco, and Ciconia are the reason for the conflicting topologies. Another possibility might be that the radiation of the lineages leading to Buteo, Falco, and Ciconia occurred within a relatively short time frame. The fossil evidence for the first appearance of the three lineages is rather vague: the earliest storklike fossil, Palaeoepphippiorhynchus, stems from the early Oligocene (37–26 MYA) of Fayum, Egypt (Olson 1985Citation ). The oldest accipitrid fossils are also from early Oligocene deposits in France. These are thought to be Buteo-like (Newton and Olsen 1990Citation ), but according to Feduccia (1996)Citation , these remains are in need of revision. Falconids have been reported from 55 MYA (del Hoyo, Elliott, and Sargatal 1994Citation ) and 48 MYA (Peters 1991Citation ), respectively, but from fragmentary fossils. The first well-documented falconids, however, were described from the Eo-Oligocene in France and England (Peters 1991Citation ). Therefore, from the fossil record, it is not possible to decide which of the three lineages split first. With respect to anatomical traits, storks differ from accipitrids and falconids mainly in numerous characteristics of skeletal bone structure, skull formation, and the arrangement of some muscles (Rea 1983Citation ). One exceptional behavioral trait which storks share with cathartids, but not with accipitrids and falconids, is that storks keep cool by squirting their legs with urine. Although the distance-based algorithms favor a topology with Buteo and Ciconia as sister taxa, a closer relationship between Buteo and Falco is suggested by the following arguments: In the CR, the most variable part of the mt genome, sequence similarity is stronger between Buteo and Falco (71.4%, 691 bp; uncorrected, gaps >=5 excluded) than between either of them and Ciconia (Buteo/Ciconia: 66.8%; Falco/Ciconia: 69.0%, 691 bp; uncorrected, gaps >=5 excluded). Furthermore, Buteo and Falco share, in contrast to Ciconia, the same derived rearrangement of the CR. Thus, the topology of the ciconiiform clade in the MP dendrogram is not only in accordance with the classical taxonomic view, but is also corroborated by structural features of the mt genome.

Usefulness of Markers
Investigations of mt genes of various vertebrates by Russo, Takezaki, and Nei (1996)Citation indicated that amino acid sequences were more informative than nucleotide sequences for reconstructing reliable trees. Our results do not conform with this, since resolution, as well as bootstrap support, decreases when amino acid sequences are used. Moreover, nd5, cyt b, and nd4 do not appear to be the most appropriate genes in our analyses. Nevertheless, the two studies differ with respect to taxonomic level: Russo, Takezaki, and Nei (1996)Citation analyzed vertebrate phylogeny, whereas we focused on avian evolution only. Moreover, we also included RNA sequences for our comparisons.

With respect to usefulness of marker genes, the results of our MP analyses of nucleotide sequences can be interpreted as follows: In general, dendrograms derived from single mt genes have low resolution and bootstrap support for expected species pairs. Whereas most genes support the species pairs Corvus/Vidua and Rhea/Struthio, the clades Gallus/Aythya and Buteo/Falco are found in the 16S and tRNA trees only. Therefore, none of the protein-coding genes can be recommended as reliable markers for phylogenetic studies at this taxonomic level. Among the concatenated protein-coding sequences, cox(1–3) (although only half as long) appears better than nd(1–6). Concatenated rRNAs plus tRNAs (4,350 bp) resolve all four expected species pairs and are thus as good as the complete coding sequence (15,742 bp). In a dendrogram calculated from the complete coding sequence except nd5 and 12S, the two genes supporting the Buteo/Ciconia clustering, the bootstrap value of the Buteo/Falco clade rose to 92, while the rest of the tree remained unchanged. A tree based on only 16S and tRNAs yielded high bootstrap support in the Buteo/Falco/Ciconia cluster but lower values for the other nodes. To summarize, the concatenated RNA genes (rRNAs, tRNAs) appear to be the favorable combination, although bootstrap support is lower than that for the total sequence.

Basal Relationships and Dating of Splits
The phylogeny presented in this study, which is based on MP analyses of mt genomes, is in accordance with several other studies based on whole mt genomes (Härlid, Janke, and Arnason 1997, 1998Citation ; Härlid and Arnason 1999Citation ; Mindell et al. 1999Citation ; Waddell et al. 1999Citation ) and nuclear sequences (Stapel et al. 1984Citation ; Caspers et al. 1997Citation ). It is also compatible with the results of Griffiths (1994)Citation , based on syringeal morphology. In comparison to the dendrogram of Mindell et al. (1999)Citation , three additional taxa were included in the present study: Buteo, Ciconia, and Corvus. Whereas in the maximum-likelihood tree of Mindell et al. (1999)Citation Falco clusters with Smithornis, in our trees Smithornis never clusters with ciconiiform species. Instead, the Ciconiiformes appear as a stable monophyletic group that is the sister group of the clade Galloanserinae/Ratitae. The Passeriformes split at the base of the avian tree, although in some of the dendrograms they do not appear to be a monophyletic group. Instead, Smithornis (a representative of the suboscines, which are considered the most basal passeriform group) splits off as the most basal lineage of the dendrogram. Our mt-based phylogeny contradicts those derived from other studies of mt as well as nuclear genes, in which ratites appear at the base of the avian tree (Groth and Barrowclough 1999Citation ; van Tuinen, Sibley, and Hedges 2000Citation ). This incongruity is not necessarily due to marker selection (nuclear/mitochondrial). One reason for it might be that the studies differ in taxon composition. For example, in the dendrogram presented by Groth and Barrowclough (1999)Citation based on the nuclear gene RAG-1, no ciconiiform and no suboscine species are represented. On the other hand, sequences of the mt genomes of birds included in the RAG-1 study (cranes, loons, penguins, hemipodes, shorebirds, rollers) have not been published so far. Another reason for the incongruity might be the different lengths of the marker sequences (e.g., 3-kb RAG vs. 15,742-bp mt genes). In our MP dendrogram, the branches of the passeriform taxa appear shorter. This may be due either to the fact that the Alligator sequence is not a suitable outgroup to root the tree or to a slower substitution rate in the Passeriformes. Thus, we cannot rule out the possibility that the basal position of Passeriformes might be caused by long-branch attraction of the ciconiiform and galloanserine clusters. Altogether, a reasonable comparison between nuclear and mt-derived phylogenies will be possible only when (1) representatives of more avian groups (with both mt and nuclear genes) have been analyzed and (2) more nuclear sequence data (different genes) are available.

Unfortunately, the fossil record allows neither corroboration nor falsification of our data concerning the position of passeriformes. When the reports about the first appearances of avian groups are compared, no clear conclusions about the succession of splits or divergence times of the various lineages can be drawn. For example, according to Feduccia (1996Citation , p. 166), the oldest fossils of Ratitae are from the Paleocene (65–53 MYA), and according to Houde and Haubold (1987)Citation , the oldest ostrich fossils are from the early Eocene (53–37 MYA), the epoch from which the oldest putative passeriform fossils (Boles 1995Citation ) as well as falconids (see above) are also dated. As the oldest neognathous fossils are from the Mesozoic (Olson 1992), there is at least no support from the fossil record for the hypothesis that the Paleognathae represent the most basal lineage.

Faced with the incomplete fossil record for birds of prey, the dating of divergences has to rely on a molecular approach. The following molecular datings are available. For the Rhea/Gallus split, Härlid, Janke, and Arnason (1997)Citation calculated 80–90 MYA, and Waddel et al. (1999)Citation calculated 92 MYA. For the divergence of Aythya/Gallus, 68 MYA has been estimated (Waddel et al.1999Citation ). According to our HKY85 distances (22.3% for Aythya/Gallus, 23.7% for Galloanserinae/Struthioniformes), these two splits should be closer. From the two different reference points, the divergence of Buteo/Falco (21.3%) can be estimated to have occurred in the late Cretaceous, either at 72–83 MYA or at 65 MYA.


    Supplementary Material
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Supplementary Material
 Acknowledgements
 References
 
The complete sequence of the mt genome of B. buteo is registered under GenBank accession number AF380305. For the analysis of the {Psi}CR, the following partial sequences have been determined: {Psi}CR of H. albicilla, AY034150; repeat units of {Psi}CR of A. chrysaetos, AY034151. Alignments of avian mt sequences can be viewed under "Sequences" at our web site at http://www.nhm-wien.ac.at/NHM/1Zoo/first_zoological_department/web/chemsyst/cuhp_24e.html.


    Acknowledgements
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Supplementary Material
 Acknowledgements
 References
 
We are grateful to Werner Mayer for critical discussions and comments on the manuscript and to two anonymous referees for numerous valuable suggestions. This work was supported by the Austrian Science Foundation (FWF) (project number P14069-BIO).


    Footnotes
 
Elizabeth Kellogg, Reviewing Editor

1 Keywords: Buteo buteo mitochondrial genome avian phylogeny gene order control and pseudo control regions Back

2 Address for correspondence and reprints: Elisabeth Haring, 1. Zoological Department, Museum of Natural History Vienna, Burgring 7, A-1014 Vienna, Austria. elisabeth.haring{at}nhm-wien.ac.at . Back


    References
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Supplementary Material
 Acknowledgements
 References
 

    Avise J. C., W. S. Nelson, C. G. Sibley, 1994 DNA sequence support for a close phylogenetic relationship between some storks and New World vultures Proc. Natl. Acad. Sci. USA 91:5173-5177[Abstract]

    Baker A. J., H. D. Marshall, 1997 Mitochondrial control region sequences as tools for understanding evolution Pp. 51–82 in D. P. Mindell, ed. Avian molecular evolution and systematics. Academic Press, London

    Bensch S., A. Härlid, 2000 Mitochondrial genomic rearrangements in songbirds Mol. Biol. Evol 17:107-113[Abstract/Free Full Text]

    Bijlsma R. G., 1997 Buteo buteo—buzzard Pp. 160–161 in W. J. M. Hagemeijer and M. J. Blair, eds. The EBCC atlas of European breeding birds. T. & A. D. Poyser, London

    Boles W. E., 1995 The world's oldest songbird Nature 374:21-22

    Brown L. H., D. Amadon, 1968 Eagles, hawks and falcons of the world Wellfleet Press, Secaucus, N.J

    Caspers G.-J., D. U. de Weerd, J. Wattel, W. W. de Jong, 1997 {alpha}-crystallin sequences support a galliform/anseriform clade Mol. Phylogenet. Evol 7:185-188[ISI][Medline]

    Clayton D. A., 1991 Replication and transcription of vertebrate mitochondrial DNA Annu. Rev. Cell Biol 7:453-478[ISI]

    Cracraft J., 1981 Toward a phylogenetic classification of the recent birds of the world (class Aves) Auk 98:681-714[ISI]

    del Hoyo J., A. Elliot, J. Sargatal, 1994 Handbook of the birds of the world Vol. 2. Lynx Edicions, Barcelona

    Desjardins P., R. Morais, 1990 Sequence and gene organization of the chicken mitochondrial genome. A novel gene order in higher vertebrates J. Mol. Biol 212:599-634[ISI][Medline]

    Dufresne C., F. Mignotte, M. Guéride, 1996 The presence of tandem repeats and the initiation of replication in rabbit mitochondrial DNA Eur. J. Biochem 235:593-600[Abstract]

    Feduccia A., 1996 The origin and evolution of birds Yale University Press, New Haven, Conn

    Griffiths C. S., 1994 Monophyly of the Falconiformes based on syringeal morphology Auk 111:787-805[ISI]

    Groth J. G., G. F. Barrowclough, 1999 Basal divergences in birds and the phylogenetic utility of the nuclear RAG-1 gene Mol. Phylogenet. Evol 12:115-123[ISI][Medline]

    Haring E., M. J. Riesing, W. Pinsker, A. Gamauf, 1999 Evolution of a pseudo-control region in the mitochondrial genome of Palearctic buzzards (genus Buteo) J. Zool. Syst. Evol. Res 37:185-194[ISI]

    Härlid A., U. Arnason, 1999 Analyses of mitochondrial DNA nest ratite birds within the Neognathae supporting a neotenous origin of ratite morphological characters Proc. R. Soc. Lond. B Biol. Sci 266:305-309[ISI]

    Härlid A., A. Janke, U. Arnason, 1997 The mtDNA sequence of the ostrich and the divergence between paleognathous and neognathous birds Mol. Biol. Evol 14:754-761[Abstract]

    ———. 1998 The complete mitochondrial genome of Rhea americana and early avian divergences J. Mol. Evol 46:669-679[ISI][Medline]

    Hasegawa M., H. Kishino, T.-A. Yano, 1985 Dating of the human-ape splitting by a molecular clock of mitochondrial DNA J. Mol. Evol 22:160-174[ISI][Medline]

    Houde P., H. Haubold, 1987 Palaeotis weigelti restudied: a small Eocene ostrich (Aves: Struthioniformes) Paleovertebrata 17:27-42

    Janke A., U. Arnason, 1997 The complete mitochondrial genome of Alligator mississippiensis and the separation between recent archosauria (birds and crocodiles) Mol. Biol. Evol 14:1266-1272[Abstract]

    Jollie M. T., 1976 A contribution to the morphology and phylogeny of the Falconiformes, part 1 Evol. Theory 1:285-298

    ———. 1977 A contribution to the morphology and phylogeny of the Falconiformes, part 2–3 Evol. Theory 2:15-300

    Kimura M., 1980 A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences J. Mol. Evol 16:111-120[ISI][Medline]

    Knight A., D. P. Mindell, 1993 Substitution bias, weighting of DNA sequence evolution, and phylogenetic position of Fea's viper Syst. Biol 42:18-31[ISI]

    König C., 1982 Zur systematischen Stellung der Neuweltgeier (Cathartidae) J. Ornithol 123:259-267[ISI]

    Kumar S., 1996 PHYLTEST: a program for testing phylogenetic hypotheses Version 2.0. Institute of Molecular Evolutionary Genetics and Department of Biology, Pennsylvania State University, University Park

    L D. L., J.-F. Duhaime, B. F. Lang, R. Morais, 1991 The transcription of DNA in chicken mitochondria initiates from one major bidirectional promoter J. Biol. Chem 266:10844-10850[Abstract/Free Full Text]

    Masuda R., M. Noro, N. Kurose, C. Nishida-Umehara, H. Takechi, T. Yamazaki, M. Kosuge, M. C. Yoshida, 1998 Genetic characteristics of endangered Japanese golden eagles (Aquila chrysaetos japonica) based on mitochondrial DNA D-loop sequences and karyotypes Zoo Biol 17:111-121[ISI]

    Mindell D. P., M. D. Sorenson, D. E. Dimcheff, 1998 Multiple independent origins of mitochondrial gene order in birds Proc. Natl. Acad. Sci. USA 95:10693-10697[Abstract/Free Full Text]

    Mindell D. P., M. D. Sorenson, D. E. Dimcheff, M. Hasegawa, J. C. Ast, T. Yuri, 1999 Interordinal relationships of birds and other reptiles based on whole mitochondrial genomes Syst. Biol 48:138-152[ISI][Medline]

    Newton I., P. Olsen, 1990 Birds of prey Merehurst Press, London

    Ojala D., J. Montoya, G. Attardi, 1981 tRNA punctuation model of RNA processing in human mitochondria Nature 290:470-474[ISI][Medline]

    Olsen S. L., 1992 Neogaeornis wetzeli Lambrecht, a cretaceous loon from Chile (Aves: Gaviidae) J. Vertebr. Paleontol 12:122-124

    Olsen P. D., 1995 Australian birds of prey Helm, Melbourne

    Olson S. L., 1985 The fossil record of birds Pp. 79–238 in D. S. Farner, J. R. King, and K. C. Parkes, eds. Avian biology. Vol. 8. Academic Press, New York

    Peters D. S., 1991 Zoogeographical relationships of the Eocene avifauna from Messel (Germany) Acta XXth Intl. Ornithol. Congr 1:572-577

    Quinn T. W., 1997 Molecular evolution of the mitochondrial genome Pp. 4–28 in D. P. Mindell, ed. Avian molecular evolution and systematics. Academic Press, London

    Quinn T. W., A. C. Wilson, 1993 Sequence evolution in and around the mitochondrial control region in birds J. Mol. Evol 37:417-425[ISI][Medline]

    Randi E., V. Lucchini, 1998 Organization and evolution of the mitochondrial DNA control region in the avian genus Alectoris J. Mol. Evol 47:449-462[ISI][Medline]

    Rea A. M., 1983 Cathartid affinities: a brief overview Pp. 26–54 in S. R. Wilbur and J. A. Jackson, eds. Vulture biology and management. University of California Press, Berkeley

    Russo C. A. M., N. Takezaki, M. Nei, 1996 Efficiencies of different genes and different tree-building methods in recovering a known vertebrate phylogeny Mol. Biol. Evol 13:525-536[Abstract]

    Saitou N., M. Nei, 1987 The neighbor-joining method: a new method for reconstructing phylogenetic trees Mol. Biol. Evol 4:406-425[Abstract]

    Sbis E., F. Tanzariello, A. Reyes, G. Pesole, C. Saccone, 1997 Mammalian mitochondrial D-loop region structural analysis: identification of new conserved sequences and their functional and evolutionary implications Gene 205:125-140[ISI][Medline]

    Seibold I., A. J. Helbig, 1995 Evolutionary history of New and Old World vultures inferred from nucleotide sequences of the mitochondrial cytochrome b gene Philos. Trans. R. Soc. Lond. B Biol. Sci 350:163-178[ISI][Medline]

    Sibley C., J. Ahlquist, 1990 Phylogeny and classification of birds of the world Yale University Press, New Haven, Conn

    Sorenson M. D., J. C. Ast, D. E. Dimcheff, T. Yuri, D. P. Mindell, 1999 Primers for PCR-based approach to mitochondrial genome sequencing in birds and vertebrates Mol. Phylogenet. Evol 12:105-114[ISI][Medline]

    Stapel O., J. A. M. Leunissen, M. Versteeg, J. Wattel, W. W. de Jong, 1984 Ratites as oldest offshoot of avian stem-evidence from {alpha}-crystallin A sequences Nature 311:257-259[ISI][Medline]

    Storer R. W., 1971 Classification of birds Pp. 1–19 in D. S. Farner and J. King, eds. Avian biology. Academic Press, New York

    Stresemann E., D. Amadon, 1979 Falconiformes Pp. 271–424 in E. Mayr and G. W. Cottrell, eds. Checklist of the birds of the world. Vol. 1. Harvard University Press, Cambridge, Mass

    Swofford D. L., 1998 PAUP*: phylogenetic analysis using parsimony (*and other methods) Version 4.0b3a. Sinauer, Sunderland, Mass

    Takezaki N., A. Rzhetsky, M. Nei, 1995 Phylogenetic test of the molecular clock and linearized trees Mol. Biol. Evol 12:823-833[Abstract]

    Thompson J. D., T. J. Gibson, F. Plewniak, F. Jeanmougin, D. G. Higgins, 1997 The ClustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools Nucleic Acids Res 25:4876-4882[Abstract/Free Full Text]

    van Tuinen M., C. G. Sibley, S. B. Hedges, 2000 The early history of modern birds inferred from DNA sequences of nuclear and mitochondrial ribosomal genes Mol. Biol. Evol 17:451-457[Abstract/Free Full Text]

    Waddell P. J., Y. Cao, M. Hasegawa, D. P. Mindell, 1999 Assessing the cretaceous superordinal divergence times within birds and placental mammals by using whole mitochondrial protein sequences and an extended statistical framework Syst. Biol 48:119-137[ISI][Medline]

    Walberg M. W., D. A. Clayton, 1981 Sequence and properties of the human KB cell and mouse L cell D-loop regions of mitochondrial DNA Nucleic Acids Res 9:5411-5421[Abstract]

    Wink M., I. Seibold, F. Lotfikhah, W. Bednarek, 1998 Molecular systematics of holarctic raptors (order Falconiformes) Pp. 29–47 in R. D Chancellor, B.-U. Meyburg, and J. J. Ferrero, eds. Holarctic birds of prey. IGRAMEX, Calamonte, Spain

Accepted for publication June 12, 2001.